Proteomic analysis of sea urchin immune responses and characterisation of highly variable immune response proteins. Macquarie University, Sydney, Australia

Proteomic analysis of sea urchin immune

responses and characterisation of highly

variable immune response proteins

Dheilly Nolwenn Marie

(B.Sc.) Licence de biologie des organismes option toxicologie (UBO, Brest)

(M.Sc.) Maîtrise de biologie des populations et écosystèmes marins options biodiversité

et biochimie, Institut Universitaire Européen de la Mer (UBO, Brest)

(M.Sc.) Master de protéomique (USTL, Lille)

Macquarie University

A thesis submitted to Macquarie University in partial fulfilment of the

requirements of the degree of Doctor of Philosophy

Department of Biological Sciences


Sydney, NSW

Australia

November 2009

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iii

TABLE OF CONTENTS

Title page i

Table of contents iii

List of Figures and Tables ix

List of abbreviations xiii

Summary xv

Declaration of authorship and originality xvii

Acknowledgements xix

CHAPTER I: General Introduction 1

1.1. Sea urchins 3

1.1.1. Echinoderms and other deuterostomes 3

1.1.2. Evolutionary considerations within the echinoderms 5

1.1.3. Classification 5

1.1.4. Sea urchin anatomy 7

1.1.5. Coelomic fluid and coelomocytes 10

1.1.6. The sea urchin genome project 14

1.2. Comparative immunology of echinoderms 16

1.2.1. Sea urchin immune response molecules 16

1.2.2. The complement system of sea urchins 17

1.2.3. The lectin-mediated complement system in sea urchins 19

1.2.4. Immunoglobulin superfamily rearrangement 19

1.3. Highly variable gene systems associated with pathogen defence 23

1.3.1. Life history strategies 24

1.3.2. Variable lymphocyte receptors (VLRs) 25

iv

1.3.3. V-region-containing chitin-binding protein (VCBPs) 26

1.3.4. Fibrinogens related proteins (FREPs) 27

1.3.5. Down’s syndrome cell adhesion molecules (Dscam) 28

1.4. The 185/333 family 30

1.4.1. Discovery 30

1.4.2. Variability of 185/333 mRNAs 31

1.4.3. 185/333 genes 35

1.4.4. Transcriptional and posttranscriptional modifications 39

1.4.5. 185/333 proteins expression and localization 40

1.5. Proteomics and sea urchins 43

1.5.1. From the static image to the dynamic image 43

1.5.2. Gel-based versus gel-free proteomics 45

1.5.3. Gel-based comparative proteomics 47

1.5.4. Gel-free comparative proteomics 49

1.5.5. Proteomics and sea urchins 50

1.6. The aims of this thesis 52

1.7. References 53

CHAPTER II: Shotgun proteomic analysis of coelomocytes from the purple sea

urchin, Strongylocentrotus purpuratus 71

2.1. Preface 73

2.2. Abstract 75

2.3. Dataset Brief 76

2.4. Acknowledgments 87

2.5. References 88

v

CHAPTER III: Proteomic analysis of sea urchin responses to bacterial injection 113

3.1. Preface 115

3.2. Abstract 117

3.3. Introduction 118

3.4. Materials and Methods 120

3.4.1. Sea urchins 120

3.4.2. Immunological challenge and sample collection 120

3.4.3. Protein extraction 120

3.4.4. Two-dimensional gel electrophoresis 121

3.4.5. Gel analysis and selection of protein spots for mass spectrometry

analysis 122

3.4.6. Peptides extraction 123

3.4.7. Nanoflow liquid chromatography – tandem mass spectrometry 123

3.4.8. Protein identification 124

3.5. Results 125

3.6. Discussion 134

3.7. References 137

CHAPTER IV: Timecourse proteomic profiling of cellular responses to

immunological challenge in sea urchins (Heliocidaris erythrogramma) 147

4.1. Preface 149

4.2. Summary 151


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4.4. Experimental procedures 155


4.4.2. Injections and sample collections 155

4.4.3. Protein extraction 156

4.4.4. One-dimensional sodium dodecyl sulfate – polyacrylamide gel

electrophoresis (1DE SDS-PAGE) 157

4.4.5. Nanoflow liquid chromatography – tandem mass spectrometry 157

4.4.6. Protein identification 158

4.4.7. Data analysis 158

4.5. Results 161

4.5.1. Reproducibility and threshold levels of shotgun MS/MS analysis 161

4.5.2. Functional classification of proteins 163

4.5.3. Identification of individual proteins affected by saline or LPS injection 167

4.6. Discussion 177


4.8. References 186

CHAPTER V: Highly variable immune-response (185/333) from the sea urchin,

Strongylocentrotus purpuratus: Proteomic analysis identifies diversity within and

between individuals. 209

5.1. Preface 211

5.2. Abstract 213




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5.4.2. Immunological challenge and sample collection 218

5.4.3. One-dimensional electrophoresis (1DE) 219

5.4.4. Two-dimensional electrophoresis (2DE) 219

5.4.5. Antibodies 220

5.4.6. Western blotting and immunostaining 220

5.4.7. Anti-185/333 ELISA 221

5.4.8. Mass spectrometry and data analysis 222

5.5. Results 225

5.5.1. One-dimensional SDS-PAGE analysis of CF proteins 225

5.5.2. Two-dimensional Western blots of CF proteins 227

5.5.3. Diversity of 185/333 proteins between individuals 229

5.5.4. MS analysis of 185/333+ proteins 233

5.5.5. 185/333 protein expression increases after LPS challenge 237

5.5.6. Diversity of 185/333 protein expression after immunological challenge 239

5.6. Discussion 243


5.8. Disclosures 250

5.9. References 251

CHAPTER VI: Ultrastructural localization of a highly variable immune response

protein (185/333) within coelomocytes and the gut of sea urchins 257

6.1. Preface 259

6.2. Summary 261



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6.4.2. Electron microscopy 266

6.4.3. Localization of 185/333 proteins 267

6.4.3.1. Anti-185/333 antisera 267

6.4.3.2. Immunofluorescence microscopy of whole cell 267

6.4.3.3. Immunofluorescence staining of semi-thin resin sections 268

6.4.3.4. Immuno-gold labelling for transmission electron microscopy 269

6.5. Results 271

6.5.1. Characterization of coelomocyte types 271

6.5.2. Immunofluorescence identification of 185/333 proteins in filopodial

amoebocytes and colorless spherule cells 273

6.5.3. Ultrastructure and 185/333 localization of coelomocytes 273

6.5.4. 185/333 proteins in the gut 281

6.5.5. Phagocytosis by coelomocytes 283

6.6. Discussion 286

6.7. References 291

CHAPTER VII: General discussion 297

7.1. General discussion 299

7.2. Caveats and future directions 306

7.3. Concluding remarks 307

7.4. References 308

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LIST OF FIGURES AND TABLES

CHAPTER I

Table 1.1: Taxonomy of the two sea urchin species studied in this thesis

Table 1.2: Coelomocytes cell types and functions in the purple sea urchin, S.

purpuratus 11

Table 1.3: General characteristics of highly variable gene systems associated with

pathogen defence in animals 22

Table 1.4: Methods of protein detection for gel-based proteomics 48

Figure 1.1: Position of echinoderms in animal phylogeny relative to the four genera of

animals from which genomes have so far been sequenced 4

Figure 1.2: Internal anatomy of the sea urchin 8

Figure 1.3:Coelomocytes in sea urchin (S. purpuratus) coelomic fluid 12

Figure 1.4: A cDNA-based alignment of 185/333 transcripts 32

Figure 1.5: Five sub-families of 185/333 proteins 33

Figure 1.6: Structure of part of the 185/333 locus 34

Figure 1.7: Repeat-based alignment of the 185/333 genes 36

Figure 1.8: Schematic representation of the consensus 185/333 protein sequence 38

Figure 1.9: Complexity of the proteome 42

Figure 1.10: Flow chart for gel-based and gel-free proteomics 44

CHAPTER II

Table 2.1: The thirty most abundant proteins identified in the coelomic fluid of 3

individual sea urchins 79

Figure 2.1: Functional classification of proteins identified from sea urchin CF 78

x

Figure 2.2: Predicted structure of the 16 scavenger receptor cysteine-rich (SRCR)

proteins identified in the coelomic fluid proteome 82

Supplementary Data 2.1: Detailed Materials and Methods 95

Supplementary Data 2.2: Proteins identified in the coelomic fluid of 3 individual sea

urchins 101

CHAPTER III

Table 3.1: Protein identification of 2DE gel spots from Figure 3.4 131

Figure 3.1: Principal components analysis of proteome profiles after the injection of

saline or bacteria 126

Figure 3.2: Number of 2DE spots that varied significantly in abundance after saline

and bacterial injections, as determined by quantitative analysis of Lava Purple

stained 2DE gels 128

Figure 3.3: Lava Purple stained 2DE maps of CF proteins following Vibrio Sp.

injection 129

Figure 3.4: Log2 normalized volumes of differentially regulated protein spots 130

Figure 3.5: Selected areas of 2DE maps showing proteins (spots 5 and 6) with

abundances that differed significantly (p<0.05) in response to the injection of

bacteria when compared to both non-injected controls and saline-injected sea

urchins 132

Supplementary Data 3.1: Mass spectrometric (LC-MS/MS) data for protein spots

isolated from 2DE gels 141

CHAPTER IV

Table 4.1: Individual proteins with significantly altered relative abundance 169

Figure 4.1: Experimental design 154

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Figure 4.2: Alteration in whole proteomes in response to injections 160

Figure 4.3: Relative abundance and functional classification of proteins 164

Figure 4.4: Consistency of the relative abundance data 168

Figure 4.5: Examples of proteins that differed in relative abundance over time but not

between treatments 174

Figure 4.6: Examples of proteins that differed in relative abundance over time and

between treatments 176

Supplementary Data 4.1: Normalized spectral abundance factors 195

CHAPTER V

Table 5.1: Mass spectrometric (LC-MS/MS) data for peptides isolated from 1DE gels

of S. purpuratus CF that match known 185/333 sequences 235

Figure 5.1: 1DE SDS-PAGE and Western blot of CF proteins from an individual sea

urchin 226

Figure 5.2: 2DE Western blot of 185/333+ proteins 228

Figure 5.3: Composite image of a 2DE Western blot 230

Figure 5.4: Enlarged region of a 2DE Western blot of CF proteins 230

Figure 5.5: Different anti-185 sera recognize subsets of 185/333 proteins 231

Figure 5.6: 1DE Western blots of CF from 13 different sea urchins sampled before (A)

and 96 h after (B) challenge with LPS 232

Figure 5.7: 1DE Western blots of CF proteins from three different sea urchins 234

Figure 5.8: The titer of 185/333 proteins in CF increases after immune challenge 238

Figure 5.9: 1DE Western blots of CF collected after sea urchins had been injected with

LPS or PG 240

Figure 5.10: 2DE Western blots of CF collected from a single sea urchin (animal 31)

that had been injected first with LPS and then 360 hours later with PG 242

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CHAPTER VI

Figure 6.1: Coelomocyte types in live cells preparation of H erythrogramma CF 270

Figure 6.2: Confocal images of 185/333-positive coelomocytes 272

Figure 6.3: Anti-185/333 immunogold stained transmission electron micrograph of a

colorless spherule cell 274


filopodial amoebocyte 276


Golgi apparatus in a filopodial amoebocyte 277

Figure 6.6: Transmission electron micrograph of 185/333-positive vesicles in

filopodial amoebocytes 278

Figure 6.7: Anti-185/333 immunogold stained transmission electron micrograph of

filopodial amoebocyte cell surface 279


gut-associated amoebocytes. 280


vesicles in anuclear bodies in gut tissue 282

Figure 6.10: Phagocytosis by petaloide amoebocytes 284


colorless spherule cells 285

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LIST OF ABBREVIATIONS

1DE; one-dimensional gel

electrophoresis

2DE; two-dimensional gel

electrophoresis

aa; amino acid

aCF; artificial coelomic fluid

ACN; acetonitrile

ANOVA; analyse of variance

ApoLp; apolipoprotein

BCP; 1-bromo-3-chloropropane

BSA; bovine serum albumine

CCT; chaperonin containing TCP1

CBD; chitin binding region

CC; coiled coil domain

CF; coelomic fluid

CID; collision induced dissociation

CMFSW-EI; calcium magnesium free

seawater with EDTA and imidazole

CR3; complement receptor 3

CT; cytoplasmic tail

Dscam; Down’s syndrome cell adhesion

molecule

DTT; dithiothreitol

ER; endoplasmic reticulum

EST; expressed sequence tag

emPAI; exponentially modified protein

abundance index

ESI; electrospray

FBS; fetal bovin serum

FDR; false discovery rate

FREP; fibrinogen-related protein

FSW; filtered seawater

GNBP; gram negative binding protein

G protein; guanine nucleotide binding

protein

GPI; glycosyl phosphatidulinositol

GPM; global proteome machine

HPLC; high performance liquid

chromatography

HSP; heat shock protein

IAA; iodoacetamide

IEF; isoelectrofocalisation

Ig; Immunoglobulin

IgSF; Immunoglobulin superfamily

IPG; immobilized pH gradient

LDL; low density lipoprotein

LLTP; large lipid transfer protein

LPS; lipopolysaccharides

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LRR; leucine-rich region

MALDI; matrix-assisted laser

desorption/ionization

MBP; mannose binding protein

MS; mass spectrometry

MS/MS; tandem mass spectrometry

MudPIT; multidimensional protein

identification technology

MW; molecular weight

MYP; major yolk protein

m/z; mass/charge ratio

nano-LC; nano-liquid chromatography

NBS; nucleotide binding site

NITR; novel immune-type receptor

NLR; NOD-like receptor

NSAF; Normalized spectral abundance

factor

PAMP; pathogen-associated molecular

pattern

PBS; phosphate buffered saline

PC; principal component

PCA; principal component analysis

PDI; protein disulfide isomerase

PG; peptidoglycan

PGRP; peptidoglycan recognition

protein

pI; isoelectric point

PMF; peptide mass fingerprinting

p.i.; post-injection

RACK; receptor of activated C kinase

RAG; recombination activator gene

SDS; sodium dodecyl sulfate

SpBf; S. purpuratus factor B

SpC3; S. purpuratus complement

component 3

SpC; spectral count

SRCR; Scavenger receptor cysteine-rich

SSH; suppressive substractive

hybridization

TdT; terminal deoxynucleotidyl

transferase

TIMP; tissue inhibitor of

metalloproteinase

TLR; Toll-like receptors

TM; transmembrane region

TEM; transmission electron microscopy

TOF; time-of-flight

VCBP; variable chitin-binding protein

VDAC; voltage dependent anion channel

vWF; von Willebrand factor

vWD; von Willebrand-factor type D

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SUMMARY

The sea urchin genome sequence identified a large repertoire of genes with

similarities to the immune response genes of vertebrates. This led to the prediction that sea

urchins have a complex immune system, involving a broad array of recognition and

effector proteins. An additional family of highly variable immune response genes (the

185/333 family) distinct from those of vertebrates has been identified in sea urchins by

transcriptomic analysis. Despite this detailed information at the genomic and

transcriptomic levels, complementary analyses have not been undertaken at the level of

proteins.

This thesis takes a proteomic approach to studying sea urchin immune systems. It

reveals a biphasic immune response involving a broad array of proteins. The initial

changes in the coelomic fluid proteome after immunological challenge involved alterations

associated with coelomocyte morphology and plasticity. These early responses did not

differ significantly between sea urchins injected with bacteria, lipopolysaccharides or

saline, suggesting that they were wounding responses. These wound reactions were

followed by a cellular response that showed more specificity to immunological challenge.

This suggests that sea urchin coelomocytes activate different cellular pathways in response

to wounding, as opposed to the presence of purified pathogen associated molecular

patterns (PAMPs) or whole bacteria.

It was also demonstrated that responses to PAMPs involved highly variable

immune response proteins, such as scavenger receptor cysteine rich proteins and 185/333

molecules. It was shown that the large suites of 185/333 proteins expressed by sea urchins

differed between individuals and were altered after challenge with different PAMPs.

185/333 proteins were localized in filopodial amoebocytes, gut-associated amoebocytes

and colorless spherule cells. The patterns of 185/333 protein expression after the injection

xvi

of pathogens suggest that they are involved in the ingestion of degenerative material within

the gut, and may be associated with wound healing and cytotoxicity in the coelomic fluid.

Overall, this thesis demonstrates the efficiency of proteomics in complementing

genomic and transcriptomic data to build a comprehensive picture of the biological

pathways involved in the inducible host defence responses of sea urchins.

xvii

DECLARATION OF AUTHORSHIP AND ORIGINALITY

The work presented in this thesis has not previously been submitted for a degree as

part of the requirements for a degree at any other university or institution. This thesis

contains only original material that has been written by me.

Any additional assistance received during the research work or in preparation of the

thesis itself has been indicated in the appropriate section. I also certify that all information

and literature sources used during the preparation of this thesis have been acknowledged.

Nolwenn M. Dheilly

Department of biological sciences


North Ryde

NSW, 2109

Australia

xviii

xix

ACKNOWLEDGEMENTS

I left France 4 years ago, wishing to enrol in a PhD and to learn to speak English. I

could have taken a postgraduate course in France but a nagging voice in my head was

telling me that if I didn't go now, I never would. Even though these two aims were fulfilled,

that's not all I remember of this wonderful experience. I immersed myself in a fascinating

foreign culture, I learned a lot at university and met wonderful people that accompanied

me through the emotional highs and lows. My time in Sydney as a PhD candidate has

been an adventure, a love-hate relationship, charged with so much energy that it would be

hard to explain. I have many people to thank for these memories that I will cherish

forever. I might not be able to thank them all within this short section but I will do my

best.

First of all, I thank A. Prof David Raftos and Dr. Sham Nair for inviting me to come

to Australia to undertake my PhD in the Marine and Freshwater Biology Laboratory of

Macquarie University. At the time, I could barely speak English but they trusted me and

believed I could learn. David was very supportive and made me feel like I was wanted in

the lab, which encouraged me study English harder. Unable to make friends with my

limited linguistic abilities, lost in this newly discovered country, his friendship was all I

had and I probably did not thank him enough for that at the time. Over the past three and

a half years, David proved to be a great supervisor and I thank him for his kindness,

accessibility and support. He is a fantastic writer and his help during the writing of my

manuscripts was extremely precious. Most of all, his presence was reassuring and helped

me throughout the hard times, always knowing that he would be there.

My co-supervisor, Dr. Sham Nair was of tremendous help too. He is always there,

always available, always supportive, always kind even if we, students, abuse his

availability much of the time. The door to his office is always open and whatever the

xx

problem is, he tries to solve it. His broad knowledge of the technologies we use is

precious. Most of all, I loved talking with him about science. Our conversations, even

when they were unrelated to my research project, always motivated me and made me want

to do more and try new things in the lab. His love of science is contagious. The ideas we

shared and the hypotheses that came out of our discussions will surely influence many of

my future research projects. From the bottom of my heart, I hope I will have the chance to

work with Sham again in the future.

Over the course of my PhD, I learned a number of new techniques and developed

others. These shaped my thesis and I have many people to thank for their help throughout.

First of all, I would like to thank our collaborator, Prof. Courtney Smith, who I finally met

this year. Even though we mostly communicated via emails, she was actively involved in

the work I undertook on 185/333 proteins. She had many ideas and inspired a number of

my experiments. Courtney's comments on my writing were also very helpful. She always

explained her comments, which helped improve all my manuscripts.

A. Prof. Paul Haynes’ unexpected help shaped the content of my thesis like few

others did. His challenging questions were at times frustrating but they always had an

insightful meaning. His knowledge in mass spectrometry was precious and also helped in

analysing my data and choosing the appropriate normalization methods. Paul, thanks a

lot for your help, without you my thesis would not have been so interesting and novel.

I would also like to thank Debra Birch and Nicole Vella for their tremendous help

when it came to microscopy. Their knowledge in microscopy techniques is extremely

valuable for Macquarie University. Microscopy is not easy; it requires patience, precision,

and time. Without the two of them, I would never have had the patience to keep going and

would not have produced such beautiful pictures that I am now proud to publish.

There are so many others that influenced me over the course of my PhD. I would

like to thank all the students that came through the lab overtime and also the staff and

xxi

students of the Australian Proteomic Analysis Facility, the Grain Foods CRC Group and

the Marine Mammals Research Group to name a few. We shared ideas, lab frustrations,

good news, chemicals, and also beers, food, coffee, laughter, and sport. Their friendship

was greatly appreciated and I wish them all the best for the future.

Because my time in Australia would have not been the same without them, I would

like to thank the new friends I made. They came from all over the world but all shared the

same qualities: a pure heart and a curiosity over foreign cultures. I learned a lot from

them and I hope I also gave them the best of myself. To name a few, I would like to thank

Andrew “Mooloo”, the best flatmate I ever had. I discovered Woolloomooloo with you and

fell in love with this area of Sydney. Thank you also to Gaetane, Esther, Chris and The

Banghlassi. I love your music guys. Thank you to all the bar flies of the Old Fitzroy. I had

the best time ever. Ellie Wilson was my first friend in Australia. I should say that we met

in the most awkward/funny way. Discovering Sydney and then Tasmania with you was a

great adventure. Mdaulin, I will miss our Sunday lunches, please come over and visit me,

wherever I am. Finally, I could never thank enough Ante Jerkovic and all his family for

their friendship, for welcoming me into their home and make me feel part of it. I feel lucky

I met the kindest people of all. Hvala mnogo.

Last but not least, I would like to thank my friends and family that I left behind

when I left France. Their love and support was my strength. Thank you for being you.

Merci a tous.

Firstly, I thank my Mum for always believing in me. Thank you for always telling

me that I could do it. Thank you also for your strength, happiness and all the fun. You are

the best example ever. I also would like to thank my forever gone Dad because he is the

one that introduced me to science and shared with me his love for the ocean. I only have

few memories of these old days but they are precious. Thank you Morgane, Pierre Yves

and Gaëtan, because growing up with you was quite a life experience in itself. You are

xxii

smart, challenging, funny, full-hearted people and I am glad that you all found somebody

that could see it and love you for it. I wish you all the best for the future.

Thank you Muriel, because asking me to be the godmother of your first child was

the best present you could ever give me. I promise you I will do my best to be somebody

special in Luna’s life, somebody she can always count on. I thank you for your friendship,

your support, your trust, and for the hours we spent on the phone sharing our experiences.

I also thank my friends Alex, Damien, Aurélien, and my symbiote Cédric. The bonds of love

that we share are so special that they will remain in my heart forever. I thank you for your

friendship and encouragements and for all the fun. See you soon boys.

Thank you all, Merci a tous.

1

CHAPTER I

General Introduction

2

3

1.1. Sea urchins

1.1.1. Echinoderms and other deuterostomes

Sea urchins are deuterostome invertebrates. “Deuterostome” comes from the Greek

“mouth second”, which refers to the initial formation of the anus by the blastopore during

development while the mouth forms later. Deuterostomes (hemichordates, echinoderms,

cephalochordates, urochordates and vertebrates) share other features, such as radial

cleavage of cells in early development and bilateral symmetry (at least during the early

stages) that further distinguish them from the protostome lineage of metazoans (mouth

first). Deuterostome divergence occurred approximately 575 million years ago in

Precambrian times [1]. Phylogenetic relationships among deuterostome animals have been

debated for many years with numerous hypotheses proposed based on both morphological

and molecular data. DNA analyses of the complete mitochondrial genome and 18S nuclear

RNA now supports the idea that hemichordates constitute a sister group to echinoderms

[2]. The position of urochordates and cephalochordates with regards to vertebrates has also

been a controversial topic. New evidence supports the conclusion that urochordates

represent the closest living relatives of vertebrates and that gene loss played a major role in

structuring the urochordate genome. Chordates are now thought to represent a

monophyletic clade (Figure 1.1).

4

Figure 1.1: Position of echinoderms in animal phylogeny relative to the four genera of

animals from which genomes have so far been sequenced (blue shading). Modified

from [3].

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1.1.2. Evolutionary considerations within the echinoderms

The name echinoderm comes from the Greek for “spiny skin”. There are around

7,000 echinoderm species, all of which inhabit marine environments. Most have a five fold

radial (pentamerous) body plan. They have several shared features that distinguish them

from other animals, such as a sea water vascular system and a calcium carbonate

endoskeleton, the stereom. Echinoderms first appeared in the fossil record around 520

million years ago during the lower Cambrian. Five taxonomic classes have been defined:

Crinoidea (sea lilies and feather stars); Asteroidea (starfishes); Ophiuroidea (basket stars

and brittle stars); Holothuroidea (sea cucumbers); and Echinoidea (sea urchins, sand

dollars and sea biscuits). Most echinoderms begin life as bilateral larvae and undergo

complex metamorphosis to form radially symmetrical adults.

1.1.3. Classification

Echinoids apparently first appeared during the Ordovician. They have imperforate,

non-crenulate tubercles, solid spines and shallow gill slits. There are numerous tropical and

temperate species. Euechinoids became the dominant echinoid form 250 million years ago,

after the great Permisian-Triassic extinction. Within the order Echninoidea there are four

families: Echinidae [4] (Echinus, Paracentrotus, parechinus, Psammechinus,

Pseudocentrotus, Colobocentrotus, Sterechinus and Loxechinus); Echinometridae [5]

(Anthocidaris, Heliocidaris, Echinometra, Heterocentrotus, Coenocentrotus and

Evehinus); Parasaleniidae [6] (Paraselinia) and Strongylocentrotidae [7] (Hemicentrotus,

Allocentrotus and Strongylocentrotus) that are principally distinguished by the character of

their pedicellariae.

6

Strongylocentrotidae and Echinometridae diverged around 35-45 million years ago

[8]. Evolution has been rapid among both lineages, leading to the direct development of

different species. Among the two genera studied in this thesis, Strongylocentrotus and

Heliocidaris (Table 1.1), the genus Heliocidaris is exclusively restricted to Australia.

Table 1.1: Taxonomy of the two sea urchin species studied in this thesis

Phylum Echinodermata Echinodermata

Eleutherozoa Eleutherozoa

Superclass Echinozoa Echinozoa

Class Echinoidea (Leske 1778) Echinoidea

Subclass Euechinoidea (Bronn 1860) Euechinoidea

Superorder Echinacea (Claus, 1876) Echinacea

Order Echinoida (Claus, 1876) Echinoida

Family Strongylocentrotidae Echinometridae

Genus Strongylocentrotus Heliocidaris

7

1.1.4. Sea urchin anatomy

The external morphology of sea urchins is characterized by a hemispheric test with

a flat oral surface and a curved anal surface (Figure 1.2). The surface of the body consists

of 20 meridional rows of thin, fused calcareous ossicles. It encloses and protects most of

the soft tissues. The endoskeleton is located in the connective tissue dermis of the body

wall and is covered by a thin epidermis. The surface of the test is covered with a large

number of articulated, movable spines and pedicelaria. The tube feet (podia) are arranged

radially forming 10 rows in 5 ambulacrae. Pairs of podia are separated by an

interambulacrum. Tube feet are used for locomotion and respiration. Aristotle’s lantern,

which is located in the center of the oral surface, is the masticatory apparatus [9] or mouth.

A persitomial membrane is located all around Aristotle’s lantern and harbors 5 pairs of

short tube feet, called buccal podia. Five pairs of small peristomial gills are located around

the margin of the peristome. These gills seem to have little respiratory role in sea urchins

[10, 11]. Farmanfarmaian [12] showed that excision of the gills does not reduce oxygen

consumption by S. purpuratus, whilst Cobb and Sneddon [11] concluded that the main

function of gills is waste removal. Aristotle’s lantern is surrounded by the peripharyngeal

peritoneum and opens into the pharynx. The pharynx extends into the oesophagus, which

turns towards the periphery and widens to become the stomach. At the end of the stomach,

the gut reverses direction forming the intestine. It completes a clockwise loop and becomes

the rectum, which extends to the anus and attaches to the outer surface of the test. The

intestine has abundant microvilli and mitochondria, and seems to be the site of absorption

of digested products. Absorption in the intestine involves active transport [13].

The nervous system of sea urchins consists of a nerve ring that lies in the peristomial

epidermis, and a radial nerve that extends along the radial canal of the water vascular

system.

8

Figure 1.2: Internal anatomy of the sea urchin. A/ Schematic representation of the

anatomy of sea urchins. Modified from [14]. B,C,D/ 3D reconstructions of selected

internal organs of S. purpuratus. B/ aboral view. C/ lateral view. D/ oral view. The color

legend specifies organ designation. Modified From [15].

9

The water vascular system is comprised of canals lined with ciliated epithelium that

connects with the exterior surface, through a cluster of pores called the madreporite. A ring

canal circles the mouth and a stone canal is linked with the anus. The axial gland is

situated along the stone canal, whilst Tiedmann’s bodies are situated interradially on the

inner side of the ring canal. Fluid in the water vascular system has similar chemical

properties to sea water, except for a higher concentration in potassium. The water vascular

system is mostly involved in supporting the locomotory tube feet, but is also involved in

gas exchange, excretion, and feeding. Free wandering coelomocytes are found within the

water vascular system.

Results obtained by Holm et al. [16] support the idea that the coelomic epithelium,

Tiedemann’s bodies and the axial organs are the hematopoietic tissues in echinoderms.

They showed that cell proliferation was rapidly induced within 4 hours post injection of

bacterial lipopolysaccharide (LPS), but that saline injection induced a delayed increase in

cell proliferation after 24 hours. The free wandering cells within the coelomic fluid

(coelomocytes) are not proliferative. However, a significant increase in the number of

circulating coelomocytes was observed by Holm et al. [16] after only 4 hours post

injection, suggesting that coelomocytes were released from the coelomic epithelium,

Tiedemann’s bodies or the axial organ.

10

1.1.5. Coelomic fluid and coelomocytes

The coelomic cavity can be divided into the oral region (near the peristomial

membrane) and the aboral region (close to the madreporite) [17]. Perivisceral coelomic

fluid resembles seawater in chemical composition. However, it contains a higher

concentration of potassium and low quantities of lipids, proteins, sugars [17, 18]. Coelomic

fluid fulfills many functions, such as excretion, locomotion, protection of the viscera and

immunological reactivity [18].

Coelomocytes populate the coelomic fluid. In S. purpuratus there are 1×106-5×106

coelomocytes per millilitre of coelomic fluid. They are comprised of at least four

coelomocyte types [19, 20], including a sub-population of phagocytic cells that can be

further subdivided into three types based on differences in morphology and gene

expression (Table 1.2) [21, 22]. Type 1 phagocytes, (also known as discoidal cells or

petaloide phagocytes), can be readily differentiated from type 2 phagocytes (also known as

polygonal cells or filopodial phagocytes) by their distinct cytoskeletal morphologies when

spread on microscopy slides (Figure 1.3) [21, 23, 24]. These two cell types can also be

separated by density gradient centrifugation, with type 1 phagocytes showing a low-

density and type 2 phagocytes having high-density [21]. The third type, small phagocytes,

is smaller with less cytoplasm than either of the two other phagocyte types [22, 25]. About

two thirds of these cells are actively phagocytic [26, 27]. Other types of coelomocytes

include vibratile cells, colorless spherule cells, and red spherule cells (also called morula

cells) (Figure 1.3). Vibratile cells are spherical and show no amoeboid movement but have

a single flagellum that may propel themselves through the coelomic fluid. The two types of

spherule cells are both amoeboid [28].

11

Table 1.2: Coelomocytes cell types and functions in the purple sea urchin, S.

purpuratus. From [29].

12

Figure 1.3: Coelomocytes in sea urchin (S. purpuratus) coelomic fluid. Polygonal

phagocytes (A) and discoidal phagocytes (B) can be differentiated by the ultrastructure of

their cytoskeleton (actin, green). C/ phagocyte. D/ red spherule cell. E/ vibratile cell. F/

colorless spherule cell. From [30].

A B C

D E F

13

A number of coelomocyte types function as mediators of the immune system [31].

Experimental observations demonstrate that they carry out many different immunological

functions: including chemotaxis, phagocytosis, encapsulation, cytotoxicity, immune gene

expression and secretion [31, 32]. As such, they are involved in the formation of cellular

clots, chemotactic accumulation at sites of injury, and allograft rejection.

Early studies of allograft rejection kinetics, and of the function of coelomocytes,

provided evidence for non-adaptive immunity in the deuterostome invertebrates [32]. Sea

urchins can reject allogeneic tissues and hence differentiate between self and non-self [33,

34]. The rejection rates for second-set allografts were accelerated relative to first-set

rejections, but they were not different from the rejection of third-party allografts [26, 35,

36]. Because similar results were obtained for clearance rates of foreign particles, the sea

urchin immune response has been thought of as non-specific and similar to the innate

immune system in higher vertebrates [31, 32].

Coelomocytes clear bacteria and other foreign substances from the coelomic cavity

with great efficiency [37-39]. In vitro, a major shape transformation of petaloide

phagocytes to a filopodial form has been observed during bacterial clearance [40]. In sea

cucumbers, this occurs after the ingestion of beads by the phagocytes [41]. Petaloide

phagocytes are believed to phagocytose foreign particles, whereas filopodial phagocytes

mostly participate in wound healing and clotting [17].

Arizza et al. [42] showed that filopodial phagocytes and colorless spherule cells are

cytotoxic and function coordinately. Phagocytes release an eliciting factor into the

coelomic fluid that activates colorless spherule cells resulting in the cytolysis of rabbit

erythrocytes and K562 tumor cells. Lin et al. [43] confirmed this result by generating

antibodies against cytotoxic coelomic fluid proteins that appeared to be specifically

localized in phagocytes and colorless spherule cells.

14

Injury increases the number of red spherule cells in the coelomic fluid [44]. These

cells have been reported to respond to injury by degranulation of their echinochrome

pigment, which is a bactericidal agent [45]. Another coelomic fluid protein, vitellogenin

exhibits hemagglutinating and antibacterial activities. Even though it is the precursor of

yolk protein in eggs, it occurs in the coelomic fluid of both male and female adults,

confirming that its function is not restricted to egg yolk. This protein is localised in

colorless spherule cells that discharged vitellogenin into the coelomic fluid in response to

stress [46].

Initial searches for immune response genes expressed in coelomocytes revealed an

increase in profilin (SpCoel1) in response to injury and LPS injection [27, 47]. It has been

suggested that this protein modulates the restructuring of the cytoskeleton during amoeboid

movement, encapsulation and clot formation. Further studies of expressed sequence tags

(ESTs) revealed that the expression of numerous other immune related proteins is also

altered by immune challenge [48]. In particular, Smith et al. [49, 50] identified

complement component C3 (SpC3; Sp064) [49] and complement factor B (SpBf; Sp152)

[50] homologues. They showed that SpC3 is specifically expressed by phagocytic

coelomocytes, confirming its importance in innate immune response.

1.1.6. The sea urchin genome project

Echinoderms have a long history as an experimental model, with some of the

discoveries made in these animals being recognized by Nobel prizes. Elie Metchnikoff’s

exploration of cellular immunity constitutes a classic example of the fundamental

biological concepts that have arisen from the study of echinoderms. For this body of work,

Metchnikoff was awarded the 1908 Nobel Prize in Medicine [51].

15

The importance of sea urchins as experimental models led to the sequencing of the

Californian purple sea urchin (Strongylocentrotus purpuratus) genome, which was

completed in 2006 [52]. The S. purpuratus genome contains more than 814 million base

pairs encoding 23,500 genes. Most of the gene families identified in this sea urchin are also

found in humans. However, the size of the gene families is often larger in humans, in part

reflecting two whole genome duplication events during vertebrate evolution. Two

unexpected exceptions to this pattern are the sensory and immune systems. The number of

sea urchin genes with putative immunological functions is ten to twenty times greater than

in humans [53]. The large expansion of innate immune receptor proteins (TLRs, NLRs and

SRCRs), together with the identification of some proteins involved in the vertebrate

adaptive immune system (Rag1/2 genes), suggests that the sea urchin immune system is far

more complex than was previously suspected.

16

1.2. Comparative immunology of echinoderms

The sea urchin genome sequence revealed the presence of an elaborate repertoire of

genes associated with immunity, most of which are more related to the vertebrate immune

gene repertoire than to the host defence molecules of other invertebrates [53]. Therefore,

the following description of echinoderm immune responses is based on comparisons with

the vertebrate immune system.

1.2.1. Sea urchin immune response molecules

Throughout the animal kingdom, the recognition of pathogens seems to be

mediated by a complex set of pattern-recognition receptors (PRRs) that bind pathogen-

associated molecular patterns (PAMPs). Five major classes of innate immune recognition

proteins are commonly observed in the animal kingdom: Toll-like receptors (TLR),

NACHT and leucine-rich repeat containing proteins (NLR), multidomain scavenger

receptor cysteine-rich (SRCR) proteins, peptidoglycan recognition proteins (PGRPs) and

Gram negative binding proteins (GNBPs). Each of these gene classes participate in

pathogen recognition through direct or indirect binding to PAMPs [54].

The S. purpuratus genome revealed a substantial expansion of many PRRs [30, 53].

Two hundred and twenty-two TLR gene models were found, which contrasts with the nine

TLR genes present in the human genome [55]. Most of the S. purpuratus TLR genes

(211/222) are more similar to each other than to homologues in other animals, suggesting

an expansion of these genes within the sea urchin lineage. Two hundred and three NLR

genes were predicted in the S. purpuratus genome, which contrasts with the 20 NLR genes

found in the human genome. The variability of NLR genes in sea urchins is thought to be

driven by gut associated pathogenesis. NLR are only expressed within the mesentery, the

17

gut and the testis of sea urchins, and are absent in all coelomocyte types [30]. A large array

of 218 sea urchin genes encode a total of 1095 SRCR domains, while in humans there are

only 16 gene models, encoding 81 SRCR domains. Among other pattern recognition

receptors, only 5 PGRP and 3 GNBP gene models were observed in the sea urchin

genome. GNBP genes are absent in vertebrates, but 4 have been found in the fruit fly

genome, suggesting that these genes were lost during chordate evolution [30, 52].

1.2.2. The complement system of sea urchins

The presence of molecules in sea urchins with clear homology to complement

components was initially revealed by investigations of the genes expressed in LPS-

activated coelomocytes [22, 32, 48]. Of the 307 expressed sequence tags (ESTs) that were

reported by Smith et al. [48], two encoded homologues of mammalian complement

components: EST064 (SpC3) is a homologue of complement component C3 [49] and

EST152 (SpBf) is a homologue of complement factor B (Bf) [50].

The presence of SpC3 and SpBf in sea urchins suggested the existence of a

complement system with similarities to the vertebrate alternative pathway. SpC3 has an

amino acid sequence with 27.9% amino acid identity to human C3 [48, 49]. It has a non

reduced molecular weight of 210 kDa, and reduces to an α-chain (130 kDa) and a β-chain

(80 kDa) that are of equivalent molecular weight to C3α and C3β from gnathostomes.

Homology of SpC3 to vertebrate C3 is concentrated around the functionally critical

thioester group and a number of other functional regions. SpC3 possesses five consensus

N-linked glycosilation sites, putative cleavage sites for factor I and C3 convertase and

cysteines in conserved positions. Functional studies have confirmed that SpC3 has an

active thioester site and opsonizes targets for phagocytosis [56, 57]. Its synthesis can be

induced by challenge with LPS in immunoquiescent sea urchins and it is specifically

18

expressed by two subpopulations of coelomocytes [22, 58]. Two other C3-like genes

(SpC3-2 and Sp-TCP1) and a C4-like gene (Sp-TCP2) have been identified in the genome

of the sea urchin [30, 59]. SpC3-2 has been found to be predominantly expressed during

larval development [59].

The deduced amino acid sequence of SpBf also shows significant similarity to

vertebrate Bf/C2 family proteins [50]. It has a mosaic structure, which includes five short

consensus repeats (SCR; as opposed to the three found in vertebrate Bf proteins), a Von

Willebrand factor domain, a serine protease domain and a conserved cleavage site for a

putative factor D protease [50]. Phylogenetic analysis of SpBf indicated that it is the most

ancient member of the vertebrate Bf/C2 family. More recently, evidence of alternative

splicing has been found [60]. The sea urchin genome revealed the presence of two

additional homologues of vertebrate Bf/C2 proteins with 5 SCRs in Sp-Bf-2 and four SCRs

in Sp-Bf-3 [30].

Together, SpBf and SpC3 may function in a similar way to the alternative pathway

in vertebrates [61]. They could result in opsonization of foreign cells or particles to

augment phagocytosis by coelomocytes. The existence of multiple homologous proteins

and/or splice variants of C3 and Bf suggests that multiple alternative pathways may exist

in sea urchins working at different times in the life cycle, in response to different

challenges, or in different cell types.

19

1.2.3. The lectin-mediated complement system in sea urchins

Evidence from tunicates and “lower” vertebrates suggests that the lectin pathway

originated earlier than the classical pathway [62-65]. Homologues of collectins, C1q (four

genes) and MBP (one gene) have also been found within the sea urchin genome. A total of

46 gene models were found containing fibrinogen domains comparable to those of ficolins.

Ficolins (collagen-fibrinogen domains) are structurally analogous to collectins and could

be involved in the activation of a lectin-like pathway [66]. However, members of

MASP/C1r/C1s, which are critical to lectin-mediated pathways, have not been identified in

sea urchins.

1.2.4. Immunoglobulin superfamily rearrangement

The sudden emergence of the entire complex of Ig/TCR/MHC mediated adaptive

immunity in ancestral jawed vertebrates has previously been correlated with the

appearance of recombination activator genes (Rag). It has been assumed that combinatorial

diversity among immunoglobulin superfamily (IgSF) genes arose with the acquisition of

Rag genes by original gene transfer of a mobile DNA element [67]. The theory was

supported by the absence of homologous Rag genes in jawless vertebrates or invertebrates.

Antibody and T-cells receptor (TCR) genes are assembled from individual variable (V),

diversity (D), and joining (J) gene segments. The Rag 1 and Rag 2 proteins are the key

mediators of this process of somatic V(D)J recombination, which also utilises terminal

deoxynucleotidyl transferase (TdT) for enhanced diversity [68, 69]. Fungmann et al. [70]

have since revealed the presence of sequences in the S. purpuratus genome with similarity

to regions of the Rag 1 and Rag 2 genes from vertebrates. This discovery suggests that the

apparent evolutionary discontinuity of genes involved in generating hypervariability in the

20

IgSF could be a consequence of genes loss or undersampling. A longer evolutionary

process may underlie the emergence of the key elements of the vertebrate adaptive immune

system.

S. purpuratus Rag 1 (SpRag1L) has 31% amino acid homology to the core region of

mouse Rag1, and similarities extend into the non-core region [70]. All three residues of the

DDE active site are conserved, as well as surrounding residues. The zinc finger B motif,

critical for the interaction with Rag 2, and most of the basic residues implicated in DNA

binding in the region of the nonamer-binding-complex (NBD) are also conserved. Finally,

a 108 aa-stretch shows significant similarity to a putative zinc-binding domain (ZBD) in

mouse Rag 1. The RING finger domain, which separates the two stretches of sequence

similarities in all known vertebrate Rag 1 proteins, is absent from the sea urchin sequence

but a repetitive coding region containing 11 repeats of an 8-aa peptide has been observed.

The S. purpuratus Rag 2 (SpRag2L) gene lies downstream of the SpRag1L gene within the

range of intergenic distances for vertebrates Rag genes (3,181 bp downstream). The first

424 aa of SpRag2L are predicted to encode a six-bladed β-propeller, which like vertebrate

Rag2, matches the β-propeller of the galactose oxidase central domain SCOP profile.

Furthermore, as for vertebrate Rag2, a C-terminal plant homeodomain (PHD) is present in

the sea urchin gene [70].

Further analysis showed that, like their vertebrate homologues, SpRag1L and

SpRag2L are co-expressed, and evidence of interaction of the two molecules to form a

heterodimer complex has been obtained by pull-down assays [70]. The ability of SpRag1L

and SpRag2L to interact with shark Rag1/2 provides additional evidence that this complex

may be functionally equivalent to the vertebrate Rag1/2 complex. Finally, similar DNA-

binding properties to the Rag1 central NBD complex were evident in the central domain of

SpRag1L when purified as a recombinant protein from Escherichia coli. SpRag1L may use

DNA as its substrate and facilitate somatic rearrangement of yet unidentified genes in the

21

sea urchin genome. The identification of the cognate target motif and the SpRag1L/2L

complex will be important for further studies.

A homologue of Terminal deoxynucleotidyl Transferase (TdT) and DNA

polymerase mu (Polµ) has also been found in the sea urchin genome [53]. Polµ appears as

a mutator (error prone) DNA polymerase potentially responsible for somatic

hypermutation of immunoglobulin genes [71]. It plays an important role in non-

homologous end joining of incompatible ends and in terminal transferase activity [72].

TdT is a DNA independante polymerase with a strict terminal transferase activity. This

enzyme increases antigen receptor diversity in gnathostomes by adding nucleotides during

the DNA joining phase of V(D)J recombination. Indeed, homologues of all enzymes

involved in DNA repair and the non-homologous end-joining pathway have been found in

the sea urchin genome, thus providing the sea urchin with the complete enzymatic

machinery required for V(D)J recombination. In addition, a total of 500 gene models

containing about 1500 Ig domains, some of which showed weak but relatively specific

identity to Ig/TCR/MHC, have been identified in the S. purpuratus genome [53]. However,

there is still no evidence for V(D)J rearranging system outside of the jawed vertebrates.

22

Table 1.3: General characteristics of highly variable gene systems associated with

pathogen defence in animals. From [73].

a includes somatic recombination and alternative splicing.

? not tested or speculative

Abbreviations: CBD, chitin binding domain; CC, coiled coil domain; CT,

cytoplasmic tail; Dscam, Down’s syndrome cell adhesion molecule; FBG, fibrinogen-like

domain; FnIII, fibronectin type III domain; FREP, fibrinogen related protein; GPI,

glycosylphosphatidylinositol; Ig, Immunoglobulin; IgSF, Immunoglobulin superfamily;

LRR, leucine rich repeat; ND, structure not determined; NBS, nucleotide binding site; TM,

transmembrane region; VCBP, variable chitin-binding protein; VLR, variable lymphocyte

receptor.

23

1.3. Highly variable gene systems associated with pathogen defence

Any receptor system showing high degrees of intra-individual variability could in

principle be a candidate recognition molecule in a pathogen-specific immune system (as

distinct from pattern recognition systems). Numerous studies investigating pathogen

specific immune responses of non-mammalian jawed vertebrates, jawless vertebrates,

protochordates and other invertebrates suggest that we may have underestimated the

diversity of such highly variable receptors [74]. Convergent evolution has given rise to

many functionally analogous immune response molecules that do not always share

evolutionary histories (Table 1.3). This is exemplified by the recent identification of highly

variable molecular systems, such as 185/333 proteins from sea urchins [75], variable

lymphocyte receptors (VLRs) from lampreys [76, 77], fibrinogen related proteins (FREPs)

from snails [78-84], Down’s syndrome cell adhesion molecules (Dscams) from insects

[78], and V-region-containing chitin-binding proteins (VCBPs) from cephalochordates and

tunicates [79] (Table 1.2). These systems are all based on high levels of molecular

variability within individuals, but are otherwise unrelated [76].

24

1.3.1. Life history strategies

The absence of adaptive immunity in invertebrates has previously been explained

by the fact that invertebrates have relatively short life spans compared with vertebrates, so

that they are less likely to encounter the same pathogen twice [80-82]. Another argument is

that invertebrates are usually small organisms with high fecundity, so that there are likely

to be numerous survivors of disease epizootics at the level of the population [83]. These

theories were mostly based on the r/K selection hypothesis and suggested that invertebrates

are r-selected (high fecundity, large populations), while vertebrates are K-selected (low

fecundity, small population). But r and K selection strategies do not neatly differentiate

invertebrates from vertebrates, and there are members with both life history strategies in

both clades [80, 81]. Some invertebrates can live for many decades, while some lower

vertebrates have short life spans and reproduce extensively [84]. For example, sea urchins

can live for over 100 years [85]. Additional selection pressures, such as the size and

density of the population, the level of eusociality within the population and the

involvement of asexual reproduction, which reduces genetic variability, are also variable

[86]. Sea urchin population structure is complex and significant fluctuations in population

size have been observed over time [87]. They are also often victims of disease outbreaks

and their disappearance from communities can have significant impacts on the entire

ecosystem of an area [88-90].

These evolutionary perspectives suggest that invertebrates would, like vertebrates,

benefit from a complex immune system. So, not surprisingly, classical immunization

experiments have now confirmed that some invertebrates can develop pathogen specific

responses [91, 92]. This pathogen specific discrimination is sometimes fine scaled,

suggesting the existence of immune processes providing both specificity and memory [93,

25

94]. However, the molecular processes responsible for pathogen-specific immunity in

these organisms are not well understood, even though a number of highly variable

recognition proteins have been identified among invertebrates. These include:

1.3.2. Variable lymphocyte receptors (VLRs)

Hagfish and lampreys are agnathans, which possess an adaptive immune system in

terms of allograft rejection and immunization [95-98]. However, their adaptive immune

responses are not due to variability of the immunoglobulin receptors used by jawed

vertebrates. Instead, they express variable lymphocyte receptors (VLR) composed of

highly diverse leucine-rich repeats (LRR) [77]. Lamprey immunized with anthrax spores

showed an increase in VLR positive lymphocytes from 4 to 98%, and an increase of 8 to

10 fold in soluble antigen-specific VLRs [99].

VLRs include a conserved signal peptide, an N-terminal LRR, up to seven internal

LRRs, a connecting peptide and a conserved C-terminus region with a GPI-anchor and a

hydrophobic tail [77]. The N-terminal and C-terminal regions are invariant. The

hypervariability of VLRs is concentrated on the concave surface of the horseshoe-shaped

molecule, where the LRR modules undertake antigen recognition. Antigen specificity is

confered by variation in the number of LRR domains and variation in their amino acid

composition [100, 101]. Lamprey and hagfish generate a potential repertoire of 1014 and

1017 unique VLRs [99], which is greater than the potential number of TCR in vertebrates

(108). A single VLR gene has been identified in lampreys, while hagfishs have two VLR

genes, designated VLR-A and VLR-B, located on the same chromosome. These two VLR

genes are made up of significantly different LRR modules [99] promoting their functional

specialization [102]. The complex somatic diversification of VLRs occurs through the

random selection of LRRs from a large bank of flanking cassettes. Diversification arranges

26

various copies of LRRs in different combinations and uses multiple sites in LRR gene

fragments for priming, a process called ‘copy choice’. It has also been demonstrated using

mutagenesis and recombination assays that AID-like cytosine deaminase may also be

involved in VLR diversification [103]. It is noteworthy that the AID/APOBECs cytosine

deaminases of vertebrates are DNA mutators acting in antigen-driven antibody

diversification processes. The recombinatorial assembly of LRRs in agnathans and of Ig in

higher vertebrates, and the involvement of cytosine deaminases, demonstrate the

convergent evolution of different strategies for generating lymphocyte-based adaptive

immune responses.

1.3.3. V-region-containing chitin-binding proteins (VCBPs)

Protochordates, such as Amphioxus, lack an antibody-based adaptive immune

system. However, a family of genes encoding secreted proteins with two immunoglobulin-

like variable (V) domains and a chitin binding domain have been identified by Cannon et

al. [104]. These molecules are designated V-region-containing chitin-binding proteins

(VCBP). Ig-like V domains are often associated with adaptive immunity, while chitin

binding domains are associated with innate immune functions. The complexity of the

VCBP family is based on multiple amino acid substitution and potential combinatorial

rearrangement that result in substantial variability among the expressed proteins within and

between indivduals [105]. The annotation of the Amphoxius genome enabled the

characterization of the entire VCBP locus [106]. Substantial allelic variation based on the

complexity of haplotypes has been observed, with multiple indels of repeats (inverted

repeats, small repeats and microsattelites) and non-coding segments. The number of

VCBPs genes can vary among individuals of the same population [106]. Investigations of

the crystallized structure of VCBP revealed that their V domains are structurally similar to

27

Ig V domains [107]. It has been suggested that VCBP constitute a transitional molecule

between innate and adaptive immunity and that they have bi-functional properties [106].

However, no functional studies have yet been undertaken, so it can only be assumed that

VCBPs are involved in pathogen defense.

1.3.4. Fibrinogens related proteins (FREPs)

Fibrinogen related proteins constitute a family of highly variable haemolymph

proteins originally found by Adema et al. [109] in the snail, Biomphalaria glabrata. This

schistosome snail produced large amounts of circulating FREPs after injection of

secretory/excretory products (SEP) derived from cultured Echinostoma paraensei

intramolluscan larvae. The secreted molecules incorporate both IgSF domains and a C-

terminal fibrinogen-β/γ domain (FBG). They can precipitate SEPs and have similar

properties to lectins [109]. Further studies revealed the large size of the FREP family with

up to 13 different genes [110]. FREPs have since been identified in a number of other

invertebrates, as well as in vertebrates [111-117]. FREPs with one or two IgSF domains

have been found, as well as truncated forms. This is consistent with alternative splicing of

full-length FREP genes [118]. A more thorough investigation of the variability of FREP3

revealed the presence of an unknown system for diversification that involves nucleotide

point mutation and/or recombinatorial diversification [119]. The frequency of point

mutation in parents is higher than that of their offspring, but the rate of recombinatorial

diversification did not differ. Multimerisation of FREPs has also been observed, with up to

24 molecules partially covalently bounded, substantially increasing the potential diversity

of these molecules [120].

It was previously thought that exposure to bacteria or wounding did not upregulate

the expression of FREPs [121, 122]. However, new evidence suggests that different

28

members of FREP gene subfamilies are differentially expressed following exposure of B.

glabrata to different trematodes [122, 123]. Anti-pathogen activities of FREPs have also

been demonstrated in other species. For instance, mosquito FREP is up-regulated after

challenge with bacteria, fungi and Plasmodium [106, 124]. Amphioxus FREP has direct

antibacterial activity revealed by binding to E. coli and S. aureus via interactions with

lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PG) [125]. FREPs

are also up-regulated (after 9 hours) in the bay scallop (Argopecten irradians) responding

to Listonella anguillarum, while recombinant A. irradians FREPs can agglutinate

erythrocytes and bacteria in a calcium dependent manner [126]. Overall, it appears that

FREPs participate in a new form of inducible, pathogen specific immune response in a

broad range of species, including molluscs and insects.

1.3 5 Down’s syndrome cell adhesion molecules (Dscam)

The Drosophila homologue of human Down’s syndrome cell adhesion molecules

(Dscam) was originally described as a guidance receptor in neuronal wiring [127]. The

Drosophila Dscam differs from its human counterparts in terms of its gene organisation.

Drosophila Dscam genes appear as clusters of variable exons flanked by constant exons.

These are subjected to mutually exclusive splicing that potentially generates as many as

38,016 different isoforms [127]. All variants of Drosophila Dscam have a conserved

architecture containing variable immunoglobulin domains and a transmembrane domain

[127]. Analysis of Dscam mRNA isoforms expressed by individual cells revealed that each

cell expresses a unique repertoire of splice variants [128].

Recently, Watson et al. [129] demonstrated the importance of Dscam in the

immune system of insects. They showed that the enginered loss of Dscam impaired the

efficiency of phagocytosis. Similarly, Dong et al. [130] showed that different splice

29

variants of Anopheles gambiae Dscams enable pathogen specific immunological

protection. Alternative splicing of different exons induced an over or under expression of

different Ig domains depending on which pathogen the host cell was exposed to. The

various A. gambiae Dscam molecules (AgDscam) produced in response to particular

pathogen also showed different adhesive characteristics and interaction specificity.

Silencing of AgDscam genes via double stranded RNA decreased survival rates after

bacterial infection [130].

The majority of non-spliced Dscams exons are extremely conserved between D.

melanogaster and A. gambiae (70-95% amino acid homology), while the spliced Ig

domain exons are far less homologous (30-70% homology), suggesting that the two types

of exons are under different selection pressure. It has been suggested that the constitutive

(non-spliced) region undertake more conserved functions, such as regulation of

intracellular signalling pathway, while the alternatively spliced Ig domains are under

constant environmental selection pressure for diversification. In support of this suggestion,

phylogenetic analysis of alternatively spliced Dscam exons from honey bees, fruit flies and

mosquitoes revealed major modifications since divergence that can be explained by

differences in life history and environment [131]. Identification of Dscam homologues in

haemocytes of the crustacean Daphnia, which are also diversified by alternative splicing,

indicates that the highly variable forms of these genes evolved from non-diversified forms

before the divergence of insects and crustaceans [132]. Comparisons between fly and

Daphnia Dscams also support the idea that the variable exons have evolved through a birth

and death process based on duplication in a nearest-neighbour scenario, meaning that

exons physically closer to each other are also more similar to each other [132].

30

1.4. The 185/333 family

1.4.1. Discovery

The 185/333 family was initially identified as uncharacterised EST from a cDNA

library of LPS-activated coelomocytes [48, 133]. They were originally designated DD185

(Genbank accession no. AF228877) and EST333 (Genbank accession no. R62011), hence

their current name 185/333. Northern blots revealed a significant up-regulation of the

DD185 and EST333 sequences in response to bacterial challenge. The significance of the

185/333 family in immune responses was further established by a suppressive substractive

hybridization (SSH) study carried out by Nair et al. [75]. cDNA substraction employed

coelomocyte RNA from an LPS-activated sea urchin, which was substracted from RNA

prepared from the same animal before challenge. Transcripts that matched DD185 and

EST333 represented up to 60% of the ESTs analysed. Re-screens showed that 185/333

transcripts constituted 6.45% of the clones in the bacterially activated coelomocyte library,

compared to only 0.09% in the non-activated coelomocyte library (a 75-fold increase).

Alignment of the 1,247 ESTs that matched 185/333 messages revealed their inherent

variability. To allow this alignment, large gaps had to be inserted, revealing blocks of

shared sequence found in multiple 185/333 sequences. These blocks were designated

“elements”. A total of 15 elements were required for the original alignment, and not all

were present in every 185/333 sequence. The authors suggested that this variability

between transcripts was due to alternative splicing.

31

1.4.2. Variability of 185/333 mRNAs

Subsequent full length cDNA sequencing allowed a thorough analysis of 185/333

diversity [132]. More optimal sequence alignments required additional gaps that defined a

total of 25 elements. On the basis of the presence or absence of these elements, 22 distinct

element patterns were identified (Figure 1.4). In addition to the element patterns, these

alignments revealed substantial nucleotide diversity reflecting indels and single nucleotide

polymorphisms (Figure 1.4). Five types of repeats were identified [134], and these were

located throughout the sequence. Within each element, single nucleotide polymorphisms

were not randomly distributed. Instead, they were located at specific sites. These positions

were not limited to element boundaries, suggesting that they do not simply result from

alternative splicing. More importantly, nucleotide substitutions were functionally

significant and not selectively neutral. This implies that diversification of 185/333

sequences is under selective pressure for variability.

Terwilliger et al. [134] also identified a correlation between element patterns and

the presence/absence or length of a specific element (element 15). The sequence groups

defined by element 15 may be representative of subfamilies of transcripts or genes,

reflecting the evolutionary diversification of 185/333 genes. This observation was

supported by the fact that sets of cDNAs with shared element patterns tend to be more

similar in terms of nucleotide sequence. Phylogenetic analysis of the transcripts collapsed

the classification of 185/333 proteins into 5 sub-families that present conserved nucleotide

substitutions, similar element patterns and conserved physico-chemical properties (Dheilly

et al., unpublished data; Figure 1.5).

32

Figure 1.4 : A cDNA-based alignment of 185/333 transcripts. Elements (numbered at

top), are represented by colored boxes. cDNAs are organized into groups (numbered at left,

identified by gray background shading) based on element 15. Group 1 is defined by sub-

element 15a, group 2 by sub-element 15b, group 3 by sub-element 15c, group 4 by sub-

element 15d, and group 5 by sub-element 15e. Groups 6 and 7 do not have element 15.

Groups are also indicated by pattern designations: group 1 is pattern A, group 2 is pattern

B, etc. Element 25 was subdivided into 3 sub-elements, 25a, -b, and -c, based on the

location of the stop codon (black vertical lines). A frame shift (white X) in element 4 of

clone D1.1 leads to an early stop codon (black vertical lines) in element 5 (black/purple).

The remainder of the D1.1 sequence is shown as smaller blocks to show that the sequence

is present but may not be translated. Eight sets of cDNAs (E2, D1, C1, 01, E3, A2, E1, C3)

are composed of multiple members that have identical element patterns (#). From [134].

33

Figure 1.5: Five sub-families of 185/333 proteins. A/ Circular representation of a

neighbour joining tree obtained with 185/333 cDNAs sequences. The same tree was

obtained using the maximum parsimony method. Bootstrap values obtained with 1000

replicates from both neighbour joining (NJ) and maximum parsimony (MP) are shown

along the branches (NJ/MP). B/ the 5 sub-families identified are compared with the groups

based on element 15 (Ex15 Figures 1.4 and Er10 Figure 1.7). Within each sub-family,

predicted proteins show homogeneous molecular weight.

!"#$%&'()*+,+

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Sub-family groups Molecular weight

Sub-family I Groups A and G 53.0 ± 2.7 kDa

Sub-family II Groups B and F 39.7 ± 1.8 kDa

Sub-family III Group C 41.1 ± 1.5 kDa

Sub-family IV Gourp D 42.0 ± 1.2 kDa

Sub-family V Group E 32.0 ± 0.1 kDa

Pattern E2.1 14.7 ± 0.03 kDa

Group 0 29.5 ± 1.5 kDa

1%

2%

34

Figure 1.6: Structure of part of the 185/333 locus. Scaffold_v2_79421 from the S.

purpuratus genome assembly (Version 2, June 15, 2006) contains four linked 185/333

genes (diagram not to scale). Each of the genes includes a single intron in the predicted

location. The element patterns of the genes based on cDNA-based alignments are

indicated. Pattern D8 was not isolated from the cloned genes. However, it is similar to

pattern D1, but contains Ex12 rather than Ex10. The orientations of the genes are indicated

by the arrows. Dinucleotides (GA; striped ovals) flank the genes, and trinucleotide repeats

(GAT; solid parallelograms) are present on the 5' side of the genes. From [135].

35

Terwilliger et al. [136] further investigated the variability of 185/333 transcripts in

response to challenge with different PAMPs (LPS, β-1,3-glucan or double stranded RNA).

The authors showed that each individual sea urchin expressed a different set of transcripts

before challenge. This set changed in response to challenge and trends in these changes

were observed between individuals. The element patterns C1 and E2.1 (Figure 1.4) were

the most commonly expressed in immunoquiescent sea urchins, while the pattern E2

(Figure 1.4) was found most often after injury, or the injection of LPS or β-1,3-glucan.

Interestingly, pattern E2.1 (65% of 185/333 messages prior to challenge) has a single

nucleotide polymorphism-generated stop codon that differentiates it from pattern E2 and it

has not been found in any of the genomic sequences [136].

1.4.3. 185/333 genes

The diversity observed among their cDNA sequences initially suggested that

185/333 genes (genomically encoded loci) have many exons independently encoding all of

the different elements, or that a large family of 185/333 genes encoding all different

variants was present. Surprisingly, genome blots performed on sperm gDNA from three

sea urchins revealed that 185/333 genes are small [134]. Further examination of the

partially assembled sea urchin genome revealed that 185/333 genes were composed of just

two exons and one intron (Figure 1.6) [135]. The first exon encoded the leader and the

second encoded elements 1-25. Because very few unique 185/333 genes were identified in

the genome, quantitative PCR (qPCR) was used to evaluate gene copy number. The results

suggested that there are about 80-120 185/333 alleles per diploid genome [134]. The

approximate age of their last common ancestor was calculated using average element

pairwise differences and indicated that the family might only exist within the echinoid

lineage.

36

Figure 1.7: Repeat-based alignment of the 185/333 genes. This alignment was optimized

for repeats, and 27 elements (colored boxes) were defined. Gaps due to missing elements

are indicated by horizontal black lines. There are 17 different exon element patterns,

which, when combined with the intron types, form a total of 21 unique gene patterns. The

frequency (Freq) of patterns indicates how often the pattern was identified. The source

animal for each pattern is indicated by the presence of a colored dot. The subtype of

element Er10 (which corresponds to Ex15, Figure 1.4) is indicated by the letter in the box.

From [135].

37

Buckley and Smith [135] sequenced 171 gDNA clones of 185/333 and obtained

121 unique sequences, meaning that 71% of the cloned sequences were unique. The gDNA

clones were classified into 33 element patterns, about 50% of which were newly described.

A phylogenetic analysis of the intron sequences was used to separate them into five major

clades (α - ε) [135]. A strong correlation was observed between exon element patterns and

intron type, suggesting the existence of different sub-families; most sequences from sub-

family I had intron type γ, sub-families III and IV had intron type α, sub-family V had

intro type δ and sub-family II had intron types α, β, or ε (Dheilly et al., unpublished data).

BAC screening revealed that few genes were intronless. Intergenic regions varied from 1.6

kb to 8 kb, and genes were flanked on both sides by a stretch of microsatellites

(approximately 15 dinucleotide GA repeats both upstream and downstream, and

trinucleotide GAT repeats at the 5' end) (Figure 1.6).

These data suggest that 185/333 represent a family of linked genes that occupy long

stretches of repeats along the genome. It has also been suggested that the dinucleotide

repeat stretches might facilitate diversification events through gene conversion,

recombination, duplication and/or deletion of intact genes [135, 137]. A total of 16

individual sea urchins have been studied at the genomic or transcriptomic level and not a

single identical sequence has been found in two different individuals.

To further define the evolution of 185/333 genes, the six different repeats identified

within 185/333 sequences [134, 135, 138] have been used to generate a new repeat-based

alignment of the gene sequences (Figure 1.7) [135]. Type 1 repeats, present in the 5’

region of the gene, seem to be arranged in tandem. This contrasts with the five other types

of repeats, which are mixed and interspersed along the 3’ region. The relatively simple

organisation of type 1 repeats permitted a thorough investigation of their molecular

evolution. Individuals contain between two and four type 1 repeats.

38

Figure 1.8: Schematic representation of the consensus 185/333 protein sequence. The

deduced protein sequence is separated into a glycine-rich region (gly-rich; orange line) and

histidine-rich region (his-rich; purple line). Symbols indicate the presence of an RGD

motif in element 7 (black circle), N-linked glycosylation sites (red circles) and O-linked

glycosylation sites (green circle), 5 types of repeats (colored diamonds: type 1 in red, type

2 in blue, type 3 in green, type 4 in purple, and type 5 in yellow), secondary structure

predictions (α-helices and β-strands), patches of acidic amino acids (red vertical bars),

patches of histidines (purple vertical bars), and 5 elements surrounded by cryptic splice

signals. All elements are drawn to scale. From [134].

39

In total, 292 type 1 repeats have been identified, corresponding to 52 unique repeats from

121 unique genes. Computational phylogenetic analysis revealed the involvement of point

mutations and gene recombination in generating the type 1 repeats [138].

This supports the hypothesis that the 185/333 family appeared through a recent

gene diversification event. It also raises the possibility that the gene family may be subject

to frequent birth and death evolution, and that the size of the family may vary between

individuals [138].

1.4.4. Transcriptional and posttranscriptional modifications

The first level of variability among 185/333 molecules appears to be generated by

recombination/duplication events. However, Buckley et al. [139] have also investigated the

potential of 185/333 genes to undertake intergenic splicing to generate element pattern

variations. Although cryptic donor splice sites were found, it appeared unlikely that

185/333 genes undergo intergenic splicing. So, Buckley et al. [140] further investigated the

presence of a second level of diversification, involving posttranscriptional modification.

To do so, they compared the sequences of genes (genomic loci) and messages (mRNA

transcripts) in individual sea urchins. The data showed that most of the nucleotide

substitutions between genes and transcripts were transitions (3:1 transition/transversion

ratio), with a strong bias towards cytidine to uridine transitions that is consistent with

cytidine deaminase activity.

40

1.4.5. 185/333 proteins expression and localization

The predicted proteins described by Terwilliger et al. [134] have a NH2-terminal

hydrophobic leader, a glycine rich domain with multiple protease cleavage sites and an

RGD motif, a histidine rich region with 11 patches of histidines varying from 2 to 17

amino-acids in length that are interspersed with Gly, Arg and Gln; and a COOH terminal

region (Figure 1.8). Conserved N-linked glycosylation sites are present in 16 locations and

there are numerous acidic patches. Seven conserved O-linked glycosylation sites can be

identified within a short region of nine amino acids. Finally, none of the predicted 185/333

proteins contain cysteines or have any discernable secondary structure other than the

hydrophobic leader containing a short β-strand followed by a short α-helical region that

extends into element 1. The deduced 185/333 proteins vary in length and sequence based

on element patterns, amino acid sequence variability and the position of translational

modification. With no known homologs, predictions of tertiary structure for these proteins

are problematic [134, 136].

Sequence comparisons suggested that 185/333 proteins could be antimicrobial

molecules or opsonins, although there is not yet any data on their function. The only

185/333 sequences that bear significant homology to molecules from other organisms are

the RGD motif and one of the histidine rich regions. The histidine rich region is similar to

histatins, a group of mammalian proteins that have powerful antibacterial and antifungal

activities [141]. The apparent lack of tertiary structure in predicted 185/333 proteins results

from their lack of cysteines, which is also a common characteristic of histatins and other

anti-microbial molecules that form a amphipathic α-helixes when they interact with target

cell surfaces [141].

41

The presence of the RGD motif in predicted 185/333 proteins suggests that they are

involved in defensive activity. RGD motifs are common among both vertebrates and

invertebrates. Their capacity to interact with cell surface integrins is thought to be a key

mechanism by which cellular effector activities, such as phagocytosis, are activated [142].

Immunocytology showed that 185/333 proteins are localized within two sub-sets of

coelomocytes: small and polygonal amoebocytes. Both cell types express 185/333 proteins

within vesicles in the perinuclear region. The expression of 185/333 proteins within small

amoebocytes was so intense that an exact localization was difficult to discern. However, by

limiting the penetration of antibodies within the cell, the authors demonstrated that

185/333 proteins are present on the surface of these cells.

42

Figure 1.9: Complexity of the proteome. One gene can produce multiple mature mRNAs

via alternative splicing of pre-mRNA transcripts. Following translation, a myriad of post-

translational modifications can produce further variations in the number and types of

protein forms. from [143].

43

1.5. Proteomics and sea urchins

1.5.1. From the static image to the dynamic image

Genome sequences have provided valuable information for comparative

immunologists. Bioinformatic technology for the detection of gene homologies has

improved so that we can now overcome problems associated with the accelerated rate at

which immune systems have evolved. However, the existence of nucleotide sequence

homologues is only a starting point for further experiments, because genes perform

different functions in different organisms. To help elucidate the function of proteins

encoded by genes, experimental characterisations at the cellular and protein levels are

required.

The two most striking features of the sea urchin genome were the diversity of

immune receptors and the presence of homologues of the genes involved in V(D)J

recombination in vertebrates. These results suggest a complex immune system [53].

However, the complexity of the immune response molecules in sea urchins observed at the

genetic level is likely to represent only a fraction of molecular diversity at the protein

level. Due to posttranslational modifications and alternative splicing, each gene may be

transcribed into 5 to 10 times more distinct mRNAs (Figure 1.9) [143]. Posttranscriptional

modifications are then likely to further increase the number of proteins expressed (Figure

1.9). Such posttranslational modifications are important regulators of activity but are

invisible to DNA-based methods. This partly explains why transcripts and proteins are not

always correlated. Therefore, proteomic analyses of immune responses are important to

confirm the biological significance of transcriptomic and genomic data.

44

Figure 1.10: Flow chart for gel-based and gel-free proteomics.

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45

Comparative immunology requires a comprehensive understanding of the

physiology of the animal and of the selection processes that it is subjected to. Natural

selection taking place during the arm’s race between hosts and pathogens puts pressure on

the phenotypes but not necessarily the genotypes of individuals. Comprehensive pictures

of phenotypes requires comparative analysis of proteomes, as well as genomes and

transcriptomes, to provide a dynamic image of biological processes.

1.5.2. Gel-based versus gel-free proteomics

Proteomes can be analysed either by using two-dimensional gel electrophoresis

(2DE) or by gel-free methods (Figure 1.10). Both techniques rely on mass spectrometric

analysis for protein identification (Figure 1.10).

2DE was first described by O’Farrell in 1975 [144]. The most popular 2DE method

separates proteins by isoelectric focusing (IEF) in the first dimension, using an

immobilized pH gradient (IPG) polyacrylamide gel, and then by molecular weight using

sodium dodecyl sulfate (SDS) electrophoresis in the second dimension. High resolution

2DE provides a powerful method for reproducible separation, visualization and

quantitation of hundreds to thousands of proteins on a single gel. The subsequent use of

mass spectrometry (MS) facilitates the identification of almost any protein spot on a 2DE

that can be visualized by Coomassie blue staining.

Mass spectrometers measure the mass/charge ratio (m/z) of ionized peptides

produced by enzymatic digestion of proteins. Two different approaches can be used for the

identification of proteins: peptide mass fingerprinting (PMF) or peptide sequencing. PMF

is achieved by matrix-assisted laser desorption/ionization (MALDI) coupled with a Time-

of-flight (TOF) mass analyzer. Peptides are identified by matching their mass/charge ratio

with the calculated masses of all predicted peptides deduced from protein databases. In

46

contrast, peptide sequencing employs electrospray ionization (ESI) and tandem mass

spectrometry (MS/MS). The peptide ion is fragmented by collision-induced dissociation

(CID) and the fragment ion is recorded. CID spectra show the masses of all ions generated

from the fragmentation of the peptide and provides information about their sequence.

Database searches are more accurate with CID spectra than with PMF. MALDI-

TOF, which is less expensive, is generally used for high throughput identification of

proteins from 2DE gels when the complete genome sequence of the species is available.

ESI-MS/MS is more accurate, but also more expensive and time consuming. However, it is

often required for the identification of proteins from species in which the genome has not

been sequenced, and for identification of post-translational modifications. ESI-MS/MS

also allows for de novo sequencing when database searches fail to definitely identify

peptides.

Although these techniques based on 2DE are more common, new proteomics tools

are being developed. Among these gel-free methods, multidimensional protein

identification technology (MudPIT) incorporates multidimensional high performance

liquid chromatography (HPLC) coupled with ESI-MS/MS for direct analyses of complex

proteomics mixtures. Alternatively, “shotgun” methods couple the separation of proteins

using one dimensional SDS-PAGE (1DE) or IEF electrophoresis with nano-LC and ESI-

MS/MS. Using only 10 to 20 runs on a mass spectrometer, shotgun proteomics can identify

hundreds of proteins simultaneously. These new proteomic methods allow for the

systematic analysis of protein identity, and more importantly allow individual proteins to

be quantified within complex protein mixtures [143].

47

1.5.3. Gel-based comparative proteomics

The reproducibility and resolution of protein separation by 2DE has improved

greatly over the past two decades. Gorg and coworkers [145] initiated the use of IPG strips

for first dimension separation and made significant improvements in sample preparation

protocols. Higher resolution of protein separation can be achieved by using several 2DE

gels with overlapping narrow pH gradients in order to reduce the occurrence of multiple

proteins within a single 2DE spot. The use of narrow pH gradients also allows for the

visualization of posttranslational modifications [146]. Methods for protein detection have

also improved significantly, allowing for a more accurate quantitation of proteins (Table

1.4).

For comparative analyses, 2DE gels are traditionally prepared from individual

experimental treatments, and compared with each other to identify proteins that show

differential abundance between treatments. Alternatively, using DIGE technology, samples

can be covalently labelled with succinimidyl esters of different cyanide dyes (Cy2, Cy3

and Cy5) (Figure 1.10). Samples from different treatments are then pooled and

electrophoresed on the same IPG strips and 2DE gels, so that differences in protein

expression can be calculated by comparing differential fluorescence intensities of the dyes.

New software for relative 2DE spot pattern analysis allow for relative quantification of

proteins visualized using all staining/labelling methods.

48

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49

1.5.4. Gel-free comparative proteomics

The original alternative to gel based comparative proteomics was the use of stable

isotope labelling coupled with shotgun methods. Isotopic labels were incorporated either in

peptide or protein samples, or during cell culture. Variations in protein abundance were

then calculated by comparing the relative intensity of the reporter ions from each sample.

The use of these techniques was, however, limited due to the high cost of the labelling

reagents (iTRAQ or ICAT) and their low throughput.

As a result of improvements in mass spectrometer performance, label-free

quantitative shotgun proteomics approaches have now been developed. Using these new

techniques relative protein abundance can be determined by either spectral counting, or

chromatographic peak intensity measurement. Spectral counts (SpC) have proven to be

more accurate than chromatographic peak intensity measurements, so that SpC is now used

preferentially [148, 149]. Shotgun proteomics and SpC allow absolute protein contents to

be estimated [150], and for relative quantitation of all proteins present within a complex

mixture. Using this technology, Zybailov et al. [148] developed methods for the relative

quantification of proteins using normalized spectral abundance factors (NSAFs). Natural

log transformations of NSAF values provide a normal distribution of protein concentration

data and allow for statistical analysis to compare the abundance of proteins between

samples.

50

1.5.5. Proteomics and sea urchins

Sea urchins have long been a model organism in reproduction and fertilization

biology. As early as 1976 [151], proteomics was used to evaluate patterns of protein

biosynthesis immediately after fertilization and up to gastrulation.

Immediately after the completion of the S. purpuratus genome sequencing project,

a number of laboratories around the world realized the opportunity to apply functional

genomics and proteomics to investigate the physiology of this invertebrate deuterostome.

First, Roux et al. [152] used a 2DE approach to investigate calcium signalling during egg

activation. Signalling components predicted by the genome were confirmed using

proteomics. They demonstrated that the initial release of calcium by the endoplasmic

reticulum after fertilisation induced multiple post-translational modifications, especially

protein phosphorylations, which coordinate egg activation. More recently, Sewell et al.

[153] used both 2DE and MudPIT to generate the first proteome map of the mature sea

urchin ovary. Twenty protein spots observed on 2DE were extracted for identification by

mass spectrometry, but only four could be unequivocally identified. Using MudPIT, a total

of 138 proteins were identified, confirming the ability of shotgun proteomics to cover a

broader proteome, and confirming that such techniques can be used to study sea urchins of

different families (Echinometridae and Strongylocentrotidae respectively)

Soon after, Mann et al. [154, 155] investigated the proteome of the test, spine and

tooth organic matrix. They took the opportunity to build a proteome map of the organic

matrix of adult sea urchins, since most previous studies had been undertaken on embryonic

spicules [156, 157]. The authors chose to use shotgun proteomics and deduced the relative

abundance of the identified proteins by calculating the exponentially modified protein

abundance index (emPAI) [150]. Over 95% of the 110 proteins identified had never been

observed within the skeletal matrices (test and spine) using traditional protein

51

characterization techniques. An additional 82 proteins were identified while studying the

tooth organic matrix.

In the most recent advance in this field, the S. purpuratus genome sequence has

rendered possible the direct identification of proteins by in-depth, high accuracy proteomic

analyses. Preliminary studies on the developing embryo, gonads and matrices of skeletal

elements have paved the way for a better understanding of sea urchin physiology. This

thesis exploits these technological advances by applying proteomics to analyse immune

responses in sea urchins.

52

1.6. The aims of this thesis

This thesis investigates the immune system of sea urchins at the protein level. So

far, our understanding has been limited to the use of genomic and transcriptomic methods,

which do not provide information on translated proteins. One hallmark of the sea urchin

genome/transcriptome is the presence of highly variable immune response gene families.

Hence, this thesis investigates whether the genomic and transcriptomic variability

corresponds at the protein level. It aims to:

- provide the first proteome map of the sea urchin immune system

- indentify proteins involved in immune responses

- clarify the extent of molecular variability among 185/333 proteins

- provide clues to the function of 185/333 proteins

The following Chapter presents the first proteome map of sea urchin coelomic fluid

and discusses the abundance of immune related proteins. The next two Chapters

investigate immune responses over time in response to challenge with LPS or whole

bacteria. Chapter Five uses proteomics to investigate variability among expressed 185/333

proteins, whilst the final “results” Chapter provides new information on the localization of

185/333 proteins and discusses their potential functions.

53

1.7. References

[1] Chen, J.-Y., Huang, D.-Y., Peng, Q.-Q., Chi, H.-M., et al., The first tunicate from the

Early Cambrian of South China. Proc Natl Acad Sci U S A 2003, 100, 8314-8318.

[2] Bromham, L. D., Degnan, B. M., Hemichordates and deuterostome evolution: robust

molecular phylogenetic support for a hemichordate + echinoderm clade. Evolution &

Development 1999, 1, 166-171.

[3] Davidson, E. H., Cameron, R. A., Arguments for sequencing the genome of the sea

urchin Strongylocentrotus purpuratus.

www.genome.gov/pages/research/sequencing/SegProposals/SeaUrchin _Genome.

prob.2002. 2002.

[4] Gray, J., An attempt to divide the Echinida, or sea eggs, into natural families. Ann.

Philos. 1825, 26, 423-431.

[5] Gray, J., Arrangement of families of Echinidea. Proc. Zool. Soc. London, III. 1855, 23,

35-39.

[6] Fell, F., Echinodermata, McGraw-Hill, New-York 1982.

[7] Gregory, J., The echinoidea, in Lankester, E.R.: A treatise on zoology, Adam and

Charles Black, London 1900.

[8] Springer, M., Tusneem, N., Davidson, E., Britten, R., Phylogeny, rates of evolution,

and patterns of codon usage among sea urchin retroviral-like elements, with

implications for the recognition of horizontal transfer. Mol Biol Evol 1995, 12, 219-

230.

[9] Klein, I., Naturalis dispositio Echinodermatum, Officina Gleditschiana, Leipzig 1734.

[10] Smith, A., Echinoid palaeobiology, George Allen and Unwin, London 1984.

[11] Cobb, J., Sneddon, E., An ultra-structural study of the gills of Echinus esculentus. Cell

Tissue Res. 1977, 182, 265-274.

54

[12] Farmanfarmaian, A., Stanford University, Stanford, Calif. 1959.

[13] Bamford, D., Echinoderm nutrition, M. Jangoux and J.M. Lawrence, Balkema,

rotterdam 1982, pp. 317-330.

[14] Ruppert, E. E., Barnes, R. D., invertebrate zoology. 6th ed., Saunders College

Publishers, Philadelphia 1994.

[15] Ziegler, A., Faber, C., Mueller, S., Bartolomaeus, T., Systematic comparison and

reconstruction of sea urchin (Echinoidea) internal anatomy: a novel approach using

magnetic resonance imaging. BMC Biology 2008, 6, 33.

[16] Holm, K., Dupont, S., Skold, H., Stenius, A., et al., Induced cell proliferation in

putative haematopoietic tissues of the sea star, Asterias rubens (L.). J Exp Biol 2008,

211, 2551-2558.

[17] Smith, V., The echinoderms, Academic Press, New York 1981.

[18] Chia, F. S., Xing, J., Echinoderm coelomocytes. Zoological studies 1996, 35, 231-

254.

[19] Boolootian, R., Geise, C., Coelomic corpuscles of echinoderms. Biol. Bull. 1958, 15,

53-56.

[20] Johnson, P., The coelomic elements of the sea urchins (Strongylocentrotus) III. In

vitro reaction to bacteria. J. Invert. Pathol. 1969, 13, 42-62.

[21] Edds, K. T., Cell biology of echinoid coelomocytes. I. Diversity and characterization

of cell types. J. Invert. Biol. 1993, 61, 173-178.

[22] Gross, P. S., Clow, L. A., Smith, L. C., SpC3, the complement homologue from the

purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations

of the phagocytic coelomocytes. Immunogenetics 2000, 51, 1034-1044.

[23] Henson, J. H., Nesbitt, D., Wright, B. D., Sholey, J. S., Immunolocalization of kinesin

in sea urchin coelomocytes: association of kinesin with intracellular organelles. J.

Cell. Sci. 1992, 103, 309-320.

55

[24] Henson, J. H., Svitkina, T. M., Burns, A. R., Hughes, H. E., et al., Two components of

actin-based retrograde flow in sea urchin coelomocytes. Mol Biol Cell 1999, 10,

4075-4090.

[25] Brockton, V., Henson, J. H., Raftos, D. A., Majeske, A. J., et al., Localization and

diversity of 185/333 proteins from the purple sea urchin - unexpected protein-size

range and protein expression in a new coelomocyte type. J Cell Sci 2008, 121, 339-

348.

[26] Smith, L. C., Davidson, E. H., The echinoid immune system and the phylogenetic

occurrence of immune mechanisms in deuterostomes. Immunol. Today 1992, 13,

356-362.

[27] Smith, L., Britten, R., Davidson, E., Lipopolysaccharide activates the sea urchin

immune system. Dev Comp Immunol 1995, 19, 217-224.

[28] Matranga, V., Pinsino, A., Celi, M., Bella, G., Natoli, A., Impacts of UV-B radiation

on short-term cultures of sea urchin coelomocytes. Mar Biol 2006, 149, 25-34.

[29] Smith, L., Rast, J., Brockton, V., Terwilliger, D., et al., The sea urchin immune

system. Invertebrate Survival Journal 2006, 3, 25-39.

[30] Hibino, T., Loza-Coll, M., Messier, C., Majeske, A., et al., The immune gene

repertoire encoded in the purple sea urchin genome. Dev Biol 2006, 300, 349-365.

[31] Smith, L. C., Davidson, E. H., The echinoderm immune system. Characters shared

with vertebrate immune systems and characters arising later in deuterostome

phylogeny. Ann. NY Acad. Sci. 1994, 712, 213-226.

[32] Gross, P. S., Al-Sharif, W. Z., Clow, L. A., Smith, L. C., Echinoderm immunity and

the evolution of the complement system. Dev Comp Immunol 1999, 23, 429-442.

[33] Hildemann, W. H., Dix, T. G., Transplantation reactions of tropical Australian

echinoderms. Transplantation 1972, 15, 624-633.

56

[34] Karp, R. D., Hildemann, W. H., Specific allograft reactivity in the seastar

Dermasterias imbricata. Transplantation 1976, 22, 434-439.

[35] Coffaro, K. A., Phylogeny of immunological memory, M.J. Manning. Elsevier / North-

Holland Biomedical Press, Amsterdam 1980, p. 77.

[36] Coffaro, K., Hinegardner, R., Immune response in the sea urchin Lytechinus pictus.

Science 1977, 197, 1389-1390.

[37] Coffaro, K. A., Clearance of bacteriophage T4 in the sea urchin Lytechinus pictus. J.

Invert. Pathol. 1978, 32, 384-385.

[38] Stabili, L., Pagliara, M., Metrangolo, M., Canicatti, C., Comparative aspects of

Echinoidea cytolysins: the cytolytic activity of Spherechinus granularis (Echinoidea)

coelomic fluid. Comp. Biochem. Physiol. 1992, 101A, 553-556.

[39] Gerardi, G., Lassegues, M., Canicatti, C., Cellular distribution of sea urchin

antibacterial activity. Biol. Cell. 1990, 70, 153-157.

[40] Edds, K., Dynamic aspects of filopodial formation by reorganization of

microfilaments. J Cell Biol 1977, 73, 479-491.

[41] Xing, J., Chia, F. S., Phagocytosis of sea cucumber amoebocytes: a flow cytometric

study. Invertebr Biol 1998, 117, 67-74.

[42] Arizza, V., Giaramita, F. T., Parrinello, D., Cammarata, M., Parrinello, N., Cell

cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus

coelomocytes. Comp Biochem Physiol A Mol Integr Physiol 2007, 147, 389-394.

[43] Lin, W., Grant, S., Beck, G., Generation of monoclonal antibodies to coelomocytes of

the purple sea urchin Arbacia punctulata: Characterization and phenotyping. Dev

Comp Immunol 2007, 31, 465-475.

[44] Matranga, V., Toia, G., Bonaventura, R., Muller, W., Cellular and biochemical

responses to environmental and experimentally induced stress in sea urchin

coelomocytes. Cell stress chaperones 2000, 5, 158-165.

57

[45] Service, M., Wardlaw, A., Echinochrome-A as a bactericidal substance in the

coelomic fluid of Echinus esculentus (L.). Comp Biochem Physiol B Biochem Mol

Biol 1984, 79, 161-165.

[46] Cervello, M., Arizza, V., Lattuca, G., Parrinello, N., Matranga, V., Detection of

vitellogenin in a subpopulation of sea urchin coelomocytes. Eur J Cell Biol 1994, 64,

314-319.

[47] Smith, L., Britten, R., Davidson, E., SpCoel1: a sea urchin profilin gene expressed

specifically in coelomocytes in response to injury. Mol Biol Cell 1992, 3, 403-414.

[48] Smith, L., Chang, L., Britten, R., Davidson, E., Sea urchin genes expressed in

activated coelomocytes are identified by expressed sequence tags. Complement

homologues and other putative immune response genes suggest immune system

homology within the deuterostomes. J Immunol 1996, 156, 593-602.

[49] Al-Sharif, W. Z., Sunyer, J. O., Lambris, J. D., Smith, L. C., Sea urchin coelomocytes

specifically express a homologue of the complement component C3. J Immunol

1998, 160, 2983-2997.

[50] Smith, L. C., Shih, C.-S., Dachenhausen, S. G., Coelomocytes express SpBf, a

homologue of factor B, the second component in the sea urchin complement system.

J Immunol 1998, 161, 6784-6793.

[51] Beck, G., Habicht, G. S., Immunity and the invertebrates. Scientific american 1996,

275, 60-66.

[52] Sodergren, E., Weinstock, G.M., Davidson, E.H., Cameron, R., A. et al. The genome

of the sea urchin Strongylocentrotus purpuratus. Science 2006, 314, 941-952.

[53] Rast, J. P., Smith, L. C., Loza-Coll, M., Hibino, T., Litman, G. W., Genomic insights

into the immune system of the sea urchin. Science 2006, 314, 952-956.

[54] Rast, J. P., Messier-Solek, C., Marine invertebrate genome sequences and our

evolving understanding of animal immunity. Biol Bull 2008, 214, 274-283.

58

[55] Du, X., Poltorak, A., Wei, Y., Beutler, B., Three novel mammalian toll-like receptors:

gene structure, expression, and evolution. European Cytokine Network 2000, 11,

362-371.

[56] Clow, L. A., Raftos, D. A., Gross, P. S., Smith, L. C., The sea urchin complement

homologue, SpC3, functions as an opsonin. J Exp Biol 2004, 207, 2147-2155.

[57] Smith, L. C., Thioester function is conserved in SpC3, the sea urchin homologue of

the complement component C3. Dev Comp Immunol 2002, 26, 603-614.

[58] Clow, L. A., Gross, P. S., Shih, C.-S., Smith, L. C., Expression of SpC3, the sea

urchin complement component, in response to lipopolysaccharide. Immunogenetics

2000, 51, 1021-1033.

[59] Shah, M., Brown, K., Smith, L., The gene encoding the sea urchin complement

protein, SpC3, is expressed in embryos and can be upregulated by bacteria. Dev

Comp Immunol 2003, 27, 529-538.

[60] Terwilliger, D., Clow, L., Gross, P., Smith, L., Constitutive expression and alternative

splicing of the exons encoding SCRs in Sp152, the sea urchin homologue of

complement factor B. Implications on the evolution of the Bf/C2 gene family.

Immunogenetics 2004, 56, 531-543.

[61] Smith, L. C., Clow, L. A., Terwilliger, D. P., The ancestral complement system in sea

urchins. Immunological Reviews 2001, 180, 16-34.

[62] Ji, X., Azumi, K., Sasaki, M., Nonaka, M., Ancient origin of the complement lectin

pathway revealed by molecular cloning of mannan binding protein-associated serine

protease from a urochordate, the Japanese ascidian, Halocynthia roretzi. Proc Natl

Acad Sci U S A 1997, 94, 6340-6345.

[63] Nakao, M., Kajiya, T., Sato, Y., Somamoto, T., et al., Lectin pathway of bony fish

complement: Identification of two homologs of the mannose-binding lectin

59

associated with MASP2 in the common carp (Cyprinus carpio). J Immunol 2006,

177, 5471-5479.

[64] Takahashi, K., Ip, W. K. E., Michelow, I. C., Ezekowitz, R. A. B., The mannose-

binding lectin: a prototypic pattern recognition molecule. Current Opinion in

Immunology 2006, 18, 16-23.

[65] Fujita, T., Evolution of the lectin-complement pathway and its role in innate

immunity. Nat Rev Immunol 2002, 2, 346-353.

[66] Fujita, T., Matsushita, M., Endo, Y., The lectin-complement pathway -- its role in

innate immunity and evolution. Immunological Reviews 2004, 198, 185-202.

[67] Agrawal, A., Eastman, Q. M., Schatz, D. G., Transposition mediated by RAG1 and

RAG2 and its implications for the evolution of the immune system. Nature 1998,

394, 744-751.

[68] Miracle, A. L., Anderson, M. K., Litman, R. T., Walsh, C. J., et al., Complex

expression patterns of lymphocyte-specific genes during the development of

cartilaginous fish implicate unique lymphoid tissues in generating an immune

repertoire. Int. Immunol. 2001, 13, 567-580.

[69] Bartl, S., Miracle, A. L., Rumfelt, L. L., Kepler, T. B., et al., Terminal

deoxynucleotidyl transferases from elasmobranchs reveal structural conservation

within vertebrates. Immunogenetics 2003, 55, 594-604.

[70] Fugmann, S. D., Messier, C., Novack, L. A., Cameron, R. A., Rast, J. P., An ancient

evolutionary origin of the Rag1/2 gene locus. Proceedings of the National Academy

of Sciences of the United States of America 2006, 103, 3728-3733.

[71] Dominguez. O., Ruiz, J., F., Lain de Lera, T., Garcia-Diaz, M., Gonzalez, M., A.,

Kirchhoff, T., Martinez-A, C., Bernard, A., Blanco, L., DNA polymerase mu (Polµ),

homologous to TdT, could act as a DNA mutator in eukaryotic cells. The EMBO

Journal 2000, 19, 1731-1742.

60

[72] Ruiz, J., F., Dominguez, O., Lain de Lera, T., Garcia-Diaz, M., Bernard, A., Blanco,

L., DNA polymerase mu, a candidate hypermutase? Philos Trans R Soc Lond B Biol

Sci 2001, 356, 99-109.

[73] Raftos, D., Raison, R. L., Evolutionary immunology: Early vertebrates reveal diverse

immune recognition strategies. Immunol Cell Biol 2008, 86, 479-481.

[74] Kurtz, J., Memory in the innate and adaptive immune systems. Microbes and Infection

2004, 6, 1410-1417.

[75] Nair, S. V., Del Valle, H., Gross, P. S., Terwilliger, D. P., Smith, L. C., Macroarray

analysis of coelomocyte gene expression in response to LPS in the sea urchin.

Identification of unexpected immune diversity in an invertebrate. Physiol Genomics

2005, 22, 33-47.

[76] Flajnik, M., Du Pasquier, L., Evolution of innate and adaptive immunity: can we draw

a line? Trends Immunol 2004, 25, 640-644.

[77] Pancer, Z., Amemiya, C., Ehrhardt, G., Ceitlin, J., et al., Somatic diversification of

variable lymphocyte receptors in the agnathan sea lamprey. Nature 2004, 430, 174-

180.

[78] Watson, F. L., Puttmann-Holgado, R., Thomas, F., Lamar, D. L., et al., Extensive

diversity of Ig-superfamily proteins in the immune system of insects. Science 2005,

309, 1874-1878.

[79] Cannon, J. P., Haire, R. N., Litman, G. W., Identification of diversified genes that

contain immunoglobulin-like variable regions in a protochordate. Nature

Immunology 2002, 3, 1200-1207.

[80] Stearns, S. C., Evolution of life-history traits - critique of theory and a review of data.

Ann. rev. Ecol. Systematics 1977, 8, 145-171.

61

[81] Getz, W. M., Metaphysiological and evolutionnary dynamics of populations

exploiting constant and interactive resources - r-K selection revisited. Evol. Ecol.

1993, 7, 287-305.

[82] Klein, J., Are invertebrates capable of anticipatory immune response? Scand. J.

Immunol. 1989, 29, 499-505.

[83] Van Valen, L. A., A new evolutionnary law. Evol. Theory 1973, 1, 1-30.

[84] Bowden, L., Dheilly, N. M., Raftos, D. A., Nair, S. V., New immune systems:

pathogen-specific host defense, life history strategies and hypervariable immune-

response genes of invertebrates. Invertebrate survival journal 2007, 4, 127-136.

[85] Ebert, T. A., Southon, J. R., Red sea urchins (Strongylocentrotus franciscanus) can

live over 100 years: confirmation with A-bomb 14 carbon. Fish bulletin

(Sacramento, Calif.) 2003, 101, 915.

[86] Stow, A., Briscoe, D., Gillings, M., Holley, M., et al., Antimicrobial defences increase

with sociality in bees. Biology Letters 2007, 3, 422-424.

[87] Sala, E., Ribes, M., Hereu, B., Zabala, M., et al., Temporal variability in abundance of

the sea urchins Paracentrotus lividus and Arbacia lixula in the northwestern

Mediterranean: comparison between a marine reserve and an unprotected area.

Marine ecology progress series 1998, 168, 135-145.

[88] Becker, P. T., Egea, E., Eeckhaut, I., Characterization of the bacterial communities

associated with the bald sea urchin disease of the echinoid Paracentrotus lividus. J

Invertebr Pathol 2008, 98, 136-147.

[89] Lester, S. E., Tobin, E. D., Behrens, M. D., Disease dynamics and the potential role of

thermal stress in the sea urchin, Strongylocentrotus purpuratus. Can. J. Fish. Aquat.

Sci. 2007, 64, 314-323.

[90] Scheibling, R. E., Hennigar, A. W., Recurrent outbreaks of disease in sea urchins

Strongylocentrotus droebachiensis in Nova Scotia: evidence for a link with large-

62

scale meteorologic and oceanographic events. Marine ecology progress series 1997,

152, 155-165.

[91] Little, T. J., Hultmark, D., Read, A. F., Invertebrate immunity and the limits of

mechanistic immunology. Nat Immunol 2005, 6, 651-654.

[92] Lemaitre, B., Reichhart, J.-M., Hoffmann, J. A., Drosophila host defense: Differential

induction of antimicrobial peptide genes after infection by various classes of

microorganisms. Proc Natl Acad Sci U S A 1997, 94, 14614-14619.

[93] Pham, L. N., Dionne, M. S., Shirasu-Hiza, M., Schneider, D. S., A specific primed

immune response in Drosophila is dependent on phagocytes. PLoS Pathogens 2007,

3, e26.

[94] Sadd, B. M., Schmid-Hempel, P., Insect immunity shows specificity in protection

upon secondary pathogen exposure. Curr Biol 2006, 16, 1206-1210.

[95] Good, R. A., Finstad, J., Litman , G. W., The biology of Lampreys, Academic, London

1972.

[96] Hildemann, W. H., Transplantation immunity in fishes: Agnathan, Chondrichthyes

and Osteichthyes. Transplant. Proc. 1970, 2, 253-259.

[97] Hagen, M., Filosa, M. F., Youson, J. H., The immune response in adult sea lamprey

(Petromyzon marinus L.): The effect of temperature. Comp Biochem Physiol A

Physiol 1985, 82, 207-210.

[98] Perey, D. Y., Finstad, J., Pollara, B., Good, R. A., Evolution of the immune response.

VI. First and second set skin homograft rejections in primitive fishes. Lab. Invest.

1968, 19, 591-597.

[99] Alder, M. N., Rogozin, I. B., Iyer, L. M., Glazko, G. V., et al., Diversity and function

of adaptive immune receptors in a jawless vertebrate. Science 2005, 310, 1970-1973.

[100] Kim, H. M., Oh, S. C., Lim, K. J., Kasamatsu, J., et al., Structural diversity of the

hagfish variable lymphocyte receptors. J Biol Chem 2007, 282, 6726-6732.

63

[101] Han, B. W., Herrin, B. R., Cooper, M. D., Wilson, I. A., Antigen recognition by

variable lymphocyte receptors. Science 2008, 321, 1834-1837.

[102] Kasamatsu, J., Suzuki, T., Ishijima, J., Matsuda, Y., Kasahara, M., Two variable

lymphocyte receptor genes of the inshore hagfish are located far apart on the same

chromosome. Immunogenetics 2007, 59, 329-331.

[103] Rogozin, I. B., Iyer, L. M., Liang, L., Glazko, G. V., et al., Evolution and

diversification of lamprey antigen receptors: evidence for involvement of an AID-

APOBEC family cytosine deaminase. Nat Immunol 2007, 8, 647-656.

[104] Cannon, J., Haire, R., Litman, G., Identification of diversified genes that contain

immunoglobulin-like variable regions in a protochordate. Nat Immunol 2002, 3,

1200-1207.

[105] Cannon, J., Haire, R., Schnitker, N., Mueller, M., Litman, G., Individual

protochordates have unique immune-type receptor repertoires. Curr Biol 2004, 14,

R465 - 466.

[106] Dishaw, L., Mueller, M. G., Gwatney, N., Cannon, J., et al., Genomic complexity of

the variable region-containing chitin-binding proteins in amphioxus. BMC Genetics

2008, 9, 78.

[107] Hernandez Prada, J., Haire, R., Allaire, M., Jakoncic, J., et al., Ancient evolutionary

origin of diversified variable regions revealed by crystal structures of an immune-

type receptor in amphioxus. Nat Immunol 2006, 7, 875-882.

[108] Litman, G., Cannon, J., Dishaw, L., Reconstructing immune phylogeny: new

perspectives. Nat Rev Immunol 2005, 5, 866-879.

[109] Adema, C. M., Hertel, L. A., Miller, R. D., Loker, E. S., A family of fibrinogen-

related proteins that precipitates parasite-derived molecules is produced by an

invertebrate after infection. Proc Natl Acad Sci U S A 1997, 94, 8691-8696.

64

[110] Léonard, P. M., Adema, C. M., Zhang, S.-M., Loker, E. S., Structure of two FREP

genes that combine IgSF and fibrinogen domains, with comments on diversity of the

FREP gene family in the snail Biomphalaria glabrata. Gene 2001, 269, 155-165.

[111] Kurachi, S., Song, Z., Takagaki, M., Yang, Q., et al., Sialic-acid-binding lectin from

the slug Limax flavus: cloning, expression of the polypeptide, and tissue location.

European Journal of Biochemistry 1998, 253, 217-222.

[112] Gokudan, S., Muta, T., Tsuda, R., Koori, K., et al., Horseshoe crab acetyl group-

recognizing lectins involved in innate immunity are structurally related to fibrinogen.

Proc Natl Acad Sci USA 1999, 96, 10086-10091.

[113] Gregorio, E. D., Spellman, P. T., Rubin, G. M., Lemaitre, B., Genome-wide analysis

of the Drosophila immune response by using oligonucleotide microarrays. Proc Nat

Acad Sci USA 2001, 98, 12590-12595.

[114] Kenjo, A., Takahashi, M., Matsushita, M., Endo, Y., et al., Cloning and

characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi. J

Biol Chem 2001, 276, 19959-19965.

[115] Dehal, P., Satou, Y., Campbell, R. K., Chapman, J., et al., The draft genome of

Ciona intestinalis: insights into chordate and vertebrate origins. Science 2002, 298,

2157-2167.

[116] Zdobnov, E. M., von Mering, C., Letunic, I., Torrents, D., et al., Comparative

genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster.

Science 2002, 298, 149-159.

[117] Holmskov, U., Thiel, S., Jensenius, J. C., Collectins and ficolins: humoral lectins of

the innate immune defense. Annual Review of Immunology 2003, 21, 547-578.

[118] Zhang, S.-M., Loker, E. S., The FREP gene family in the snail Biomphalaria

glabrata: additional members, and evidence consistent with alternative splicing and

FREP retrosequences. Dev Comp Immunol 2003, 27, 175-187.

65

[119] Zhang, S., Adema, C., Kepler, T., Loker, E., Diversification of Ig superfamily genes

in an invertebrate. Science 2004, 305, 251-254.

[120] Zhang, S.-M., Yong Zeng, Loker, E. S., Expression profiling and binding properties

of fibrinogen-related proteins (FREPs), plasma proteins from the schistosome snail

host Biomphalaria glabrata. Innate Immunity 2008, 14, 175-189.

[121] Adema, C. M., Hertel, L. A., Loker, E. S., Evidence from two planorbid snails of a

complex and dedicated response to digenean (echinostome) infection. Parasitology

1999, 119, 395-404.

[122] Hertel, L. A., Adema, C. M., Loker, E. S., Differential expression of FREP genes in

two strains of Biomphalaria glabrata following exposure to the digenetic trematodes

Schistosoma mansoni and Echinostoma paraensei. Dev Comp Immunol 2005, 29,

295-303.

[123] Zhang, S.-M., Nian, H., Zeng, Y., DeJong, R. J., Fibrinogen-bearing protein genes in

the snail Biomphalaria glabrata: Characterization of two novel genes and expression

studies during ontogenesis and trematode infection. Developmental & Comparative

Immunology 2008, 32, 1119-1130.

[124] Dong, Y., Dimopoulos, G., Anopheles fibrinogen-related proteins provide expanded

pattern recognition capacity against bacteria and Malaria parasites. J. Biol. Chem.

2009, 284, 9835-9844.

[125] Fan, C., Zhang, S., Li, L., Chao, Y., Fibrinogen-related protein from amphioxus

Branchiostoma belcheri is a multivalent pattern recognition receptor with a

bacteriolytic activity. Mol Immunol 2008, 45, 3338-3346.

[126] Zhang, H., Wang, L., Song, L., Song, X., et al., A fibrinogen-related protein from

bay scallop Argopecten irradians involved in innate immunity as pattern recognition

receptor. Fish Shellfish Immunol 2009, 26, 56-64.

66

[127] Schmucker, D., Clemens, J., Shu, H., Worby, C., et al., Drosophila Dscam is an axon

guidance receptor exhibiting extraordinary molecular diversity. Cell 2000, 101, 671-

684.

[128] Neves, G., Zucker, J., Daly, M., Chess, A., Stochastic yet biased expression of

multiple Dscam splice variants by individual cells. Nature genetics 2004, 36, 240-

246.

[129] Watson, F., Puttmann-Holgado, R., Thomas, F., Lamar, D., et al., Extensive

diversity of Ig-superfamily proteins in the immune system of insects. Science 2005,

309, 1874-1878.

[130] Dong, Y., Taylor, H., Dimopoulos, G., AgDscam, a hypervariable immunoglobulin

domain-containing receptor of the Anopheles gambiae innate immune system. PLoS

Biol 2006, 4, e229.

[131] Crayton, M., Powell, B., Vision, T., Giddings, M., Tracking the evolution of

alternatively spliced exons within the Dscam family. BMC Evolutionary Biology

2006, 6, 16.

[132] Brites, D., McTaggart, S., Morris, K., Anderson, J., et al., The Dscam homologue of

the crustacean Daphnia is diversified by alternative splicing like in insects. Mol Biol

Evol 2008, 25, 1429-1439.

[133] Rast, J., Pancer, Z., Davidson, E., New approaches towards an understanding of

deuterostome immunity. Curr Top Microbiol Immunol 2000, 248, 3-16.

[134] Terwilliger, D., Buckley, K., Mehta, D., Moorjani, P., Smith, L., Unexpected

diversity displayed in cDNAs expressed by the immune cells of the purple sea

urchin, Strongylocentrotus purpuratus. Physiol Genomics 2006, 26, 134-144.

[135] Buckley, K., Smith, L., Extraordinary diversity among members of the large gene

family, 185/333, from the purple sea urchin, Strongylocentrotus purpuratus. BMC

Mol Biol 2007, 8, 68.

67

[136] Terwilliger, D., Buckley, K., Brockton, V., Ritter, N., Smith, L. C., Distinctive

expression patterns of 185/333 genes in the purple sea urchin, Strongylocentrotus

purpuratus: an unexpectedly diverse family of transcripts in response to LPS, beta-

1,3-glucan, and dsRNA. BMC Mol Biol 2007, 8, 16.

[137] Rogaev, E. I., Simple human DNA-repeats associated with genomic hypervariability,

flanking the genomic retroposons and similar to retroviral sites. Nucl. Acids Res.

1990, 18, 1879-1885.

[138] Buckley, K. M., Munshaw, S., Kepler, T. B., Smith, L. C., The 185/333 gene family

is a rapidly diversifying host-defense gene cluster in the purple sea urchin

Strongylocentrotus purpuratus. J Mol Biol 2008, 379, 912-928.

[139] Buckley, K., Florea, L., Smith, L. C., A method for identifying alternative or cryptic

donor splice sites within gene and mRNA sequences. Comparisons among sequences

from vertebrates, echinoderms and other groups. BMC Genomics 2009, 10, 318.

[140] Buckley, K. M., Terwilliger, D. P., Smith, L. C., Sequence variations in 185/333

messages from the purple sea urchin suggest posttranscriptional modifications to

increase immune diversity. J Immunol 2008, 181, 8585-8594.

[141] Tsai, H., Bobek, L. A., Human salivary histatins: promising anti-fungal therapeutic

agents. Critical Reviews in Oral Biology & Medicine 1998, 9, 480-497.

[142] Johansson, M. W., Cell adhesion molecules in invertebrate immunity.

Developmental & Comparative Immunology 1999, 23, 303-315.

[143] Peng, J., Gygi, S. P., Proteomics: the move to mixtures. Journal of Mass

Spectrometry 2001, 36, 1083-1091.

[144] O'Farrell, P. H., High resolution two-dimensional electrophoresis of proteins. J. Biol.

Chem. 1975, 250, 4007-4021.

68

[145] Rabilloud, T., Adessi, C., Guiraudel, A., Lunardi, J., Improvement of the

solubilization of proteins in two-dimensional electrophoresis with immobilized pH

gradients. Electrophoresis 1997, 18, 307-316.

[146] Hoving, S., Voshol, H., van Oostrum, J., Towards high performance two-

dimensional gel electrophoresis using ultrazoom gels. Electrophoresis 2000, 21,

2617-2621.

[147] Cristea, I. M., Gaskell, S. J., Whetton, A. D., Proteomics techniques and their

application to hematology. Blood 2004, 103, 3624-3634.

[148] Zybailov, B. L., Florens, L., P., W. M., Quantitative shotgun proteomics using a

protease with broad specificity and normalized spectral abundance factors. Mol

Biosyst 2007, 3, 354-360.

[149] Old, W. M., Meyer-Arendt, K., Aveline-Wolf, L., Pierce, K. G., et al., Comparison

of label-free methods for quantifying human proteins by shotgun proteomics. Mol

Cell Proteomics 2005, 4, 1487-1502.

[150] Ishihama, Y., Oda, Y., Tabata, T., Sato, T., et al., Exponentially modified protein

abundance index (emPAI) for estimation of absolute protein amount in proteomics

by the number of sequenced peptides per protein. Mol Cell Proteomics 2005, 4,

1265-1272.

[151] Brandhorst, B. P., Two-dimensional gel patterns of protein synthesis before and after

fertilization of sea urchin eggs. Dev Biol 1976, 52, 310-317.

[152] Roux, M. M., Townley, I. K., Raisch, M., Reade, A., et al., A functional genomic

and proteomic perspective of sea urchin calcium signaling and egg activation.

Developmental Biology 2006, 300, 416-433.

[153] Sewell, M. A., Eriksen, S., Middleditch, M. J., Identification of protein components

from the mature ovary of the sea urchin Evechinus chloroticus (Echinodermata:

Echinoidea). Proteomics 2008, 8, 2531-2542.

69

[154] Mann, K., Poustka, A., Mann, M., The sea urchin (Strongylocentrotus purpuratus)

test and spine proteomes. Proteome Science 2008, 6, 22.

[155] Mann, K., Poustka, A., Mann, M., In-depth, high-accuracy proteomics of sea urchin

tooth organic matrix. Proteome Science 2008, 6, 33.

[156] Wilt, F. H., Killian, C. E., Livingston, B. T., Development of calcareous skeletal

elements in invertebrates. Differentiation 2003, 71, 237-250.

[157] Wilt, F. H., Matrix and mineral in the sea urchin larval skeleton. Journal of

Structural Biology 1999, 126, 216-226.

70

71

CHAPTER II

Shotgun proteomic analysis of coelomocytes from the purple sea urchin,

Strongylocentrotus purpuratus.

Submitted as a Dataset Brief to

Proteomics

Author contributions:

Haynes PA2 - Experimental design - Technical support

Smith LC3 - Sample preparation

Raftos DA1 - Projet supervision

Nair SV1 – Experimental design - Project supervision

1Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia.

2Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109,

Australia

3Department of Biological Sciences, George Washington University, Washington, DC, 20052, USA.

72

73

2.1. Preface

As outlined in Chapter 1 of this thesis, there is already extensive genomic and

trancriptomic data on the immune system of sea urchins. These predicted a complex

immune system that with some similarities to vertebrate immune responses. Coelomocytes

appear to be the main effector cells of sea urchins immune responses. However, little is

known about the proteins expressed by coelomocytes. The objective of this Chapter was to

draw an initial proteome map of the sea urchin coelomic fluid as a first step in assessing

changes in that proteome after immunological challenge.

Remarks: The following Chapter has been submitted to Proteomics as a Dataset

Brief. As such, it has been prepared accordingly to the following instructions for authors:

“Dataset briefs describing novel proteomic datasets of specific types of samples, such as organisms,

tissues,organelles,andcells.ThesedatasetscanbegeneratedwithanyproteomicPlatformincluding

two‐dimensionalgels,massspectrometryorproteinarrays.Animportantcriterionisthatthedataset

containsasignificantnumberofidentifiedproteinsthatwillbenefitfurtherresearchonthatparticular

sampletype.Themanuscriptsshouldbearthewords“DatasetBrief”immediatelyabovethetitleonthe

firstpage.Theyshouldnotbesubdividedintotitledsectionsbutwritteninacontinuousstyle.Dataset

briefsshouldnotexceed2500words(includingreferencesandFigurelegends)andcontainnomore

than 3 display elements (Figures and tables). Authors are encouraged to submit supporting

information,suchasannotatedtwo‐dimensionalgelimagesandtablesofproteinidentifications,which

willonlyappearonline.”

74

75

2.2. Abstract

Recent genome sequencing and transcriptomic analyses have revealed a complex

immune system in sea urchins that appears to be mediated by coelomocytes (blood cells).

However, the broad array of proteins that comprise the coelomocyte proteome remains

unknown. Here, we identified 319 proteins present in sea urchin coelomocytes. Proteins

involved in the coelomocyte cytoskeleton were the most abundant. This is consistent with

the capacity of coelomocytes for pseudopodium formation and phagocytosis. A number of

immune response proteins were also identified, including a preponderance of complement

components and scavenger receptor cysteine rich (SRCR) proteins. The relative abundance

of dual oxidase 1, apextrin, echinonectin, major yolk protein, apolipoprotein B,

ceruloplasmin, ferritin and transferrin suggest that these proteins may also be involved in

host defense. Overall, the function of many coelomocytes proteins could ultimately be

related to anti-pathogen defense, which supports the role of coelomocytes as the primary

immune mediators of sea urchins.

76

2.3. Dataset Brief

The last common ancestor of the deuterostomes lived during the Cambrian period,

520-530 million years ago [1]. This ancestor gave rise to echinoderms, of which sea

urchins (class Echinodidea) constitute a major taxon [2]. Their phylogenetic position, as

well as their importance in the studies of early deuterostome development, were the

motivations for sequencing the genome of the purple sea urchin, Strongylocentrotus

purpuratus [3]. That genome sequence confirmed the kinship between echinoderms and

chordates, but also revealed some unexpected results. Among these surprises was the

complexity of immune response genes in sea urchins [3-5].

Given this complexity, it is essential to analyze the functional immunome of sea

urchins at the protein level. The current study uses large scale shotgun proteomics to

provide the first assessment of the sea urchin coelomocyte proteome. Shotgun proteomics

allows the majority of proteins in a particular cell type (coelomocytes in our study) to be

identified and characterized simultaneously, providing a comprehensive picture of these

cells at the protein level. In this study, we identify 319 proteins expressed in coelomocytes

and discuss their putative functions with an emphasis on their role in immunological

reactivity.

Three adult S. purpuratus were challenged with lipopolysaccharide (LPS) as

described previously [6-11] (For detailed descriptions of methodology see Supplementary

Data 2.1). Coelomic fluid (CF) proteins were extracted and separated on SDS-PAGE gels

and digested with trypsin before being analyzed by nanoLC-MS/MS using a LTQ-XL ion-

trap mass spectrometer. MS/MS spectra were searched against a local database that

contained all 44,037 Strongylocentrotus protein sequences held by NCBI as of April 2008.

The relative abundance of identified proteins (FDR< 0.1%) were calculated based on

normalized spectral abundance factor (NSAF) values as described previously [12]. For

77

each protein, k, the sum (S) of all spectral counts obtained from the three sea urchins was

calculated, and the corresponding NSAFS deduced. NSAFS values were used as a measure

of protein abundance. The NSAFi values for each of the individual sea urchins were

strongly correlated with these NSAFS values (R2, 0.85-0.92), suggesting that NSAFS values

were representative of protein abundances within the three CF samples. The natural log of

the NSAFs values were plotted against the number of proteins obtained, which showed a

normal distribution with a mean of -4.78 and a standard deviation of ± 0.62.

Shotgun proteomic analysis identified 319 distinct coelomocyte proteins with high

confidence (Table 2.1, for detailed description of results, see Supplementary Data 2.2).

Most (284 of 319) of the proteins were predicted from the genome of S. purpuratus, but

had not been previously identified at the protein level using non-genomic methods.

Hypothetical proteins predicted in the S. purpuratus genome without a known or putative

function accounted for 48 of the 319 proteins. Protein BLAST and conserved domain

searches against the entire NCBI database showed strong matches to homologous proteins

in all but 16 of these cases.

Proteins were grouped into 11 functional categories based on the annotations for

the corresponding genes in the S. purpuratus genome or other homologies. Figure 2.1

shows the number of proteins that fell within defined functional categories and the relative

abundance (NSAFs values) of each category within the total coelomocyte proteome.

Proteins involved in cell structure, shape and mobility were the most common in the

coelomocyte proteome, including 52 distinct proteins that accounted for 53% of all NSAFs

values. These included the cytoskeletal actins (CyIIb and CyIIIb), profilin, fascin, cofilin,

gelsolin, myosin, microtubule associated protein, coronin, tubulin, α-actinin and collagen.

This agrees with physiological studies, which have shown that phagocytic coelomocytes

represent up to 82% of the cells in the CF [13, 8]. They are highly mobile cells that can

quickly develop extended filopodia and lamellipodia [14] and are avidly phagocytic [15].

78

Figure 2.1: Functional classification of proteins identified from sea urchin CF. The

relative abundance of each category was based on either (A) the total number of proteins in

each functional category, or (B) the normalized spectral abundance factors (NSAFs)

obtained from the 3 individual sea urchins for each protein. The total number of proteins

included in this analysis is 319, as detailed in Supplementary Data 2.1.

B

NSAFs

A

Number of

Proteins

Cell structure, shape and mobility

Cell adhesion

Immune response

Lysosomes, proteases and peptidases

Intracellular transport

Exchanger and ATPases

Cell signaling

Stress response, detoxification

Energy metabolism

Nucleic acid and protein metabolism and processing

Cell proliferation, reproduction and development

Others

Unknown

79

Table 2.1: The thirty most abundant proteins identified in the coelomic fluid of 3

individual sea urchins 24 hours after injection of lipopolysaccharide. NSAFs values

correspond to the normal spectral abundance factor of the sum of rI from the three

individuals. Protein numbers correspond to those in Supplementary Data 2.2. Proteins

that had previously been identified are shown in bold. Proteins predicted by genome

annotation are shown in normal font. The function of hypothetical proteins was deduced

from Blast and conserved domains searches and is shown in brackets and underlined.

Prot Nb

Accession number Protein identification MW

(kDa) pI NSAFs

1 gi|47551037| cytoskeletal actin CyIIb . 41.8 5.3 1.6E-03

2 gi|115956638| PREDICTED: similar to cytoskeletal actin CyIIb . 41.8 5.3 1.3E-03 3 gi|47550921| actin . 41.8 5.3 1.2E-03

4 gi|115949854| PREDICTED: similar to cytoskeletal actin . 41.7 5.3 1.1E-03

5 gi|47551035| cytoskeletal actin CyIIIb . 41.8 5.2 8.5E-04

6 gi|1703135| Actin, cytoskeletal 3A (Actin, cytoskeletal IIIA). class: standard . 41.9 5.4 6.0E-04

7 gi|115943916| PREDICTED: similar to actin . 41.8 5.3 5.5E-04

85 gi|47551123| major yolk protein 153.9 7 4.1E-04

86 gi|115970375| PREDICTED: similar to melanotransferrin/EOS47 . 79.1 4.5 4.1E-04

8 gi|115971461| PREDICTED: similar to actin . 42 5.6 3.1E-04 114 gi|47551235| amassin . 56.4 4.7 3.0E-04

53 gi|115970610| PREDICTED: hypothetical protein, partial (complement control protein) . 33.4 4.7 2.8E-04

192 gi|47551061| H4 histone protein . 11.4 11.2 2.7E-04 304 gi|115971048| PREDICTED: hypothetical protein . 35.3 4.7 2.2E-04

9 gi|115961140| PREDICTED: similar to gelsolin . 41.2 5.1 2.2E-04

54 gi|115618101| PREDICTED: similar to complement component C3, partial . 24.2 4.9 2.1E-04

10 gi|47551153| profilin . 15.3 6.1 2.1E-04

55 gi|47551023| complement component C3 . 186.1 4.9 1.9E-04

144 gi|115685173| PREDICTED: similar to Low density lipoprotein receptor-related protein 6 . 35.2 7 1.9E-04

115 gi|115963381| PREDICTED: similar to amassin-2, partial . 9.8 4.4 1.8E-04

11 gi|47551049| fascin . 54.9 5.5 1.7E-04 56 gi|115950487| PREDICTED: similar to arylsulfatase isoform 2 . 63.6 5.5 1.7E-04

12 gi|115955758| PREDICTED: similar to related to cofilin . 18.9 5.2 1.7E-04

116 gi|118601062| amassin-2 . 53.9 4.4 1.4E-04

13 gi|115955651| PREDICTED: similar to Col protein . 44.3 4.8 1.3E-04

117 gi|115955254| PREDICTED: similar to alpha macroglobulin . 157.7 4.9 1.2E-04

57 gi|115974487| PREDICTED: hypothetical protein, partial (Cub and sushi multiple domains) . 93.7 4.9 1.1E-04

230 gi|115956476| PREDICTED: similar to apextrin . 49.5 4.3 9.8E-05

58 gi|115934253| PREDICTED: similar to scavenger receptor cysteine-rich protein . 62.8 6 9.5E-05

305 gi|115963910| PREDICTED: hypothetical protein, partial . 41.8 4.7 9.3E-05

80

Coelomocyte proteins that matched molecules directly involved in immune

responses represented 8.7% of the proteome (32 coelomocyte proteins). The most abundant

of these proteins were members of the complement cascade (Table 2.1). They included two

homologues of complement component C3, which comprised 2.3% of the total NSAFs

values. Complement protein Bf and a complement control protein were also identified

along with a complement-related long precursor that had similarities to factors H and I,

both of which are complement regulatory proteins [16]. Additional complement-like

proteins with CUB and SUSHI domains, which are found in a variety of complement

related proteins [17-19], were also found. The co-expression of SpBf and SpC3 supports

previous work that described a basic complement system in S. purpuratus [18-21]. The

identification of additional complement proteins, and putative complement related

proteins, suggests that the integrated complement system of coelomocytes may be more

extensive than previously thought.

Other immune response proteins that we identified included scavenger receptor

cysteine rich proteins (SRCRs). In mammals, members of the SRCR family are cell surface

receptors that play an important role in innate immunity [22]. There are an estimated 1,200

distinct SRCR-like domains encoded in the S. purpuratus genome [3-4]. A previous study

[23] has shown that coelomocytes transcribe numerous SRCR genes through the swapping

of exons that encode these domains, thus yielding different patterns of SRCR expression in

each individual sea urchin. Our study identified 16 proteins with similarities to SRCRs

(Figure 2.2). Of these, eight were similar to the previously characterized sea urchin SRCR

proteins that are similar to mammalian SRCR1, SRCR5, SRCR7 variant 2, SRCR12 or

SRCR20 [23], whilst the remaining eight SRCRs were encoded by S. purpuratus genes

with domain structures that are unique to sea urchins (Figure 2.2).

81

Stress response and detoxification proteins identified in coelomocytes included 29

distinct proteins, which accounted for 7.7 % of the NSAFS values. The most abundant of

these proteins included homologues of tranferrin, melanotransferrin, major yolk protein,

ferritin, and ceruloplasmin. Major yolk protein and melanotransferrin, were the 8th and 9th

most abundant proteins in coelomocytes respectively (Table 2.1). Transferrin, which is

involved in iron metabolism and transport [24], promotes lymphocyte proliferation in

vertebrates [25] and stimulates nitric oxide responses in macrophages [26]. Ceruloplasmin

is the major copper transporter (ferroxidase) in vertebrate blood, where it also plays a role

in iron metabolism and acts as an intercellular adhesion molecule [27-28]. In sea urchins,

major yolk protein is a transferrin-like molecule that transports iron, but may have other

activities [29-30]. Ferritin sequesters iron in the coelomic fluid of sea stars so that it is

unavailable to support bacterial proliferation, thereby acting as an acute phase protein

during immune responses [31] in a manner similar to vertebrates [32-33]. The abundance

of these putative iron-binding proteins in sea urchins suggest that they may play a critical

role in their immune systems, providing defense against infection via cell proliferation,

activating nitric oxide responses or depleting iron.

Homologues of dual oxidase 1 and dual oxidase maturation factor were also

relatively abundant in coelomocytes. Dual oxidases are involved in the production of

reactive oxygen species in organisms as diverse as yeast and mammals [34]. They help

control microbial activities in the gut of Drosophila melanogaster [35] and are involved in

the production of hydrogen peroxide in vertebrate macrophages during host defense

reactions [36]. In sea urchin CF, they might also be involved in post-phagocytic

intracellular killing mechanisms.

82

2153aa

gi 47551157

RGD

840 aa

precursor, partial: gi 115949177

gi 47550953

749 aa

partial: gi 115752650

437 aa

973 aa

gi 47550951

1036aa

gi 47551161

RGD

528aa

gi 47551167

short: gi 115977060

391 aa

SRCR similar to SRCR protein type 12 precursor, partial: gi 115973483

405aa

842aa

SRCR hyothetical protein: gi 115944063

SRCR similar to SRCR protein precursor, partial: gi 115936114

872aa

1363 aa

SRCR similar to SRCR protein type 12 precursor: gi 115968906

866 aa

SRCR similar to deleted in malignant brain tumor 1: gi 115975788

582aa

SRCR similar to SRCR rich protein: gi 115934253

RGD

2083aa


RGD

SpSRCR12

SpSRCR20

SpSRCR7.2

SpSRCR1

SpSRCR5

764 aa


Von willebrand factor repeats

Transmembrane

Short consensus repeats

SRCR repeats

Extracellular matrix like domain

CUB domain

HYR domain

Identified peptides

83

Figure 2.2: Predicted structure of the 16 scavenger receptor cysteine-rich (SRCR)

proteins identified in the coelomic fluid proteome. The predicted SRCR proteins have

between 1 and 20 SRCR domains. The non-SRCR domains shown are: Von Willebrand

factor repeats, transmembrane domains, short consensus repeats, CUB domains and HYR

domains. The position of the peptides identified by shotgun mass spectrometry within

each protein is given below each protein model. For each protein, a minimum of 2 unique

peptides were obtained. The length of the protein is given as the total number of amino

acids to the right of the sequence. Eight of these SRCRs identified had previously been

characterized and classified into five classes: SpSRCR12, SpSRCR20, SpSRCR5,

SpSRCR7.2, and SpSRCR1. For the remaining 8 SRCRs, the predicted protein description

is provided above the sequence.

84

Thirty coelomocyte proteins were putatively involved in cell adhesion and

accounted for 7.3% of the total NSAFs values. The most abundant of these were amassins

(Table 2.1), which are proteins with olfactomedin domains that are known to be involved

in cell adhesion during the clotting of CF. The expression of amassins is enhanced after

LPS injection [7, 37]. Other cell adhesion proteins identified were similar to Von

Willebrand factor (vWF), annexins, and α2-macroglobulin. These proteins may be involved

the sequestration of pathogens and potentially their opsonization.

Of the 15 proteins classified as lysosymes, proteases or peptidases, those that were

similar to serine proteases and thrombin were relatively abundant. In mammals, these

proteins are also involved in the coagulation cascade, including the serine proteases,

factors XII, XI, IX, X and VII, thrombin and plasmin [38]. Serine proteases also have a

special role linking the complement and coagulation pathways [39]. These proteins may

form a complex network of factors involved in CF clotting and coagulation. Other cell

adhesion molecules may also play a role in these reactions and stimulate phagocytosis.

This is illustrated by the presence of proteins that are similar to cadherin, selectin, talin and

a galectin-like protein. Selectins have C-type lectin domains that are activated during

inflammatory responses and mediate the calcium dependent binding of cadherins [40].

Galectins are a family of large carbohydrate-binding proteins found in a range of species

that participate in opsonization of pathogens [41-44].

Proteins putatively involved in cell proliferation, reproduction and development

accounted for 2.5% (17 of 319 proteins) of the coelomocyte proteome. Numerous variants

of echinonectin were identified in this category. Echinonectin is a highly variable protein

in sea urchin species with 162 unique protein sequences in the NCBI database for S.

purpuratus alone. Echinonectin, which has been described as an adhesion protein

expressed during embryonic development, incorporates domains with similarities to

coagulation factors V and VIII [45]. A protein similar to apextrin was also relatively

85

abundant in coelomocytes. Apextrin, which is also involved in cell adhesion, was initially

identified in the secretory vesicles of sea urchin eggs [46]. Interestingly, the expression of

apextrin is induced during acute immune responses of the protochordate, Amphioxus [47].

Forty-eight coelomocyte proteins were putatively involved in intracellular

signaling. However, most were present in low abundance and this category constituted

only 6.7% of the total NSAFs. Homologues of calcium binding proteins, such as calponin,

calcistorin and calcium binding protein p22, were the most commonly identified

intracellular signaling proteins suggesting that calcium is an important intermediate

messenger in coelomocytes [48-50]. Proteins putatively involved in GTP-based signaling

systems were also relatively abundant in S. purpuratus coelomocytes. Overall, the

diversity of proteins falling within this functional category suggested that coelomocytes

have an extensive capacity to modulate intracellular activity. In other species, homologues

of these proteins have roles in regulating cellular immune responses, including oxidative

killing mechanisms and the induction of antimicrobial activity.

We also identified proteins with similarities to apolipoprotein B (ApoB), an

apolipophorin (ApoLp) precursor of the large lipid transfer protein (LLTP) superfamily

and proteins homologous to low density lipoprotein (LDL) receptor-related proteins 4 and

6. Avarre et al. [51] noted that LLTP family members containing von Willebrand-factor

type-D (vWD) domains tend to be involved in clotting and defense reactions. Insect

ApoLps in particular are involved in inactivation of LPS by formation of insoluble

aggregates and activation of immune responses [52-54]. The data suggest an important role

for lipid metabolism in coelomocytes that might be involved in the induction of

immunological responses [55-56].

Overall, our shotgun proteomic analysis revealed a complex network of proteins in

S. purpuratus coelomocytes that is focused on cellular plasticity and host defense. This

agrees with previous transcriptome analysis of LPS-challenged sea urchins by Nair et al.

86

[7]. Transcripts for the majority of the proteins identified in the current study were also

identified by subtractive cDNA probing of an arrayed coelomocyte cDNA library [7]. The

advantage of our shotgun proteomic analysis is that it is quantitative. It allowed us to build

a model of cellular activities at the protein level, based on the relative abundance of the

proteins in sea urchin CF. So, our proteomic data provides a framework for the

development of realistic models of cellular activity.

From this quantitative analysis, it is clear that proteins involved in a dynamic actin-

based cytoskeleton are the most abundant within coelomocytes. The high capacity of

coelomocytes for amoeboid movement and pseudopodia formation [57-58] can be

explained by the fact that sea urchins have a low pressure, open circulatory system in

which cells have to rely on amoeboid movement to exert their key functions. In this

context, it has been shown that coelomocytes can actively migrate from the coelomic

cavity to the peristomial connective tissue at sites of injury [57]. Our data also support the

role of coelomocytes in important host defense mechanisms including clotting,

phagocytosis, coagulation and iron sequestration. Phagocytic coelomocytes, which are the

most abundant cells in echinoderm CF, have a high capacity for pseudopodium formation

and phagocytosis [56-57]. Pathogen recognition receptors (eg. SRCRs and C3) that

facilitate phagocytosis were abundant in coelomocytes as were proteins associated with

phagocytosis or post-phagocytic intracellular killing mechanisms.

The commonality in our data is that the function of most proteins in coelomocytes

could ultimately be related to immune defense mechanisms. This provides a framework for

future investigations that will provide a broader picture of the coordinated functions of

these proteins in the immunological defense of sea urchins.

87

2.4. Acknowledgments

This study was funded in part by an Australian Research Council Discovery grant to

DA Raftos and LC Smith (DP0880316). NM Dheilly is the recipient of an iMURS

postgraduate scholarship from Macquarie University. The research has been facilitated by

access to the Australian Proteome Analysis Facility established under the Australian

Government’s Major National Research Facilities Program. PA Haynes acknowledges

financial support from the NSW Government Office of Science and Medical Research in

the form of a Biofirst Fellowship and would like to thank Paul Mas for continued support

and encouragement.

88

2.5. References

[1] Peterson, K., J., Lyons, J., B., Nowak, K., S., Takacs, C., M. et al., Estimating

metazoan divergence times with a molecular clock. Proc. Natl. Acad. Sci. USA

2004, 101, 6536-6541.

[2] Littlewood, D., T., J., Smith, A., B., A., Combined morphological and molecular

phylogeny for sea urchins (Echnoidea: Echinodermata). Philos. Trans. R. Soc.

Lond. B. Biol. Sci. 1995, 347, 213-234.

[3] Sodergren, E., Weinstock, G., M., Davidson, E., H., Cameron, R., A. et al., The

genome of the sea urchin Strongylocentrotus purpuratus. Science 2006, 314, 941-

952.

[4] Hibino, T., Loza-Coll, M., Messier, C., Majeske, A., J. et al., The immune gene

repertoire encoded in the purple sea urchin genome. Dev. Biol. 2006, 300, 349–365.

[5] Rast, J., P., Smith, L., C., Loza-Coll, M., Hibino, T., Litman, G., W., Genomic insights


[6] Gross, P., S., Al-Sharif, W., Z., Clow, L., A., Smith, L., C., Echinoderm immunity and

the evolution of the complement system. Dev. Comp. Immunol. 1999, 23, 429-442.

[7] Nair, S., V., Del Valle, H., Gross, P., S., Terwilliger, D., P., Smith, L., C., Macroarray


Identification of unexpected immune diversity in an invertebrate. Physiol.

Genomics 2005, 22, 33-47.

[8] Gross, P., S., Clow, L., A., Smith, L., C., SpC3, the complement homologue from the

purple sea urchin, Strongylocentrotus purpuratus, is expressed in two

subpopulations of the phagocytic coelomocytes. Immunogenet. 2000, 51, 1034-

1044.

89

[9] Clow, L., A., Gross, P., S., Shih, C., S., Smith, L., C., Expression of SpC3, the sea

urchin complement component, in response to lipopolysaccharide. Immunogenet.

2000, 51, 1021-1033.

[10] Smith, L., C., Chang, L., Britten, R., J., Davidson, E., H., Sea urchin genes expressed

in activated coelomocytes are identified by expressed sequence tags. Complement


homology within the deuterostomes. J. Immunol. 1996, 156, 593-602.

[11] Smith, L., C., Britten, R., J., Davidson, E., H., Lipopolysaccharide activates the sea

urchin immune system. Dev. Comp. Immunol. 1995, 19, 217-224.

[12] Zybailov, B., L., Florens, L., Washburn, M., P., Quantitative shotgun proteomics

using a protease with broad specificity and normal spectral abundance factors. Mol.

Biosyst. 2007, 3, 354-360.

[13] Smith, L., C., Rast, J., P., Brockton, V., Terwilliger, D., P. et al., The sea urchin

immune system. Invertebr. Survival J. 2006, 3, 25-39.

[14] Edds, K., T., Chambers, C., Allen, R., D., Coelomocyte motility. Cell motil. cytoskel.

2005, 3, 113-121.

[15] Yui, M., A., Bayne, C., J., Echinoderm immunology: bacterial clearance by the sea

urchin Strongylocentrotus purpuratus. J. Biol. Bull. 1983, 165, 473-486.

[16] Multerer, K., A., Smith, L., C., Two cDNAs from the purple sea urchin,

Strongylocentrotus purpuratus, encoding mosaic proteins with domains found in

factor H, factor I and complement components C6 and C7. Immunogenet. 2004, 56,

89-106.

[17] Kirkitadze, M., D., Barlow, P., N., Structure and flexibility of the multiple domain

proteins that regulate complement activation - immunobiology of complement.

Immunol. Rev. 2001, 180, 146-161.

90

[18] Bork, P., Beckmann, G., The CUB domain: a widespread module in developmentally

regulated proteins. J. Mol. Biol. 1993, 231, 539-545.

[19] Gaboriaud, C., Rossi, V., Bally, I., Arlaud, G., J., Fontecilla-Camps, J., C., Crystal

structure of the catalytic domain of human complement C1s: a serine protease with

a handle. EMBO J. 2000, 19, 1755-1765.

[20] Smith, L., C., Clow, L., A., Terwilliger, D., P., The ancestral complement system in

sea urchins. Immunol. Rev. 2001, 180, 16-34.

[21] Smith, L., C., Shih, C.-S., Dachenhausen, S., G., Coelomocytes express SpBf, a

homologue of factor B, the second component in the sea urchin complement

system. J. Immunol. 1998, 161, 6784-6793.

[22] Yamada, Y., Doi, T., Hamakubo, T., Kodama, T., Scavenger receptor family proteins:

roles for atherosclerosis, host defence and disorders of the central nervous system.

Cell. Mol. Life Sci. 1997, 54, 628–640.

[23] Pancer, Z., Dynamic expression of multiple scavenger receptor cysteine-rich genes in

coelomocytes of the purple sea urchin. Proc. Natl. Acad. Sci. USA 2000, 97, 13156-

13161.

[24] Baker, E., N., Lindley, P., F., New perspectives on the structure and function of

transferrins. J. Inorg. Biochem. 1992, 47, 147-160.

[25] Brock, J., H., de Sousa, M., Immunoregulation by iron-binding proteins. Immunol.

today 1986, 7, 30-31.

[26] Stafford, J., L., Belosevic, M., Transferrin and the innate immune response of fish:

identification of a novel mechanism of macrophage activation. Dev. Comp.

Immunol. 2003, 27, 539-554.

[27] Sekyere, E., Richardson, D., R., The membrane-bound transferrin homologue

melanotransferrin: roles other than iron transport? FEBS letters 2000, 48, 11-16.

91

[28] Fox, P., L., The copper-iron chronicles: the story of an intimate relationship.

Biometals 2003, 16, 9-40.

[29] Yokota, Y., Unuma, T., Moriyama, A., Yamano, K., Cleavage site of a major yolk

protein (MYP) determined by cDNA isolation and amino acid sequencing in sea

urchin, Hemicentrotus pulcherrimus. Comp. Biochem and Physiol. Part B:

Biochem. And Mol. Biol. 2003, 135, 71-81.

[30] Brooks, J., M., Wessel, G., M., The major yolk protein in sea urchins is a transferrin-

like, iron binding protein. Dev. Biol. 2002, 245, 1-12.

[31] Beck, G., Ellis, T., W., Habicht, G., S., Schluter, S., F., Marchalonis, J., J., Evolution

of the acute phase response: iron release by echinoderms (Asterias forbesi)

coelomocytes, and cloning of an echinoderm ferritin molecule. Dev. Comp.

Immunol. 2002, 26, 11-26.

[32] Brock, J., H., Mulero, V., Cellular and molecular aspects of iron and immune

function. Proc. Natl. Acad. Sci. 2000, 59, 537-540.

[33] Ganz, T., Iron in innate immunity: starve the invaders. Curr. Opin. Immunol. 2009,

21, 63-67.

[34] Donkó, A., Péterfi, Z., Sum, A., Leto, T., Geiszt1, M., Dual oxidases. Philos. Trans.

R. Soc. Lond. B. Biol. Sci. 2005, 360, 2301–2308.

[35] Ha, E., M., Oh, C.-T., Bae, Y., S., Lee1, W., J., A direct role for dual oxidase in

Drosophila gut immunity. Science 2005, 310, 847-850.

[36] Forman, H., J., Torres, M., Reactive oxygen species and cell signaling – respiratory

burst in macrophage signaling. Am. J. Respir. Crit. Care. Med. 2002, 166, 54-58.

[37] Hillier, B., J., Vacquier, V., D., Amassin, an olfactomedin protein, mediates the

massive intercellular adhesion of sea urchin coelomocytes. J. Cell Biol. 2003, 160,

597-604.

92

[38] Davie, E., W., Fujikawa, K., Kiesel, W., The coagulation cascade: initiation,

maintenance, and regulation. Biochem. 1991, 30, 10363-10370.

[39] Amara, U., Albers, S., Gebhard, F., Bruckner, U., B., Huber-Lang, M., Crosstalk of

the complement and coagulation system. Fed. Am. Soc. Exp. Biol. J. 2008, 22,

673.4

[40] Aplin, A., E., Howe, A., Alahari, S., K., Juliano, R., L., Signal transduction and signal

modulation by cell adhesion receptors: the role of integrins, cadherins,

immunoglobulin-cell adhesion molecules, and selectins. Pharmacol. Rev. 1998, 50,

197-263.

[41] Barondes, S., H., Cooper, D., N., W., Gitt, M., A., Leffler, H., Galectins. Structure

and function of a large family of animal lectins. J. Biol. Chem. 1994, 269, 20807-

20810.

[42] Kima, J., Y., Kima, Y., M., Chob, S., K., Choic, K., S., Choa, M., Noble tandem-

repeat galectin of Manila clam Ruditapes philippinarum is induced upon infection

with the protozoan parasite Perkinsus olseni. Dev. Comp. Immunol. 2008, 32, 1131-

1141.

[43] Tasum, S., Vasta, G., R., A galectin of unique domain organization from hemocytes

of the eastern oyster (Crassostrea virginica) is a receptor for the protictan parasite

Perkinsus marinus. J. Immunol. 2007, 179, 3086-3098.

[44] Vasta, G., R., Quensenberry, M., Ahmed, H., O’Leary, N., C-type lectins and

galectins mediate innate and adaptive immune functions: their roles in the

complement activation pathway. Dev. Comp. Immunol. 1999, 23, 401-420.

[45] Alliegro, M., C., Alliegro, M., A., A., Echinonectin is a Del-1-like molecule with

regulated expression in sea urchin embryos. Gene Expr. Patterns 2007, 7, 651-656.

93

[46] Haag, E., S., Sly, B., J., Adrews, M., E., Raff, R., A., Apextrin, a novel extracellular

protein associated with larval ectoderm evolution in Heliocidaris erythrogramma.

Dev. Biol. 1999, 211, 77-87.

[47] Huang, G., Liu, H., Han, Y., Fan, L. et al., Profile of acute immune response in

Chinese amphioxus upon Staphylococcus aureus and Vibrio parahaemolyticus

infection. Dev. Comp. Immunol. 2007, 31, 1013-1023.

[48] Berridge, M., J., Inositol trisphosphate and calcium signaling. Ann. NY Acad. Sci.

2006, 766, 31-43.

[49] Lebeche, D., Kaminer, B., Characterization of a calsequestrin-like protein from sea-

urchin eggs. Biochem. 1992, 287, 741-747.

[50] Feske, S., Calcium signaling in lymphocyte activation and disease. Nat. Rev.

Immunol. 2007, 7, 690-702.

[51] Avarre, J.-C., Lubzens, E., Babin, P., J., Apolipocrustacein, formerly vitellogenin, is

the major egg yolk precursor protein in decapod crustaceans and is homologous to

insect apolipophorin II/I and vertebrates apolipoprotein B. BMC Evol. Biol. 2007,

7, 3-12.

[52] Kin, J., H., Je, H., J., Park, S., Y., Lee, I., H. et al., Immune activation of

apolipophorin-III and its distribution on hemocyte from Hyphantria cunea. Insect

Biochem. Mol. Biol. 2004, 34, 1011-1023.

[53] Cheon, H., M., Shin, S., W., Bian, G., Park, J., H., Raikhel, A., S., Regulation of lipid

metabolism genes, lipid carrier protein lipophorin, and its receptor during immune

challenge in the mosquito Aedes aegypti. J. Biol. Chem. 2006, 281, 8426-8435.

[54] Ma, G., Hay, D., Li, D., Asgari, S., Schmidt, O., Recognition and inactivation of LPS

by lipophorin particles. Dev. Comp. Immunol. 2006, 30, 619-626.

94

[55] Miller, Y., I., Chang, M.-K., Binder, C., J., Shaw, P., X., Witztum, J., L., Oxidized

low density lipoprotein and innate immune receptors. Curr. Opin. Lipido. 2003, 14,

437-445.

[56] Van den Elzen, P., Garg, S., Leon, L., Brigl, M. et al., Apolipoprotein-mediated

pathways of lipid antigen presentation. Nature 2005, 437, 906-910.

[57] Edds, K., T., The formation and elongation of filopodia during transformation of sea

urchin coelomocytes. Cell Mot. 1980, 1, 131-140.

[58] Mangiaterra, M., B., B., C., D., Silva, J., R., M., C., Induced inflammatory process in

the sea urchin Lytechinus variegates. Inv. Biol. 2001, 120, 178-184.

95

Supplementary Data 2.1: Detailed Materials and Methods

1. Sea urchins and sample collection

Three adult S. purpuratus were supplied by Marinus Scientific Inc. (Long Beach,

CA) after collection from the coast of southern California (USA). They were maintained

in the laboratory as described previously [1-3]. Purple sea urchins become

immunoquiescent after long-term housing in the laboratory (greater than eight months

without significant disturbance [4]). So, prior to coelomic fluid (CF) sample collection,

the three sea urchins were challenged by injecting 2 µg of lipopolysaccharide (LPS)

(Sigma Aldrich, St. Louis, MO) per ml of CF, as previously described [3-6]. The injection

of LPS has been shown to reverse immunoquiescence and return sea urchins to an

immunologically active state [4]. These conditions were designed to mimic previous

studies of coelomocyte structure, function and gene expression [1, 4-7]. Twenty four

hours after injection, 23-gauge needles attached to 1 ml syringes were inserted through the

peristomeum into the coelomic cavity of each sea urchin and CF was withdrawn, without

anticoagulant, from each of the three sea urchins. The CF was immediately expelled into a

1 ml tube and mixed with 100 µl of urea sample buffer (2.4 M Tris-HCl pH 6.8; 0.25%

SDS; 4 M urea; 20% glycerol) and stored at -70°C until use. Samples were lyophilized

and the extracted proteins were resuspended in sodium dodecyl sulfate (SDS) sample

buffer (0.05 M Tris HCl, 10% glycerol, 10% SDS, 1% DTT). Total protein content of

each sample was determined using Bradford reagent (Biorad, Hercules, CA).

96

2. One-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis (1DE)

Coelomic fluid proteins in SDS sample buffer (10 µg per well) were separated on

7.5% Bis-Tris polyacrylamide gels at 180 V for 1 hour [8]. After electrophoresis, proteins

were visualized using Coomassie Blue [9]. The gels were washed twice in sterile Milli-Q

water (10 minutes per wash), before individual lanes were cut into 16 slices of equal sizes

from top to bottom. Proteins in each slice were reduced, alkylated and subjected to trypsin

digestion as previously described [10].

3. Nanoflow liquid chromatography – tandem mass spectrometry

The tryptic digest extracts from 1DE gel slices were analyzed by nanoLC-MS/MS

using a LTQ-XL ion-trap mass spectrometer (Thermo, CA, USA) according to Breci et al.

[11]. Reversed phase columns were packed in-house to approximately 7 cm (100 µm i.d.)

using 100 Å, 5 mM Zorbax C18 resin (Agilent Technologies, CA, USA) in a fused silica

capillary with an integrated electrospray tip. A 1.8 kV electrospray voltage was applied

via a liquid junction up-stream of the C18 column. Samples were injected onto the C18

column using a Surveyor autosampler (Thermo, CA, USA). Each sample was loaded onto

the C18 column followed by an initial wash step with buffer A (5% (v/v) ACN, 0.1% (v/v)

formic acid) for 10 minutes at 1 µL min-1. Peptides were subsequently eluted from the

C18 column with 0%-50% Buffer B (95% (v/v) ACN, 0.1% (v/v) formic acid) over 58

minutes at 500 nL min-1, followed by 50%-95% Buffer B over 5 minutes at 500 nL min-1.

The column eluate was directed into a nanospray ionization source of the mass

spectrometer.

97

Spectra were scanned over the range 400–1500 amu. Automated peak recognition,

dynamic exclusion, and tandem MS of the top six most intense precursor ions at 35%

normalization collision energy were performed using the Xcalibur software (version 2.06)

(Thermo, CA, USA).

4. Protein identification

Raw data files were converted to mzXML format and processed through the Global

Proteome Machine (GPM) software using version 2.1.1 of the X!Tandem algorithm, freely

available from www.thegpm.org [12-13]. For each experiment, the 16 fractions were

processed sequentially with output files for each individual fraction. A merged, non-

redundant output file was then generated for protein identification with log(e) values less

than -1.

MS/MS spectra were searched against a combined Strongylocentrotus database

created with sequences downloaded from NCBI. This FASTA format database contained

44,037 protein sequences comprising all S. purpuratus predicted protein sequences and

identified proteins held by NCBI as of April 2008. The database also incorporated a list of

common human and trypsin peptide contaminants. The search was also performed against

a reversed sequence database to evaluate the false discovery rate (FDR) [14]. Search

parameters included MS and MS/MS tolerances of ± 2 Da and ± 0.2 Da, tolerance of up to

3 missed tryptic cleavages and K/R-P cleavages. Fixed modifications were set for

carbamidomethylation of cysteine and variable modifications were set for oxidation of

methionine.

98

Only proteins that were present in at least two out of the three sea urchins analyzed, that

had log(e) values < -9, and that yielded at least four spectral counts over the three samples

were retained for subsequent analysis. After this filtering, no reversed peptide sequences

were identified, at a protein level FDR of < 0.1%.

5. Quantitative proteomic analysis

Protein abundance data were calculated based on normalized spectral abundance

factor (NSAF) values as described previously [14]. For each sample, i, the number of

spectral counts (SpC) identifying a protein, k, was divided by the molecular weight of the

protein in kDa to give and (SpC/MW)k value. (SpC/MW)k values were then divided by the

sum of (SpC/MW) for all (N) proteins in the experiment to give the NSAFi values for each

protein and CF sample (eqn (1))

(1)

For each protein, k, the sum S of all spectral counts obtained from the three sea

urchins was calculated, and the corresponding NSAFS deduced. NSAFS values were used

as a measure of protein abundance. The NSAFi values for each of the individual sea

urchins were strongly correlated with these NSAFS values (R2, 0.85-0.92), suggesting that

NSAFS values were representative of protein abundance. The natural log of the NSAFs

values were plotted against the number of proteins obtained which showed a normal

distribution with a mean of -4.78 and a standard deviation of ± 0.62. For each protein, k,

the sum of the log(e) value obtained from the three sea urchins were calculated and the

corresponding Sum(log(e)) values deduced.

99

6. References for Detailed Materials and Methods

[1] Nair, S., V., Del Valle, H., Gross, P., S., Terwilliger, D., P., Smith, L., C., Macroarray


Identification of unexpected immune diversity in an invertebrate. Physiol.

Genomics 2005, 22, 33-47.

[2] Gross, P., S., Clow, L., A., Smith, L., C., SpC3, the complement homologue from the


subpopulations of the phagocytic coelomocytes. Immunogenet. 2000, 51, 1034-

1044.

[3] Clow, L., A., Gross, P., S., Shih, C., S., Smith, L., C., Expression of SpC3, the sea

urchin complement component, in response to lipopolysaccharide. Immunogenet.

2000, 51, 1021-1033.

[4] Gross, P., S., Al-Sharif, W., Z., Clow, L., A., Smith, L., C., Echinoderm immunity and

the evolution of the complement system. Dev. Comp. Immunol. 1999, 23, 429-442.

[5] Smith, L., C., Chang, L., Britten, R., J., Davidson, E., H., Sea urchin genes expressed in



homology within the deuterostomes. J. Immunol. 1996, 156, 593-602.

[6] Smith, L., C., Britten, R., J., Davidson, E., H., Lipopolysaccharide activates the sea

urchin immune system. Dev. Comp. Immunol. 1995, 19, 217-224.

[7] Smith, L., C., Rast, J., P., Brockton, V., Terwilliger, D., P. et al., The sea urchin

immune system. Invertebr. Survival J. 2006, 3, 25-39.

[8] Laemmli, U., K., Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 1970, 227, 680-685.

100

[9] Candiano, G., Bruschi, M., Musante, L., Santucci, L. et al., Blue silver: a very sensitive

colloidal coomassie G-250 staining for proteome analysis. Electrophoresis 2004,

25, 1327-1333.

[10] Dheilly, N., M., Nair, S., V., Smith, L., C., Raftos, D., A., Highly variable immune-

response proteins (185/333) from the sea urchin, Strongylocentrotus purpuratus:

proteomic analysis identifies diversity within and between individuals. J. Immunol.

2009, 182, 2203-2212.

[11] Breci, L., Hattrup, E., Keeler, M., Letarte, J. et al., Comprehensive proteomics in

yeast using chromatographic fractionation, gas phase fractionation, protein gel

electrophoresis and isoelectric focusing. Proteomics 2005, 5, 2018-2028.

[12] Craig, R., Beavis, R., C., A., method for reducing the time required to match protein

sequences with tandem mass spectra. Rapid Commun. Mass Spectrom. 2003, 17,

2310-2316.

[13] Craig, R., Beavis, R., C., TANDEM: matching proteins with tandem mass spectra.

Bioinformatics 2004, 20, 1466-1467.

[14] Zybailov, B., L., Florens, L., Washburn, M., P., Quantitative shotgun proteomics

using a protease with broad specificity and normal spectral abundance factors. Mol.

Biosyst. 2007, 3, 354-360.

101

Supplementary Data 2.2: Proteins identified in the coelomic fluid of 3 individual sea

urchins, conducted 24 hours after injection of lipopolysaccharide. The database search

was conducted using MudPIT merged results. rI corresponds to the total number of

spectral counts. The log(e) value indicates the probability that a putative peptide sequence

corresponding to a mass spectrum arose stochastically. The lower the log(e) value, the

more significant the assignment of the peptide sequence to the mass spectrum. # indicates

the number of unique peptides identified. NSAFs values correspond to the normal spectral

abundance factor of the sum of rI from the three individuals. Proteins that had previously

been identified are shown in bold. Proteins predicted by genome annotation are shown in

normal font. The function of hypothetical proteins was deduced from Blast and conserved

domain searches, and are shown in brackets and underlined.

102

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tein

.42

.35

28-5

1.4

340

-52

215

-39.

32

6.6E

-05

18

gi|115951109|

PR

ED

ICT

ED

: si

mil

ar t

o t

ensi

lin .

32.7

631

-110

1016

-76.

87

11-4

1.8

56.

0E-0

5

19

gi|115977085|

PR

ED

ICT

ED

: si

mil

ar t

o a

lpha-

1 t

ubuli

n .

50.2

58

-26.

53

56-1

434

22-6

3.2

75.

8E-0

5

20

gi|115960585|

PR

ED

ICT

ED

: si

mil

ar t

o t

ubuli

n, al

pha

2 i

sofo

rm 1

.46

.45

8-2

1.9

150

-134

620

-47.

72

5.7E

-05

21

gi|115976308|

PR

ED

ICT

ED

: si

mil

ar t

o L

ym

phocy

te c

yto

soli

c pro

tein

1, par

tial

.9.

55

3-1

3.8

23

-14.

32

8-1

5.9

25.

0E-0

5

22

gi|115929203|

PR

ED

ICT

ED

: si

mil

ar t

o E

NS

AN

GP

00000011796 (

alpha

acti

nin

)10

45

32-1

3414

80-3

0528

25-9

0.4

104.

5E-0

5

23

gi|115945717|

PR

ED

ICT

ED

: si

mil

ar t

o E

nab

led h

om

olo

g (

Dro

sophil

a) .

44.9

1023

-108

1020

-107

93.

3E-0

5

24

gi|115751610|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

acti

n b

indin

g p

rote

in)

26.4

65

-36

219

-75.

32

3.1E

-05

25

gi|115939656|

PR

ED

ICT

ED

: si

mil

ar t

o A

DP

-rib

osy

lati

on f

acto

r 1 .

20.6

65

-13.

81

7-4

0.3

55

-13.

42

2.8E

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26

gi|47550983|

nu

clea

r in

term

edia

te f

ilam

ent

pro

tein

.64

.66

4-2

1.9

334

-122

1315

-77.

39

2.8E

-05

27

gi|115936071|

PR

ED

ICT

ED

: si

mil

ar t

o A

rp2/3

com

ple

x s

ubunit

.20

.39

6-2

13

10-5

1.6

52.

7E-0

5

28

gi|115960968|

PR

ED

ICT

ED

: si

mil

ar t

o s

ea u

rchin

Arp

3 (

SU

Arp

3)

.47

.16

4-2

73

19-9

2.2

97

-39

52.

2E-0

5

29

gi|115963096|

PR

ED

ICT

ED

: si

mil

ar t

o C

G15792-P

D, par

tial

(m

yosi

n)

133

61

-81

58-2

1718

13-9

7.1

91.

8E-0

5

30

gi|115958283|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

69420 p

rote

in (

acti

n r

elat

ed p

rote

in)

19.6

95

-19.

33

3-1

1.2

21

-4.3

11.

6E-0

5

31

gi|115974054|

PR

ED

ICT

ED

: si

mil

ar t

o f

ibro

pel

lin I

a .

24.7

65

-19.

92

4-1

7.7

32

-21.

12

1.5E

-05

32

gi|115630732|

PR

ED

ICT

ED

: si

mil

ar t

o m

yosi

n, hea

vy p

oly

pep

tide

10, non-m

usc

le .

915

1-1

.61

32-1

4113

4-1

2.7

21.

4E-0

5

33

gi|115944135|

PR

ED

ICT

ED

: si

mil

ar t

o s

ea u

rchin

Arp

2 (

SU

Arp

2)

.44

.47

6-3

6.2

48

-48.

95

4-2

4.2

31.

4E-0

5

34

gi|115931813|

PR

ED

ICT

ED

: si

mil

ar t

o L

IM a

nd S

H3 p

rote

in .

27.2

85

-18.

63

3-1

2.1

23

-11.

52

1.4E

-05

se

a u

rch

in 1

se

a u

rch

in 2

se

a u

rch

in 3

Cel

l st

ruct

ure

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ap

e an

d m

ob

ilit

y*

103

Pro

t

Nb

Acc

essi

on

nu

mb

erP

rote

in i

den

tifi

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on

MW

(kD

a)pI

rIlo

g(e)

#rI

log(

e)#

rIlo

g(e)

#N

SA

Fs

35

gi|115961398|

PR

ED

ICT

ED

: si

mil

ar t

o C

apzb

-pro

v p

rote

in (

F a

ctin

cap

pin

g p

rote

in b

eta)

27.4

53

-5.8

17

-48

51

-2.3

11.

4E-0

5

36

gi|115948375|

PR

ED

ICT

ED

: si

mil

ar t

o C

G10540-P

A i

sofo

rm 1

(F

act

in C

appin

g p

rote

in a

lpha)

30.6

89

-41.

44

3-1

0.8

21.

3E-0

5

37

gi|115939472|

PR

ED

ICT

ED

: si

mil

ar t

o E

NS

AN

GP

00000023777 (

CD

C42, ce

ll d

ivis

ion c

ycl

e 42

)21

.37

2-9

.12

6-2

9.9

41.

3E-0

5

38

gi|115971193|

PR

ED

ICT

ED

: si

mil

ar t

o R

ac1 p

rote

in .

21.6

95

-17.

32

3-1

1.8

21.

3E-0

5

39

gi|115930179|

PR

ED

ICT

ED

: si

mil

ar t

o g

elso

lin .

40.3

64

-27.

33

6-3

7.5

54

-29

31.

2E-0

5

40

gi|115974358|

PR

ED

ICT

ED

: si

mil

ar t

o L

OC

398551 p

rote

in i

sofo

rm 1

(A

DP

rib

osy

lati

on f

acto

r)17

.85

1-2

14

-21.

91

9.9E

-06

41

gi|115964477|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

acti

n r

elat

ed p

rote

in)

40.8

95

-33

44

-21

32

-11.

82

9.1E

-06

42

gi|115933962|

PR

ED

ICT

ED

: si

mil

ar t

o g

elso

lin .

40.8

52

-15.

62

3-1

4.5

24

-19.

42

7.5E

-06

43

gi|115975440|

PR

ED

ICT

ED

: si

mil

ar t

o m

oes

in .

67.4

51

-4.7

15

-18.

33

3-9

.62

4.5E

-06

44

gi|166795321|

ad

vil

lin

.93

.18

3-1

2.8

26

-12.

12

3-1

3.7

24.

4E-0

6

45

gi|115925936|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

84047 p

rote

in (

dre

bri

n l

ike

isofo

rm)

345

1-1

.91

2-1

3.2

21

-1.4

14.

0E-0

6

46

gi|115950170|

PR

ED

ICT

ED

: si

mil

ar t

o l

ong m

icro

tubule

-ass

oci

ated

pro

tein

1A

; lo

ng M

AP

1A

.22

14

3-1

8.7

314

-34.

54

7-3

4.9

43.

7E-0

6

47

gi|115974031|

PR

ED

ICT

ED

: si

mil

ar t

o r

etic

ulo

n 4

iso

form

B2 .

105

91

-2.4

12

-14.

82

3-1

4.8

21.

9E-0

6

48

gi|115960644|

PR

ED

ICT

ED

: si

mil

ar t

o m

yosi

n h

eavy c

hai

n .

224

65

-38.

15

5-5

0.6

51.

5E-0

6

49

gi|115939091|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

83833 p

rote

in (

leth

al g

iant

larv

ae h

om

olo

gue)

98.5

71

-1.6

13

-19.

43

1.4E

-06

50

gi|115945231|

PR

ED

ICT

ED

: si

mil

ar t

o K

IAA

0587 p

rote

in (

NC

R a

ssoci

ate

pro

tein

)12

86

2-9

21

-6.7

12

-14.

22

1.3E

-06

51

gi|115945063|

PR

ED

ICT

ED

: si

mil

ar t

o f

ibro

surf

in, par

tial

.20

04

1-6

.11

6-3

0.9

31.

2E-0

6

52

gi|115925414|

PR

ED

ICT

ED

: si

mil

ar t

o K

IAA

0727 p

rote

in (

myosi

n)

118

92

-9.9

22

-6.5

11.

2E-0

6

1.5

E-0

3

53

gi|115970610|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(co

mple

men

t co

ntr

ol

pro

tein

)33

.45

54-1

181

179

-167

340

-90.

52

2.8E

-04

54

gi|115618101|

PR

ED

ICT

ED

: si

mil

ar t

o c

om

ple

men

t co

mponen

t C

3, par

tial

.24

.25

55-6

5.7

554

-60

443

-57.

44

2.1E

-04

55

gi|47551023|

com

ple

men

t co

mp

on

ent

C3 .

186

534

3-5

3247

456

-598

5025

1-4

1236

1.9E

-04

56

gi|115950487|

PR

ED

ICT

ED

: si

mil

ar t

o a

ryls

ulf

atas

e is

ofo

rm 2

.63

.66

127

-170

1613

9-1

4712

56-1

1411

1.7E

-04

57

gi|115974487|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(C

ub a

nd s

ush

i m

ult

iple

dom

ains)

93.7

560

-145

1319

5-1

8116

49-1

039

1.1E

-04

58

gi|115934253|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

.62

.86

14-7

7.2

814

9-1

4812

13-3

8.7

49.

5E-0

5

59

gi|115940254|

PR

ED

ICT

ED

: si

mil

ar t

o c

ycl

ophil

in .

17.6

99

-31.

23

17-6

5.3

714

-43.

35

7.7E

-05

60

gi|115619038|

PR

ED

ICT

ED

: si

mil

ar t

o a

ryls

ulf

atas

e .

60.8

578

-206

1820

-91.

89

34-8

0.6

77.

3E-0

5

61

gi|47550953|

scaven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

.77

.14

40-5

3.8

632

-62.

57

9-4

1.8

53.

5E-0

5

62

gi|118421783|

Sp

185/3

33 .

23.8

111

-1.3

121

-60.

21

3.2E

-05

63

gi|47550955|

DD

104 p

rote

in, u

pre

gu

late

d u

pon

bact

eria

l ch

all

enge

an

d t

rau

ma .

31.3

516

-21

39

-13.

12

3-1

2.2

23.

0E-0

5

64

gi|47551161|

scaven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

.11

04

21-4

1.9

558

-40.

25

17-1

9.9

33.

0E-0

5

65

gi|115752650|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

, par

tial

.44

.84

15-2

1.2

119

-24.

21

2.6E

-05

66

gi|47551167|

scaven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

typ

e 5 .

58.2

417

-48.

75

10-5

35

12-2

8.6

42.

3E-0

5

67

gi|115944063|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

SR

CR

)94

.66

7-2

0.2

344

-68.

27

1.8E

-05

68

gi|115977060|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

type

5 p

recu

rsor

.42

.95

13-2

6.6

110

-43.

71

1.8E

-05

Imm

un

e re

spon

se*

se

a u

rch

in 1

se

a u

rch

in 2

se

a u

rch

in 3

104

Pro

t

Nb

Acc

essi

on

nu

mb

erP

rote

in i

den

tifi

cati

on

MW

(kD

a)pI

rIlo

g(e)

#rI

log(

e)#

rIlo

g(e)

#N

SA

Fs

69

gi|115968904|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

type

12 p

recu

rsor

.82

.84

6-1

8.7

334

-38.

94

3-1

2.1

21.

8E-0

5

70

gi|47825406|

com

ple

men

t re

late

d-l

on

g p

recu

rsor

. 20

45

23-7

1.4

744

-116

1220

-93.

19

1.4E

-05

71

gi|115968659|

PR

ED

ICT

ED

: si

mil

ar t

o r

ham

nose

-bin

din

g l

ecti

n (

SA

L),

par

tial

.32

.48

8-3

1.9

44

-15.

52

1.3E

-05

72

gi|47551047|

com

ple

men

t fa

ctor

B .

91.2

66

-20.

23

13-5

6.7

710

-38.

35

1.1E

-05

73

gi|115940188|

PR

ED

ICT

ED

: si

mil

ar t

o p

epti

dylp

roly

l is

om

eras

e B

iso

form

2 (

cycl

ophil

in)

.23

.49

4-6

.91

3-1

1.3

21.

0E-0

5

74

gi|115936114|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

pre

curs

or,

par

tial

.94

415

-27.

33

9-3

1.4

44

-3.6

11.

0E-0

5

75

gi|115975788|

PR

ED

ICT

ED

: si

mil

ar t

o d

elet

ed i

n m

alig

nan

t bra

in t

um

ors

1 (

SR

CR

)89

.44

6-1

0.6

213

-22.

93

7.3E

-06

76

gi|47550951|

scaven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

vari

an

t 2

105

517

-54.

56

5-2

8.1

47.

1E-0

6

77

gi|115973483|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

type

12 p

recu

rsor,

par

tial

.45

.35

5-2

8.6

14

-12.

42

6.9E

-06

78

gi|115949177|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

type

12 p

recu

rsor,

par

tial

.90

.95

8-3

3.6

410

-77.

48

6.8E

-06

79

gi|115950182|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

type

12 p

recu

rsor

.22

25

8-3

6.3

411

-44

66

-26.

84

3.8E

-06

80

gi|115968906|

PR

ED

ICT

ED

: si

mil

ar t

o s

caven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

type

12 p

recu

rsor

.14

75

3-5

.41

7-1

6.6

21

-3.6

12.

5E-0

6

81

gi|115960292|

PR

ED

ICT

ED

: si

mil

ar t

o c

om

ple

men

t re

late

d-l

ong p

recu

rsor,

par

tial

.70

.16

1-1

.81

4-1

1.1

22.

5E-0

6

82

gi|115966254|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

SA

M d

om

ain a

nd H

D d

om

ain c

onta

inin

g)

61.2

91

-1.9

13

-13.

22

2.3E

-06

83

gi|115970612|

PR

ED

ICT

ED

: si

mil

ar t

o C

UB

and S

ush

i m

ult

iple

dom

ains

1 v

aria

nt,

par

tial

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.65

2-1

02

3-2

.61

2.0E

-06

84

gi|47551157|

scaven

ger

rec

epto

r cy

stei

ne-

rich

pro

tein

typ

e 12

226

54

-28.

34

4-3

1.1

42

-10.

12

1.5E

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1.3

E-0

3

85

gi|47551123|

majo

r yolk

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154

776

5-1

065

9276

3-9

5668

335

-539

494.

1E-0

4

86

gi|115970375|

PR

ED

ICT

ED

: si

mil

ar t

o m

elan

otr

ansf

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n/E

OS

47 .

79.1

523

8-4

8936

511

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3820

1-3

4526

4.1E

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87

gi|115685450|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

68486 p

rote

in, par

tial

(phosp

hoglu

conat

e deh

ydro

gen

ase

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95

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4.6

38

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22

7-2

4.8

29.

1E-0

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88

gi|115944169|

PR

ED

ICT

ED

: si

mil

ar t

o d

ual

oxid

ase

1 i

sofo

rm 1

.18

66

32-1

4916

189

-441

4341

-172

194.

8E-0

5

89

gi|115972586|

PR

ED

ICT

ED

: si

mil

ar t

o 7

1 K

d h

eat

shock

cognat

e pro

tein

.71

.75

16-1

059

46-1

8816

31-1

4413

4.4E

-05

90

gi|115968538|

PR

ED

ICT

ED

: si

mil

ar t

o f

erri

tin .

19.6

83

-9.3

28

-24.

93

13-3

24

4.1E

-05

91

gi|115944173|

PR

ED

ICT

ED

: si

mil

ar t

o E

NS

AN

GP

00000006016 i

sofo

rm 1

(dual

oxid

ase

mat

ura

tion f

acto

r)48

.75

6-3

7.8

443

-82.

67

6-1

3.8

23.

8E-0

5

92

gi|115926107|

PR

ED

ICT

ED

: si

mil

ar t

o f

erro

xid

ase

(EC

1.1

6.3

.1)

pre

curs

or

- ra

t .

115

525

-122

1234

-150

1435

-125

112.

8E-0

5

93

gi|115963949|

PR

ED

ICT

ED

: si

mil

ar t

o f

erro

xid

ase

(EC

1.1

6.3

.1)

pre

curs

or

- ra

t .

39.2

54

-6.2

126

-67.

66

1-2

.21

2.7E

-05

94

gi|115926010|

PR

ED

ICT

ED

: si

mil

ar t

o g

luta

thio

ne

per

oxid

ase,

par

tial

.21

73

-14

211

-43.

85

1-6

.51

2.4E

-05

95

gi|115926884|

PR

ED

ICT

ED

: si

mil

ar t

o C

G7820-P

A (

carb

onic

anhydra

se a

lpha

ver

tebra

te l

ike)

29.7

67

-29

49

-43.

25

5-3

3.6

52.

4E-0

5

96

gi|115963947|

PR

ED

ICT

ED

: si

mil

ar t

o c

erulo

pla

smin

.52

.84

35-6

9.9

61

-7.6

12.

3E-0

5

97

gi|115972590|

PR

ED

ICT

ED

: si

mil

ar t

o h

eat

shock

pro

tein

70 i

sofo

rm 1

.53

.96

20-6

81

16-5

6.5

12.

3E-0

5

98

gi|115893581|

PR

ED

ICT

ED

: si

mil

ar t

o h

eat

shock

pro

tein

pro

tein

.60

621

-84

416

-59.

21

2.1E

-05

99

gi|115954976|

PR

ED

ICT

ED

: si

mil

ar t

o h

eat

shock

pro

tein

pro

tein

.68

.66

17-6

3.2

116

-62.

71

1.6E

-05

100

gi|115937420|

PR

ED

ICT

ED

: ca

tala

se .

58.2

81

-4.9

118

-78.

58

5-3

6.9

41.

4E-0

5

101

gi|115956665|

PR

ED

ICT

ED

: si

mil

ar t

o h

eat

shock

pro

tein

pro

tein

.68

.36

6-4

0.7

117

-63.

41

1.1E

-05

Str

ess

resp

on

se, d

etoxif

icati

on

*

se

a u

rch

in 1

se

a u

rch

in 2

se

a u

rch

in 3

105

Prot

Nb

Accessio

n

nu

mb

er

Prote

in i

den

tifi

cati

on

MW

(kD

a)pI

rIlo

g(e)

#rI

log(

e)#

rIlo

g(e)

#N

SA

Fs

102

gi|115815329|

PR

ED

ICT

ED

: si

mil

ar t

o C

ct6a

pro

tein

, par

tial

(ch

aper

onin

conta

inin

g T

CP

1)

276

5-2

4.8

12

-11.

31

9.0E

-06

103

gi|115891388|

PR

ED

ICT

ED

: si

mil

ar t

o h

eat

shock

90 k

Da

pro

tein

, par

tial

.57

.65

2-1

1.7

212

-39.

25

8.3E

-06

104

gi|115954867|

PR

ED

ICT

ED

: si

mil

ar t

o C

hap

eronin

conta

inin

g T

CP

1, su

bunit

4 (

del

ta)

.57

.16

1-1

.71

8-6

0.9

73

-12.

82

7.1E

-06

105

gi|115944450|

PR

ED

ICT

ED

: si

mil

ar t

o c

hap

eronin

subunit

8 t

het

a57

.45

7-4

5.2

54

-19.

93

6.6E

-06

106

gi|115661720|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

suci

nat

e se

mia

ldeh

yde

deh

ydro

gen

ase)

52.3

61

-5.3

17

-45.

15

1-4

.21

5.8E

-06

107

gi|115945316|

PR

ED

ICT

ED

: si

mil

ar t

o C

holi

ne

deh

ydro

gen

ase

.66

.29

8-4

6.6

61

-1.4

14.

7E-0

6

108

gi|115964822|

PR

ED

ICT

ED

: si

mil

ar t

o m

itoch

ondri

al c

hap

eronin

Hsp

56 .

62.2

54

-33.

84

2-9

.42

3.4E

-06

109

gi|115924889|

PR

ED

ICT

ED

: si

mil

ar t

o C

hap

eronin

conta

inin

g T

CP

1, su

bunit

5 (

epsi

lon)

.59

.86

3-1

8.4

32

-2.5

12.

9E-0

6

110

gi|115930256|

PR

ED

ICT

ED

: si

mil

ar t

o C

hap

eronin

conta

inin

g T

CP

1, su

bunit

3 (

gam

ma)

.60

.28

3-1

0.9

22

-8.7

22.

9E-0

6

111

gi|115956641|

PR

ED

ICT

ED

: si

mil

ar t

o L

OC

495278 p

rote

in (

chap

eronin

conta

inin

g T

CP

1)

627

3-1

6.8

21

-1.4

12.

3E-0

6

112

gi|115958481|

PR

ED

ICT

ED

: si

mil

ar t

o 7

0 k

Da

hea

t sh

ock

pro

tein

pre

curs

or

.76

.25

1-3

.81

3-1

7.8

21.

9E-0

6

113

gi|115959568|

PR

ED

ICT

ED

: si

mil

ar t

o g

luta

thio

ne

reduct

ase

.13

36

2-1

22

3-1

1.6

21.

3E-0

6

1.3

E-0

3

114

gi|47551235|

am

assin

.56

.45

146

-183

1918

8-2

2822

171

-163

173.

0E-0

4

115

gi|115963381|

PR

ED

ICT

ED

: si

mil

ar t

o a

mas

sin-2

, par

tial

.9.

84

17-1

8.6

115

-19.

91

19-1

8.8

11.

8E-0

4

116

gi|118601062|

am

assin

-2 .

53.9

460

-142

1381

-141

1378

-110

101.

4E-0

4

117

gi|115955254|

PR

ED

ICT

ED

: si

mil

ar t

o a

lpha

mac

roglo

buli

n .

158

527

9-5

2444

220

-337

2860

-171

171.

2E-0

4

118

gi|115963192|

PR

ED

ICT

ED

: si

mil

ar t

o A

nnex

in A

4 (

Annex

in I

V)

(Lip

oco

rtin

IV

) (E

ndonex

in I

)

(Chro

mobin

din

-4)

(Pro

tein

II)

(P

32.5

) (P

lace

nta

l34

.86

22-1

0010

44-1

4713

18-7

38

8.2E

-05

119

gi|115851666|

PR

ED

ICT

ED

: si

mil

ar t

o C

G7002-P

A, par

tial

(V

on w

ille

bra

nd f

acto

r)27

.36

19-3

0.3

227

-26.

21

3-1

2.6

16.

1E-0

5

120

gi|115944296|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

Gal

ecti

n)

31.5

1013

-58.

16

36-8

4.4

82

-3.5

15.

5E-0

5

121

gi|118601058|

am

assin

4 .

52.6

422

-63.

58

35-8

8.2

828

-34.

71

5.5E

-05

122

gi|115720465|

PR

ED

ICT

ED

: si

mil

ar t

o s

elec

tin-l

ike

pro

tein

, par

tial

.35

.55

14-5

26

21-6

0.8

712

-25.

33

4.5E

-05

123

gi|115956497|

PR

ED

ICT

ED

: si

mil

ar t

o t

etra

span

in f

amil

y p

rote

in .

26.2

47

-23

38

-6.6

111

-13.

82

3.4E

-05

124

gi|118601054|

am

assin

-3 .

53.5

416

-46.

92

15-4

0.1

121

-40.

15

3.3E

-05

125

gi|115970015|

PR

ED

ICT

ED

: si

mil

ar t

o v

on W

ille

bra

nd f

acto

r .

275

956

-286

1215

9-5

8625

2.6E

-05

126

gi|115970125|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

139263 p

rote

in (

Annex

in)

61.9

811

-67.

57

16-4

4.9

512

-37.

54

2.1E

-05

127

gi|115663188|

PR

ED

ICT

ED

: si

mil

ar t

o t

etra

span

in, par

tial

.21

.54

4-6

.21

6-1

6.1

21

-2.8

11.

7E-0

5

128

gi|115786699|

PR

ED

ICT

ED

: si

mil

ar t

o a

lpha-

2-m

acro

glo

buli

n, par

tial

.40

.14

7-1

82

13-1

0.6

11.

7E-0

5

129

gi|115960842|

PR

ED

ICT

ED

: si

mil

ar t

o t

alin

.27

46

4-2

5.3

363

-291

2626

-175

171.

1E-0

5

130

gi|115963196|

PR

ED

ICT

ED

: si

mil

ar t

o A

nnex

in A

5 .

32.5

57

-41.

34

2-8

.52

2-7

.92

1.1E

-05

131

gi|115949647|

PR

ED

ICT

ED

: si

mil

ar t

o a

lpha

mac

roglo

buli

n, par

tial

.64

.36

12-2

8.4

17

-25.

11

1.0E

-05

132

gi|115950639|

PR

ED

ICT

ED

: si

mil

ar t

o v

incu

lin, par

tial

.39

.58

1-2

.71

1-2

.31

9-1

3.6

29.

4E-0

6

133

gi|115924509|

PR

ED

ICT

ED

: si

mil

ar t

o c

alpai

n B

.69

.15

5-3

8.5

412

-45.

25

8.4E

-06

134

gi|115949077|

PR

ED

ICT

ED

: si

mil

ar t

o i

nte

gri

n a

lpha

1 .

42.6

42

-9.5

27

-18.

62

1-3

.81

7.9E

-06

Cell

ad

hesio

n*

se

a u

rch

in 1

se

a u

rch

in 2

se

a u

rch

in 3

106

Pro

t

Nb

Acc

essi

on

nu

mb

erP

rote

in i

den

tifi

cati

on

MW

(kD

a)pI

rIlo

g(e)

#rI

log(

e)#

rIlo

g(e)

#N

SA

Fs

135

gi|47551105|

inte

gri

n b

eta L

su

bu

nit

.87

.95

6-3

3.3

412

-64

62

-14.

52

7.7E

-06

136

gi|115967683|

PR

ED

ICT

ED

: si

mil

ar t

o N

CA

M-1

40 (

Neu

ral

cell

adhes

ion m

ole

cule

)98

.44

10-3

9.7

47

-31.

54

4-1

1.1

27.

2E-0

6

137

gi|47551115|

inte

gri

n b

eta G

su

bu

nit

.85

.55

4-2

0.1

211

-60.

36

6.0E

-06

138

gi|115975224|

PR

ED

ICT

ED

: si

mil

ar t

o G

-cad

her

in .

302

418

-90.

79

22-9

5.5

97

-37.

54

5.3E

-06

139

gi|115945577|

PR

ED

ICT

ED

: si

mil

ar t

o p

uta

tive

cell

adhes

ion p

rote

in S

ym

32 .

54.5

62

-9.1

11

-21

4-1

9.7

34.

3E-0

6

140

gi|115906323|

PR

ED

ICT

ED

: si

mil

ar t

o v

incu

lin .

44.9

61

-1.4

11

-7.1

13

-10.

71

3.8E

-06

141

gi|115910910|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(F

ibro

nec

tin

)23

65

3-1

02

17-4

7.4

52.

9E-0

6

142

gi|47551111|

inte

gri

n b

eta-C

su

bu

nit

.89

.35

1-3

.21

3-1

1.3

21

-2.4

11.

9E-0

6

143

gi|115975625|

PR

ED

ICT

ED

: si

mil

ar t

o p

lexin

A2 .

186

54

-13.

32

3-8

.72

1.3E

-06

1.2

E-0

3

144

gi|115685173|

PR

ED

ICT

ED

: si

mil

ar t

o L

ow

den

sity

lip

opro

tein

rec

epto

r-re

late

d p

rote

in 6

.35

.27

65-1

334

68-1

303

60-1

142

1.9E

-04

145

gi|115964065|

PR

ED

ICT

ED

: si

mil

ar t

o a

den

yly

l cy

clas

e-as

soci

ated

pro

tein

.21

.15

1-2

.41

44-6

3.7

613

-31.

33

9.3E

-05

146

gi|115964067|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

cap a

den

yla

te c

ycl

ase

asso

ciat

ed p

rote

in)

298

1-7

.11

53-1

6415

15-7

7.7

98.

0E-0

5

147

gi|115973265|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

79770 p

rote

in (

rho G

DP

dis

soci

atio

n i

nhib

itor)

22.7

513

-65.

37

34-1

0610

2-1

3.8

27.

3E-0

5

148

gi|115976887|

PR

ED

ICT

ED

: si

mil

ar t

o S

TA

RT

dom

ain c

onta

inin

g p

rote

in .

27.3

59

-32.

74

33-6

4.4

714

-50.

75

6.9E

-05

149

gi|115958629|

PR

ED

ICT

ED

: si

mil

ar t

o L

ow

-den

sity

lip

opro

tein

rec

epto

r-re

late

d p

rote

in 4

pre

curs

or

(Mult

iple

epid

erm

al g

row

th f

acto

r-li

ke

dom

ains

129

658

-163

1693

-200

2089

-186

186.

3E-0

5

150

gi|115976312|

PR

ED

ICT

ED

: si

mil

ar t

o C

G8649-P

C (

calp

onin

)51

.86

14-6

3.6

746

-192

1615

-47.

85

4.9E

-05

151

gi|115940166|

PR

ED

ICT

ED

: si

mil

ar t

o a

poli

popro

tein

B .

609

822

2-9

4285

600

-178

9#

4.6E

-05

152

gi|115929815|

PR

ED

ICT

ED

: si

mil

ar t

o N

ucl

eosi

de

dip

hosp

hat

e kin

ase

fam

ily p

rote

in .

19.2

76

-30.

64

13-5

96

6-1

52

4.4E

-05

153

gi|47551041|

ER

calc

isto

rin

.54

.94

43-1

7516

7-4

9.6

53.

1E-0

5

154

gi|115968621|

PR

ED

ICT

ED

: si

mil

ar t

o c

alponin

.17

.510

3-1

1.4

25

-19.

72

8-3

6.6

53.

1E-0

5

155

gi|115970013|

PR

ED

ICT

ED

: si

mil

ar t

o a

poli

popro

tein

B .

202

640

-140

314

2-3

6815

2-9

.42

3.1E

-05

156

gi|115936803|

PR

ED

ICT

ED

: si

mil

ar t

o h

eter

otr

imer

ic g

uan

ine

nucl

eoti

de-

bin

din

g p

rote

in b

eta

subunit

isofo

rm 3

.37

.76

9-4

9.3

512

-51

69

-52.

15

2.7E

-05

157

gi|115940411|

PR

ED

ICT

ED

: si

mil

ar t

o 1

4-3

-3-l

ike

pro

tein

2 .

31.7

53

-13.

91

13-7

0.5

77

-32.

54

2.5E

-05

158

gi|115945715|

PR

ED

ICT

ED

: si

mil

ar t

o R

as-r

elat

ed p

rote

in O

RA

B-1

.20

.65

3-1

5.3

28

-43.

35

3-1

4.3

22.

3E-0

5

159

gi|160623362|

pu

tati

ve

14-3

-3 e

psi

lon

iso

form

.24

.15

3-2

6.5

39

-23

34

-20.

72

2.2E

-05

160

gi|115944489|

PR

ED

ICT

ED

: si

mil

ar t

o C

G2082-P

A (

este

rase

lip

ase)

37.6

77

-38.

64

7-3

4.2

48

-25.

53

2.0E

-05

161

gi|115940258|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

lipid

raf

t as

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ICT

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PR

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PR

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ICT

ED

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PR

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: si

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ar t

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23.1

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53

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PR

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ED

: si

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it .

40.3

53

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PR

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ICT

ED

: hypoth

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87

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169

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PR

ED

ICT

ED

: hypoth

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20.1

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170

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PR

ED

ICT

ED

: si

mil

ar t

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tein

ME

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23.5

95

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171

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PR

ED

ICT

ED

: si

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onse

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al p

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n N

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172

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PR

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ICT

ED

: si

mil

ar t

o a

poli

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n pre

curs

or

pro

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.40

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68-3

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173

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PR

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ICT

ED

: hypoth

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tial

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opto

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15.7

51

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13

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12

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174

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PR

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ICT

ED

: si

mil

ar t

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AB

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sofo

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.23

.76

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175

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PR

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ICT

ED

: si

mil

ar t

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tor

2 .

47.5

62

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176

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PR

ED

ICT

ED

: si

mil

ar t

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AC

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308

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31

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11

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177

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PR

ED

ICT

ED

: si

mil

ar t

o a

den

yla

te k

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e 2 i

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.27

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31

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6

178

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PR

ED

ICT

ED

: si

mil

ar t

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6

179

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PR

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ICT

ED

: si

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22.2

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180

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PR

ED

ICT

ED

: si

mil

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o E

NS

AN

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181

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PR

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ICT

ED

: si

mil

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ase

I .

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PR

ED

ICT

ED

: si

mil

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24.4

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184

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PR

ED

ICT

ED

: si

mil

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23

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PR

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ICT

ED

: si

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186

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187

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PR

ED

ICT

ED

: si

mil

ar t

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, par

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.57

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6

188

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189

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PR

ED

ICT

ED

: si

mil

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kin

ase

.56

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190

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PR

ED

ICT

ED

: si

mil

ar t

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PR

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ICT

ED

: si

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PR

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ED

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PR

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PR

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ED

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PR

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ICT

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PR

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ICT

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PR

ED

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ED

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ICT

ED

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206

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PR

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PR

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PR

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PR

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PR

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ICT

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PR

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PR

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ICT

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PR

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ICT

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PR

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PR

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PR

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ICT

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PR

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tin, par

tial

.49

.46

67-9

8.7

818

-50.

96

1-1

.81

5.9E

-05

234

gi|115899605|

PR

ED

ICT

ED

: si

mil

ar t

o e

chin

onec

tin, par

tial

.56

.75

18-4

6.4

234

-56.

73

3.1E

-05

235

gi|115945705|

PR

ED

ICT

ED

: si

mil

ar t

o E

H-d

om

ain-c

onta

inin

g p

rote

in 1

(T

esti

lin)

(hPA

ST

1)

.62

.27

36-1

6015

17-8

7.8

102.

9E-0

5

236

gi|115924200|

PR

ED

ICT

ED

: si

mil

ar t

o s

ecre

ted l

ecti

n h

om

olo

g;

HeE

L-1

.20

.24

4-1

7.7

15

-19.

93

1.5E

-05

237

gi|115952980|

PR

ED

ICT

ED

: si

mil

ar t

o e

chin

onec

tin, par

tial

.18

.35

3-2

.51

3-9

.62

1.1E

-05

238

gi|115945319|

PR

ED

ICT

ED

: si

mil

ar t

o p

rim

ary m

esen

chym

e sp

ecif

ic p

rote

in M

SP

130-r

elat

ed-1

.56

.85

11-4

3.2

53

-19.

93

5-1

4.7

21.

1E-0

5

239

gi|115955360|

PR

ED

ICT

ED

: si

mil

ar t

o a

pex

trin

.62

.35

2-5

.61

12-6

5.4

52

-3.1

18.

7E-0

6

240

gi|115948485|

PR

ED

ICT

ED

: si

mil

ar t

o m

oll

usk

-der

ived

gro

wth

fac

tor;

MD

GF, par

tial

.60

.17

4-1

8.7

37

-33.

44

6.3E

-06

241

gi|115964625|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

nm

rA l

ike

fam

ily d

om

ain c

onta

inin

g )

32.5

62

-5.4

13

-20.

83

5.4E

-06

242

gi|115960970|

PR

ED

ICT

ED

: si

mil

ar t

o p

roli

fera

tion-a

ssoci

ated

pro

tein

1 .

44.8

64

-38

42

-2.2

14.

7E-0

6

243

gi|115956777|

PR

ED

ICT

ED

: si

mil

ar t

o E

CM

18 .

267

417

-57.

47

17-4

6.5

44.

3E-0

6

244

gi|115968740|

PR

ED

ICT

ED

: si

mil

ar t

o S

epti

n 6

.50

.36

4-1

3.7

22

-13.

62

4.2E

-06

245

gi|115770276|

PR

ED

ICT

ED

: si

mil

ar t

o z

onad

hes

in

89.3

46

-22.

13

4-1

8.9

33.

9E-0

6

246

gi|115957151|

PR

ED

ICT

ED

: si

mil

ar t

o v

itel

logen

in .

244

64

-21.

33

4-1

9.3

23

-21.

73

1.5E

-06

2.8

E-0

4

247

gi|115935979|

PR

ED

ICT

ED

: si

mil

ar t

o v

olt

age-

dep

enden

t an

ion c

han

nel

2 .

30.4

621

-90

934

-105

1028

-86.

49

9.2E

-05

248

gi|115966189|

PR

ED

ICT

ED

: si

mil

ar t

o H

(+)-

tran

sport

ing A

TP

ase

bet

a su

bunit

.55

.95

16-1

1411

48-1

9415

15-9

8.1

104.

8E-0

5

249

gi|115941346|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(A

TP

ase

H+

tra

nsp

ort

ing

)8.

45

3-1

4.1

24

-8.5

14

-16.

72

4.4E

-05

250

gi|47551121|

mit

och

on

dri

al A

TP

syn

thase

alp

ha s

ub

un

it p

recu

rsor

.59

.68

17-7

6.7

832

-145

139

-55.

76

3.3E

-05

251

gi|115956824|

PR

ED

ICT

ED

: si

mil

ar t

o S

olu

te c

arri

er f

amil

y 2

5 (

mit

och

ondri

al c

arri

er;

aden

ine

nucl

eoti

de

tran

sloca

tor)

, m

ember

433

.310

10-5

6.3

78

-23.

73

5-2

7.8

42.

3E-0

5

252

gi|115710920|

PR

ED

ICT

ED

: si

mil

ar t

o N

a+/K

+ A

TP

ase

alpha

subunit

.11

45

3-2

4.2

327

-139

131

-7.6

19.

2E-0

6

253

gi|115940494|

PR

ED

ICT

ED

: si

mil

ar t

o M

GC

69168 p

rote

in (

solu

te c

arri

er f

amil

y, m

itoch

ondri

al)

77.1

92

-13.

52

15-8

9.3

92

-10.

52

8.3E

-06

254

gi|115926312|

PR

ED

ICT

ED

: si

mil

ar t

o L

OC

446923 p

rote

in (

H t

ransp

ort

ing A

TP

synth

ase)

22.8

104

-17.

83

1-3

.71

7.7E

-06

255

gi|115926329|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

solu

te c

arri

er f

amil

y 8

sodiu

m c

alci

um

exch

anger

)48

.45

8-4

9.8

51

-3.1

16.

4E-0

6

256

gi|115939138|

PR

ED

ICT

ED

: si

mil

ar t

o S

lc25a3

-pro

v p

rote

in (

solu

te c

arri

er f

amil

y 2

5 m

ember

3)

39.1

92

-7.7

13

-10.

22

1-6

.11

5.2E

-06

257

gi|115926327|

PR

ED

ICT

ED

: si

mil

ar t

o N

a/C

a ex

chan

ger

.40

.46

3-1

2.5

22

-17

24.

3E-0

6

258

gi|115697801|

PR

ED

ICT

ED

: si

mil

ar t

o N

icoti

nam

ide

nucl

eoti

de

tran

shydro

gen

ase

.11

36

4-1

3.8

21

-4.6

12

-13.

62

2.1E

-06

259

gi|115924511|

PR

ED

ICT

ED

: si

mil

ar t

o E

NS

AN

GP

00000007239 (

myosi

n +

atp

bin

din

g c

asse

tte

AB

C)

304

61

-3.5

14

-24.

24

5.8E

-07

2.9

E-0

4

260

gi|115964543|

PR

ED

ICT

ED

: si

mil

ar t

o t

ransa

ldola

se .

33.4

620

-90.

17

21-9

1.9

820

-97.

49

6.2E

-05

261

gi|115738231|

PR

ED

ICT

ED

: si

mil

ar t

o g

lyce

rald

ehydep

hosp

hat

e deh

ydro

gen

ase

isofo

rm 1

.36

.57

26-1

059

20-1

0810

15-5

5.8

55.

6E-0

5

262

gi|115944251|

PR

ED

ICT

ED

: si

mil

ar t

o t

ransk

etola

se i

sofo

rm 1

.66

.16

3-1

1.2

231

-140

1416

-58

62.

6E-0

5

263

gi|115929324|

PR

ED

ICT

ED

: si

mil

ar t

o g

luco

se-6

-phosp

hat

e 1-d

ehydro

gen

ase,

par

tial

.43

.38

27-1

1913

4-1

93

2.4E

-05

En

ergy m

etab

oli

sm*

Exch

an

ger

an

d A

TP

ase

s*

se

a u

rch

in 1

se

a u

rch

in 2

se

a u

rch

in 3

110

Pro

t

Nb

Acc

essi

on

nu

mb

erP

rote

in i

den

tifi

cati

on

MW

(kD

a)pI

rIlo

g(e)

#rI

log(

e)#

rIlo

g(e)

#N

SA

Fs

264

gi|115972829|

PR

ED

ICT

ED

: si

mil

ar t

o g

luco

se-6

-phosp

hat

e is

om

eras

e .

60.1

75

-34.

24

16-6

4.6

66

-40.

75

1.5E

-05

265

gi|115959412|

PR

ED

ICT

ED

: si

mil

ar t

o f

ruct

ose

-bip

hosp

hat

e al

dola

se .

39.3

77

-39.

84

10-5

0.4

51.

5E-0

5

266

gi|115663344|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(phosp

hoglu

cose

iso

mer

ase)

12.8

62

-5.2

13

-11.

61

1.4E

-05

267

gi|115939485|

PR

ED

ICT

ED

: si

mil

ar t

o m

alat

e deh

ydro

gen

ase

.35

.26

5-3

2.2

38

-56.

66

1-1

.71

1.3E

-05

268

gi|115680328|

PR

ED

ICT

ED

: si

mil

ar t

o c

yto

soli

c m

alat

e deh

ydro

gen

ase

.36

.26

3-8

29

-46.

75

1-8

.61

1.2E

-05

269

gi|115955959|

PR

ED

ICT

ED

: si

mil

ar t

o G

luco

sam

ine-

6-p

hosp

hat

e is

om

eras

e (G

luco

sam

ine-

6-p

hosp

hat

e

dea

min

ase)

(G

NP

DA

) (G

lcN

6P

dea

min

ase)

32.1

62

-16.

32

4-1

2.3

26.

5E-0

6

270

gi|115924009|

PR

ED

ICT

ED

: si

mil

ar t

o M

ethylt

hio

aden

osi

ne

phosp

hory

lase

.28

64

-16.

22

1-2

.21

6.3E

-06

271

gi|115945027|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in i

sofo

rm 1

(in

tegra

l m

embra

ne

pro

tein

1

oli

gosa

cchar

ylt

ransf

eras

e co

mple

x)

82.8

713

-50.

16

2-1

1.4

26.

2E-0

6

272

gi|115931669|

PR

ED

ICT

ED

: si

mil

ar t

o a

ldeh

yde

deh

ydro

gen

ase

1A

2 i

sofo

rm 1

.53

.36

3-1

6.2

36

-34.

54

5.8E

-06

273

gi|115961332|

PR

ED

ICT

ED

: si

mil

ar t

o g

luta

mat

e deh

ydro

gen

ase

1 .

61.3

75

-27.

83

4-1

1.4

25.

1E-0

6

274

gi|115970392|

PR

ED

ICT

ED

: si

mil

ar t

o i

ndep

enden

t phosp

hogly

cera

te m

uta

se .

47.6

65

-22.

63

1-1

.91

4.4E

-06

275

gi|115926187|

PR

ED

ICT

ED

: si

mil

ar t

o H

ydro

xyac

yl-

Coen

zym

e A

deh

ydro

gen

ase/

3-k

etoac

yl-

Coen

zym

e A

thio

lase

/enoyl-

Coen

zym

e A

75.9

94

-18.

13

3-1

22

2-8

.52

4.0E

-06

276

gi|115958742|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(la

ctat

e deh

ydro

gen

ase

)36

.36

3-1

2.1

21

-2.2

13.

9E-0

6

277

gi|115968074|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

aldeh

yde

deh

ydro

gen

ase

)61

.15

2-1

0.4

14

-30.

73

3.4E

-06

278

gi|115978426|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

dih

ydro

lipoyll

ysi

n S

succ

inylt

ransf

eras

e)

557

3-1

2.4

22

-9.4

23.

2E-0

6

279

gi|84688617|

insu

lin

rec

epto

r p

recu

rsor

.12

05

1-2

.71

3-9

.71

1.2E

-06

2.3

E-0

4

280

gi|115971190|

PR

ED

ICT

ED

: si

mil

ar t

o s

erin

e pro

teas

e .

12.3

513

-35.

64

10-2

7.6

31

-1.5

16.

6E-0

5

281

gi|115769879|

PR

ED

ICT

ED

: si

mil

ar t

o e

ndoder

min

, par

tial

.8.

66

3-4

.91

4-5

.81

2-1

0.5

23.

5E-0

5

282

gi|115959123|

PR

ED

ICT

ED

: si

mil

ar t

o L

pa-

pro

v p

rote

in, par

tial

(T

hro

mbin

)57

.95

4-1

7.8

222

-89.

29

20-4

15

2.7E

-05

283

gi|115958627|

PR

ED

ICT

ED

: si

mil

ar t

o t

hro

mbin

.72

.25

6-1

2.5

29

-44.

15

32-7

18

2.2E

-05

284

gi|115971224|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

amin

opep

tidas

e)

55.3

61

-3.9

115

-84.

98

7-4

6.2

51.

4E-0

5

285

gi|115938941|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

lyso

som

al m

embra

ne

gly

copro

tein

)51

.96

11-4

5.2

57

-8.5

11.

2E-0

5

286

gi|115958371|

PR

ED

ICT

ED

: si

mil

ar t

o l

yso

zym

e .

15.4

93

-13.

32

2-1

1.9

21.

1E-0

5

287

gi|115925688|

PR

ED

ICT

ED

: si

mil

ar t

o C

ND

P d

ipep

tidas

e 2 (

met

allo

pep

tidas

e M

20 f

amil

y)

.52

.55

6-3

2.8

46

-23.

93

7.9E

-06

288

gi|115970724|

PR

ED

ICT

ED

: si

mil

ar t

o V

alosi

n c

onta

inin

g p

rote

in, par

tial

.59

.65

11-8

6.3

72

-3.4

17.

5E-0

6

289

gi|115940918|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

amin

opep

tidas

e li

ke

1)

55.9

87

-54.

45

2-5

.51

5.6E

-06

290

gi|115955224|

PR

ED

ICT

ED

: si

mil

ar t

o L

OC

494800 p

rote

in (

cath

epsi

n)

31.6

62

-3.4

13

-10.

72

5.6E

-06

291

gi|115975288|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

Thro

mbosp

ondin

)10

35

7-2

5.2

31

-5.2

16

-29.

34

4.6E

-06

292

gi|115924999|

PR

ED

ICT

ED

: si

mil

ar t

o t

issu

e in

hib

itor

of

met

allo

pro

tein

ase

TIM

P32

.37

3-1

9.7

31

-31

4.4E

-06

293

gi|115774731|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in, par

tial

(le

ukotr

iene

A4 h

ydro

lase

)63

.45

4-1

3.1

21

-1.4

12.

8E-0

6

294

gi|115953033|

PR

ED

ICT

ED

: si

mil

ar t

o A

min

opep

tidas

e puro

myci

n s

ensi

tive

.87

.45

1-3

.61

3-1

5.4

22

-3.5

12.

3E-0

6

Lyso

som

es, p

rote

ase

s an

d p

epti

dase

s*

se

a u

rch

in 1

se

a u

rch

in 2

se

a u

rch

in 3

111

Pro

t

Nb

Acc

essi

on

nu

mb

erP

rote

in i

den

tifi

cati

on

MW

(kD

a)pI

rIlo

g(e)

#rI

log(

e)#

rIlo

g(e)

#N

SA

Fs

1.1

E-0

4

295

gi|115936009|

PR

ED

ICT

ED

: si

mil

ar t

o E

NS

AN

GP

00000009431 (

floti

lin

)46

.96

22-1

5914

36-1

9217

14-8

9.4

95.

2E-0

5

296

gi|115931510|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

sort

ing n

exin

)39

.39

4-2

2.3

313

-69.

16

10-4

5.3

52.

3E-0

5

297

gi|115963658|

PR

ED

ICT

ED

: m

ajor

vau

lt p

rote

in .

95.9

65

-26.

14

31-1

3114

3-8

.92

1.4E

-05

298

gi|115928607|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

floti

lin

)76

.65

4-2

1.6

326

-116

121.

3E-0

5

299

gi|115950948|

PR

ED

ICT

ED

: si

mil

ar t

o A

dap

tor-

rela

ted p

rote

in c

om

ple

x 2

, bet

a 1 s

ubunit

, par

tial

.28

.59

1-3

.61

5-4

0.7

47.

3E-0

6

300

gi|115968951|

PR

ED

ICT

ED

: hypoth

etic

al p

rote

in (

adap

tor

pro

tein

com

ple

x A

P2

)49

.910

2-2

.91

3-1

4.9

23.

5E-0

6

9.1

E-0

5

301

gi|115925590|

PR

ED

ICT

ED

: si

mil

ar t

o m

uci

n 5

, par

tial

.56

.45

30-7

8.2

751

-80.

67

14-4

14

5.7E

-05

302

gi|115955764|

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112

113

CHAPTER III

Proteomic analysis of sea urchin responses to bacterial injection

In preparation for submission to

Journal of Proteomics


Bove U1 – Experimental design - Technical support

Haynes PA2 – Technical support

Nair SV1 – Project supervision

Raftos DA1 – Experimental design – Project supervision

1 Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia

2 Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109,

Australia

114

115

3.1. Preface

After describing the fundamental proteome of the coelomic fluid of sea urchins

(Chapter 2), the next aim of this thesis was to identify proteins involved in immune

responses. The goal of this Chapter was to identify immune response proteins that are

specifically expressed in response to the injection of bacteria. To do this, the proteomes of

coelomocytes from naïve sea urchins were compared to those of sea urchins injected with

saline or bacteria by two-dimensional electrophoresis (2DE).

116

117

3.2. Abstract

Echinoderms evolved early in the deuterostome lineage, and as such constitute model

organisms for comparative physiology and immunology. The sea urchin genome sequence

revealed a complex repertoire of genes with similarities to the immune response genes of

other species. To complement these genomic data, we investigated the responses of sea

urchins to the injection of bacteria using a comparative proteomics approach. The relative

abundance of many proteins was altered in response to the injection of both bacteria and

saline, suggesting their involvement in wounding response, while others were differentially

altered in response to bacteria only. The identities of 15 proteins that differed in relative

abundance were determined by mass spectrometry. These proteins revealed a significant

modification in energy metabolism in coelomocytes towards the consumption of glutamate

and the production of NADPH after injection, as well as an increased concentration of cell

signalling molecules, such as heterotrimeric guanine nucleotide binding protein. The

injection of bacteria specifically increased the abundance of apextrin and calreticulin,

suggesting that these two proteins are involved in the sequestration or inactivation of

bacteria.

118

3.3. Introduction

Sea urchins are deuterostome invertebrates, and their position within this

phylogenetic lineage provides a unique view point to study the evolution of immunological

responses. These animals can live up to 100 years [1] and are abundant in the marine

environment. Sea urchins also have a large number of blood cells (coelomocytes), and can

survive multiple rounds of immunological challenge and collection of coelomic fluid,

permitting long-term challenge experiments. As such, sea urchins are ideal model

organisms for the study of early deuterostome immune responses.

Coelomocytes populate the coelomic fluid and fulfil many functions, including

protection of the viscera and cellular immunity [2]. In S. purpuratus there are between

1×106-5×106 coelomocytes per mL of coelomic fluid. About two thirds of these cells are

actively phagocytic [3, 4]. Other cell types carry out antibacterial, cytotoxic and clotting

functions [5]. Coelomocytes clear bacteria from the coelomic fluid with high efficiency [6,

7]. In vitro, phagocytes undergo substantial morphological transformations in response to

the presence of bacteria, and red spherule cells release a bactericidal agent, echinochrome

A [8]. Both transcriptome and protein studies have revealed the activation of numerous

immune-related genes in coelomocytes that are similar to those of vertebrates in response

to the injection of lipopolysaccharide (LPS). These immune response genes and proteins

include complement component C3, complement factor Bf and profilin homologues [3, 4,

9, 10].

Recently, the sea urchin genome sequencing project revealed the presence of an

elaborate repertoire of genes associated with immunity, most of which are closely related

to those of vertebrates. It also showed that the size of many immune response gene

families is greatly expanded in sea urchins relative to other deuterostomes [11]. The

pattern recognition receptors of sea urchins include 222 Toll-like receptor genes, 203

119

NACHT and leucine rich repeat containing (NLR) genes, 1095 SRCR domains distributed

among 218 gene models, and 46 genes containing fibrinogen domains [12]. Sea urchins

also express 185/333 genes, which appear to be restricted to echinoderms. 185/333 genes

constitute an additional family of highly variable immune response genes [13]. Overall, the

genome sequence predicted a complex immune system in sea urchins.

In the present study, we used proteomics to assess changes in protein abundances

after injecting saline or bacteria into sea urchins. Alterations in the cellular responses of

coelomocytes to the injection of bacteria were examined using a comparative two-

dimensional electrophoresis (2DE) approach. We observed substantial differences in

protein concentrations in response to the injection of saline and Vibrio sp. The identities of

15 proteins involved in these responses were determined by mass spectrometry, and the

potential functions of these proteins are discussed in detail.

120

3.4. Materials and Methods

3.4.1. Sea urchins

H. erythrogramma were collected from Camp Cove in Sydney Harbour. They were

housed in a recirculating sea water facility at Macquarie University as described previously

[14]. The sea urchins were left undisturbed in the aquaria for 12 months. Previous work

has shown that sea urchins become immunoquiescent after long-term housing of greater

than 8 months without significant disturbance [5, 14]. Immunoquiescence can be reversed

by injecting microbes, or in response to injury [14-16].

3.4.2. Immunological challenge and sample collection

Three sea urchins were injected with 500 µL of heat killed Vibrio sp. (VPSYS2; EF

S84094; OD600nm = 0.5 [17]) in sterile artificial CF (aCF; [18]). Three more animals

were injected with 500 µL of aCF, and a further three non-injected control animals were

left unchallenged. Injections were made by inserting a 23-gauge needle attached to a 1 mL

syringe through the peristomium into the coelomic cavity. Previous studies have shown

that microbial challenges elevate gene expression levels within 24 hours [13, 19]. To

mimic these conditions, the 12 sea urchins were left undisturbed for 20 hours after

injection before CF (approx 15 mL per animal) was withdrawn by dissecting the oral face.

3.4.3. Protein extraction

The CF was immediately mixed with an equal volume of calcium-magnesium-free

sea water with EDTA and imidazole (CMFSW-EI; 10 mM KCl, 7 mM Na2SO4, 2.4 mM

NaHCO3, 460 mM NaCl, 70 mM EDTA and 50 mM imidazole, pH 7.4) on ice to prevent

121

clotting, and centrifuged at 12000 × g for 2 minutes. The cell free CF was discarded and

the pellet of cells was resuspended in 700 µl of TriReagent (Sigma Aldrich). The

suspension was vortexed for 10 minutes before adding 70 µl of 1-bromo-3-chloropropane

(BCP). The solution was then held at room temperature for 10-15 minutes and centrifuged

at 12000 × g for 15 minutes at 4°C. The phenol chloroform phase was extracted and mixed

with 3 volumes of acetone and 200 µl of ethanol before centrifugation at 12000 × g for 10

minutes at 4°C. The supernatant was discarded and the protein pellet was resuspended in

phosphate buffered saline (PBS; 8.06 mM sodium phosphate, 1.94 mM potassium

phosphate, 2.7 mM KCl and 0.137 M NaCl, pH7.4). Re-suspended proteins were stored (-

80°C ) for further analysis.

3.4.4. Two-dimensional gel electrophoresis

The proteins were prepared for 2DE electrophoresis using 2D clean up kits (GE

Healthcare) according to the manufacturer’s instructions and were re-suspended in 2DE

sample buffer (8 M urea, 4% CHAPS, 60 mM dithiothreitol [DTT]). The total protein

content of each sample was determined with 2-D Quant kits (GE Healthcare).

Isoelectrofocusing was performed on an IPGphor IEF system (Amersham Biosciences).

Immobilized pH gradients (IPG) gel strips (11 cm, pH 3-6 and pH 5-8, GE Healthcare)

were equilibrated overnight with 100 µg of CF proteins in a final volume of 200 µL of

rehydration buffer containing 0.5 % immobiline (8 M urea, 2% CHAPS, 50 mM DTT and

0.5% carrier ampholytes; Immobiline GE Healthcare). Isoelectrofocusing was performed at

20°C in four stages: calibration (100 V for 3 hours), active rehydration (300 V for 1 hours),

a voltage ramping (up to 8000 V for 8 hours), and focusing (8000 V for 11.5 hours), to

obtain a total of 120-130,000 Vh. The current was limited to 45 µA per strip. After

isoelectric focusing, the IPG strips were reduced (1% DTT, 15 minutes) and alkylated

122

(2.5% iodoacetamide, IAA, 15 minutes) before separation in the second dimension by

SDS-PAGE. The proteins on the IPG strips were separated in the second dimension by

SDS-PAGE using 8-16% Tris-HCl precast polyacrylamide gels (Criterion Gel System,

BioRad). After electrophoresis, protein spots on the gels were visualized with Lava Purple

(also known as Deep Purple; FLUOROtechnics, GE-Healthcare) [20].

3.4.5. Gel analysis and selection of protein spots for mass spectrometry analysis

Digital gel images (Typhoon UV scanner) were analysed using Progenesis (Non-

Linear Dynamics). Spot detection and background subtraction were performed on all gel

images. Artefacts (dust particles or streaks) were removed by manual editing. Where

appropriate, spots were split manually into separate entities. A total of 385 protein spots

were identified. The gel images were then analysed for differences in spot intensity. A

normalization procedure was employed to allow for variation in total protein loading onto

different gels. Total spot volume was calculated for each gel and each spot was ascribed a

normalized spot volume as a proportion of this total value. Log2 normalized spot volumes

were then used to compare all spots present on all 12 sea urchin proteome maps in each of

the isoelectric point ranges (pI 3-6 and 5-8). We then performed a principal component

analysis (PCA) using XL stat 2009 to identify global proteome changes in response to

challenge. T tests (p<0.05) were employed to identify significant differences in the relative

abundance of individual proteins between treatments (non-injected, saline-injected and

bacteria-injected).

123

3.4.6. Peptide extraction

Proteins that differed significantly in relative abundance between treatments (T test,

p<0.05) were excised from 2DE gels and transferred to sterile Eppendorf tubes. The gel

pieces were washed briefly with 100 mM ammonium carbonate (NH4HCO3) and de-

stained in a solution of 25 mM NH4HCO3, and 50% acetonitrile (ACN) for 3 times for 10

minutes, before being dehydrated with 100% ACN for 5 minutes. The solution was

discarded and the gel pieces air-dried. After de-staining, the proteins were reduced with 10

mM DTT in 100 mM NH4HCO3 for 45-60 minutes at 56°C and alkylated with 55 mM IAA

in 100 mM NH4HCO3 for 45 minutes in the dark at room temperature. The gel pieces were

washed again with 100 mM NH4HCO3 for 5 minutes, then twice with 25 mM NH4HCO3,

50% ACN for 5 minutes, before being dehydrated with 100% ACN as above. They were

then left to air dry before rehydration in trypsin solution (12.5 ng/µl in 50 mM NH4HCO3,

Promega, WI.) for 30 minutes at 4°C. Additional 50 mM NH4HCO3 was added and the

proteins were digested at 37ºC overnight. The resulting tryptic peptides were extracted

from the gel slices by washing 2 times with 2% formic acid in 50% acetonitrile. The

extracts were combined and concentrated to 10 µl by vacuum centrifugation.

3.4.7. Nanoflow liquid chromatography – tandem mass spectrometry

Mass spectrometric analyses were performed at the Australian Proteome Analysis

Facility (APAF; Macquarie University). The tryptic digest extracts from 2DE gel slices

were analyzed by nanoLC-MS/MS using a LTQ-XL ion-trap mass spectrometer (Thermo

Dynamics) according to Breci et al. [21]. Reversed phase columns were packed in-house

to approximately 7 cm (100 µm i.d.) using 100 Å, 5 mM Zorbax C18 resin (Agilent

Technologies) in a fused silica capillary with an integrated electrospray tip. A 1.8 kV

124

electrospray voltage was applied via a liquid junction up-stream of the C18 column.

Samples were injected onto the C18 column using a Surveyor autosampler (Thermo

Dynamics). Each sample was loaded onto the C18 column followed by an initial wash

step with buffer A (5% (v/v) ACN, 0.1% (v/v) formic acid) for 10 minutes at 1 µL min-1.

Peptides were subsequently eluted from the C18 column with 0%-50% Buffer B (95%

(v/v) ACN, 0.1% (v/v) formic acid) over 58 minutes at 500 nL min-1 followed by 50%-

95% Buffer B over 5 minutes at 500 nL min-1. The column eluate was directed into a

nanospray ionization source of the mass spectrometer. Spectra were scanned over the

range 400–1500 amu. Automated peak recognition, dynamic exclusion, and tandem MS of

the top six most intense precursor ions at 35% normalization collision energy were

performed using the Xcalibur software (version 2.06) (Thermo Dynamics).

3.4.8. Protein identification

Raw files were converted to mzXML format and processed through Global

Proteome Machine (GPM) software using version 2.1.1 of the X!Tandem algorithm

(www.thegpm.org) [22, 23]. MS/MS spectra were searched against a combined

Strongylocentrotus database created with sequences downloaded from NCBI. This

FASTA format database contained 44,037 protein sequences comprising all S. purpuratus

predicted protein sequences and characterized proteins held by NCBI as of April 2008.

The database also incorporated a list of common human and trypsin peptide contaminants.

Search parameters included MS and MS/MS tolerances of ± 2 Da and ± 0.2 Da, tolerance

of up to 3 missed tryptic cleavages and K/R-P cleavages. Fixed modifications were set for

carbamidomethylation of cysteine and variable modifications were set for oxidation of

methionine.

125

3.5. Results

A total of 384 protein spots were identified, of which 166 spots were detected on 2DE gels

using pI 3-6 strips and 268 spots were present on 2DE gels in the pI 5-8 range. We initially

investigated the effect of saline and bacteria injections by principal components analysis. A

first step was to define the number of principal components (PC) to extract. A screeplot

was generated to determine if a clear break point was present (Figure 3.1A). The Eigen

values in Figure 3.1A correspond to the proportion of variance for each PC, while the

upper line shows the cumulative variance explained by the 8 components. The PCs were

sorted in decreasing order of variance so that the most representative PCs were listed first.

In this dataset, the first three PCs explained more of the variance in the data (31.3%, 17.7%

and 15.4% respectively) than all subsequent PCs. These three first PCs accounted for 64%

of the total variance (Figure 3.1A). Figure 3.1B shows a 3D score plot obtained using these

three PCs. In this plot, sea urchin samples with similar protein abundance profiles appear

closer in space, whereas significantly different samples appear more distant from each

other. Samples from saline-injected and bacteria-injected sea urchins clustered together

with high PC1 values, and low PC2 and PC3 values. In contrast, samples from non-

injected sea urchins all had low PC1 values and variable PC2 and PC3 values. Thus, PC1

was deemed to represent variations in the proteome due to the wound produced by the

needle during the injection, the injection of aCF, and/or the increase in CF volume

resulting from injection.

126

Figure 3.1: Principal components analysis of proteome profiles after the injection of

saline or bacteria. A/ Screeplot showing the Eigen values (proportion of variance) for

each principal component (PC; histogram) and the cumulative proportion of variances

(curve) explained by consecutive principal components. B/ 3D score plot using the first

three PCs shown in A. The Figure shows cumulative data for all of the protein spots

observed on 2DE gels from the CF of sea urchins non-injected, saline-injected and

bacteria-injected.

0

20

40

60

80

100

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1

1.5

2

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3

PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8

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Axis X = PC1 Axis Y = PC3

Axis Z = PC2

!

!

Non injected

Saline injected

Bacteria injected

PC1

PC3

PC2

!"

#"

127

The analysis of individual proteins on 2DE maps of saline-injected sea urchins

revealed 24 proteins with differential concentrations relative to non-injected controls,

whereas 42 proteins were altered in response to the injection of bacteria (Figure 3.2).

Seventeen spots corresponding to the most abundant of these differentially regulated

proteins were excised from the gels and subjected to in-gel trypsin digestion and MS

analysis (Figure 3.3). For 12 of these spots, at least two unique peptide matches to the

same protein in the S. purpuratus database were discerned (Table 3.1, Supplementary Data

3.1, Figure 3.3) so that identities could be assigned to these proteins. In the case of spot 4,

which was less abundant in response to saline and bacterial injection (Figure 3.4), two

different matches were obtained with high confidence for the same spot (Table 3.1,

Supplementary Data 3.1). One match corresponded to cytoskeletal actin CyIIb

(gi47551037), whilst the second was to a succinate CoA ligase (gi115975381). It is likely

that the match to CyIIb came from contamination from the neighboring spot 3. All of the

spots in proximity to spot 3 (spots 1, 2, 4, 7), had peptides matching CyIIb. Cytoskeletal

actin CyIIb (spot 3; gi47551037) was the most abundant protein in all samples.

PCA suggested that the most substantial alterations to the proteome were similar

after saline and bacterial injections. Of these proteins, which are putatively involved in

wounding responses, 10 protein spots were extracted and 6 proteins were identified. Most

of these proteins decreased in abundance in response to wounding. These included proteins

similar to vacuolar (V-type) H(+)-ATPase B subunit isoform 1 (spot 1; gi115935893) and

JIA crystalline, ADP-ribosylglycohydrolase (spot 16; gi115955510), a putative 14-3-3

epsilon isoform (spot 8; gi160623362) and a potential carbonic anhydrase alpha (spot 17;

gi115926884). The proteins that increased equally in abundance after both saline and

bacteria injection included a cytosolic malate dehydrogenase (spot 12; gi115680328) and a

protein similar to a heterotrimeric guanine nucleotide binding protein beta subunit (spot

13; gi115936805).

128

Figure 3.2: Number of 2DE spots that varied significantly in abundance after saline

and bacterial injections, as determined by quantitative analysis of Lava Purple

stained 2DE gels. This Venn Diagram shows the number of proteins that differed

significantly between treatments (T test, p<0.05); non-injected controls versus saline-

injected (C vs S), saline-injected versus bacteria-injected (S vs B) and non-injected

controls versus bacteria-injected (C vs B).

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129

Figure 3.3: Lava Purple stained 2DE maps of CF proteins following Vibrio Sp.

injection. One hundred micrograms of proteins were loaded on 2DE gels (IEF on pH 3-6,

top, and 5-8, bottom, 11 cm IPG strips followed by 8-16% SDS-Page). Spot detection was

performed using Lava Purple staining. Labeled spots were analysed by quantitative

Progenesis analysis to identify proteins that vary significantly in relative abundance (T test,

p<0.05) after saline or bacterial injection. Those 17 spots were extracted for identification

by mass spectrometry.

2

1

7

4

5

6

8 9

10

3 6

11

12 13 14

15

16 17

8 5

8-1

6%

SD

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pI

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130

Log 2

nor

mal

ized

vol

umes

131

Figure 3.4: Log2 normalized volumes of differentially regulated protein spots. Spot

numbers correspond to those allocated in Figure 3.3 and Table 3.1. NI: non-injected; S:

saline injection; B: bacterial injection. The letters a, b and c indicate significantly different

log2 normalized volumes between treatments (T test, P<0.05). Like letters are not

significantly different (T test, P<0.05). Bars = SEM, n = 3.

Table 3.1: Protein identification of 2DE gel spots from Figure 3.4. The Genbank

accession numbers of the identified proteins are shown. Matching peptides are detailed in

Supplementary Data 3.1. Parameter definitions are: log(e), the probability that a protein

match arises stochastically. The lower the log(e) value, the more significant the protein

identification. %, percentage of coverage of the sequence. #, number of unique peptides

identified. Total, total number of peptide sequences corresponding to a mass spectrum. Mr,

predicted molecular weight of the protein.

Ref. Spot

GenBank Accession

No. Description log(e) % # Total Mr 1 gi115935893 PREDICTED: similar to vacuolar (V-type) H(+)-

ATPase B subunit isoform 1 -254.3 40 19 55 54.1

2 gi115945943 PREDICTED: similar to ENSANGP00000011972 (rab GDP dissociation inhibitor)

-53.7 15 6 10 49

3 gi47551037 cytoskeletal actin CyIIb -144 27 12 28 41.8 4 gi47551037 cytoskeletal actin CyIIb -124.6 26 11 22 41.8 gi115975381 PREDICTED: hypothetical protein (succinate CoA

ligase, ADP forming, beta subunit) -109.3 23 9 16 49

5 gi115956476 PREDICTED: similar to apextrin -121 17 14 65 49.5 6 gi47550939 calreticulin -210.2 28 19 36 48.8 8 gi160623362 putative 14-3-3 epsilon isoform -26.6 15 3 4 24.1 11 gi115972829 PREDICTED: similar to glucose-6-phosphate

isomerase -98.8 23 10 21 60.1

12 gi115680328 PREDICTED: similar to cytosolic malate dehydrogenase

-67.7 13 7 18 36.2

13 gi115936805 PREDICTED: similar to heterotrimeric guanine nucleotide-binding protein beta subunit isoform 2

-102.2 21 9 23 37.5

16 gi115955510 PREDICTED: similar to J1A crystallin (ADP-ribosylglycohydrolase)

-141.5 16 12 31 33.9

17 gi115926884 PREDICTED: similar to CG7820-PA (carbonic anhydrase alpha)

-55.8 13 6 9 29.7

132

Figure 3.5: Selected areas of 2DE maps showing proteins (spots 5 and 6) with

abundances that differed significantly (p<0.05) in response to the injection of bacteria

when compared to both non-injected controls and saline-injected sea urchins.

133

In addition to the major shift in protein abundances in response to wounding, 42

proteins had significantly altered abundance in bacteria-injected samples when compared

to either non-injected controls or saline-injected sea urchins. Of these, 7 had significantly

different concentrations compared with both non-injected and saline-injected samples.

Among the proteins identified by mass spectrometry, a molecule similar to Rab GDP

dissociation inhibitor (spot 2; GDI; gi115945943) and another similar to glucose-6-

phosphate isomerase (spot 11; gi115972829) had significantly (p<0.05) reduced

concentrations in presence of bacteria relative to the other treatments. However, other

proteins that were low in abundance or absent in unchallenged sea urchins and after saline

injection, were more abundant after the injection of bacteria. These included spots 5 and 6

shown in Figure 3.5, which were significantly more abundant in response to bacterial

injection. These two proteins matched apextrin (spot 5; gi115956476) and calreticulin

(spot 6; gi47550939).

134

3.6. Discussion

This study employed a 2DE based comparative proteomic analysis to identify

proteins involved in the response of sea urchin coelomocytes to the injection of bacteria. It

identified two discrete components within the host defense responses of sea urchins,

reflecting different pathways involved in the responses to wounding and bacterial

injection.

Injection alone induced a consistent response among all sea urchins regardless of the

presence or absence of bacteria. Numerous proteins varied in concentration after injection,

which resulted in a major shift in the first principal component when proteomes were

compared by PCA. This shift seemed to include a significant modification of energy

metabolism towards the consumption of glutamate and the production of NADPH

necessary for the production of free radicals such as O-2 or NO [13, 24, 25]. This is

reflected by the significant alteration in abundance of a vacuolar H(+)-ATPase and a

protein similar to cytosolic malate dehydrogenase. Proteins altered by wounding also

included those involved in cell signalling, which could ultimately regulate various cell

functions, including antimicrobial activity, wound healing, and actin restructuring [24-26].

These signalling molecules included a ADP-ribosylglycohydrolase, a putative 14-3-3

epsilon isoform, and a heterotrimeric guanine nucleotide binding protein. The changes

evident in the concentration of these proteins might reflect major cytoskeletal and

functional modification of coelomocytes, allowing the recruitement of coelomocytes to the

site of injury for wound repair [29-31].

In addition to this wounding response, the injection of bacteria, as opposed to just

saline, induced discrete modifications in coelomocyte proteome. The more substantial

decrease in glucose-6-phosphate isomerase after bacterial injection supports the previously

implied shift in metabolism towards the production of NADPH, which may be essential for

135

phagocytosis. The abundance of Rab GDP dissociation inhibitor was also altered more

substantially by bacterial injection, compared to saline. GDI regulates vesicular transport

through interaction with Rab GTPase and is necessary for phagocytosis. These changes

detected in both glucose-6-phosphate isomerase and Rab GDP dissociation inhibitor

suggest that the presence of bacteria triggers additional cellular pathways involved in

phagocytosis.

Two proteins (apextrin and calreticulin) that were absent or in very low abundance in

non-injected controls or saline-injected animals were substantially up-regulated in the

presence of bacteria. This suggests that they both have important functions in the

sequestration or neutralization of pathogens. Apextrin has been described as a membrane

attack complex/perforin domain (MAC/PF) protein [27, 28]. However, it is not yet known

whether this protein has any lytic activity. Apextrin was initially identified in secretory

vesicles within sea urchin eggs and is involved in cell adhesion during embryonic

development [29]. Interestingly, the expression of apextrin is also induced during acute

immune responses of the protochordate, Amphioxus [30]. As many as 56 unique sequences

of proteins similar to apextrin have been recorded on NCBI for S. purpuratus alone,

suggesting that this protein might be involved in a range of functions including embryonic

development [29] and neutralization of pathogens [30].

The significant increase in calreticulin suggests that calcium regulation also plays an

important role during bacterially induce processes. Calreticulin is an important

multifunctional calcium (Ca2+)-binding protein involved in the regulation of intracellular

Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage [31]. Its increase in

abundance supports previous studies, which showed the importance of calcium signalling

in immune cells [32, 33]. Calreticulin is a multifunctional protein and might also be

involved at other levels of the immune response process. Human calreticulin is expressed

on the surface of activated T lymphocytes in association with MHC molecules [34], and is

136

released from lytic granules with perforins [35]. This supports previous studies that

demonstrated the calcium dependent antibacterial and cytotoxic activity of sea urchins

coelomocytes [36].

Despite our ability to identify a range of proteins involved in apparently discrete

responses to wounding and the presence of bacteria, the current study was limited to

assessing just a single time point after challenge. We are now using more high throughput

proteomic techniques to investigate a broader time scale during sea urchin immune

responses.

137

3.7. References

[1] Ebert, T. A., Southon, J. R., Red sea urchins (Strongylocentrotus franciscanus) can live

over 100 years: confirmation with A-bomb 14 carbon. Fish bulletin (Sacramento,

Calif.) 2003, 101, 915.

[2] Chia, F. S., Xing, J., Echinoderm coelomocytes. Zoological studies 1996, 35, 231-254.



[4] Smith, L., Britten, R., Davidson, E., SpCoel1: a sea urchin profilin gene expressed

specifically in coelomocytes in response to injury. Mol Biol Cell 1992, 3, 403-414.

[5] Gross, P. S., Al-Sharif, W. Z., Clow, L. A., Smith, L. C., Echinoderm immunity and the

evolution of the complement system. Dev Comp Immunol 1999, 23, 429-442.

[6] Gerardi, G., Lassegues, M., Canicatti, C., Cellular distribution of sea urchin

antibacterial activity. Biol. Cell. 1990, 70, 153-157.

[7] Stabili, L., Pagliara, M., Metrangolo, M., Canicatti, C., Comparative aspects of

Echinoidea cytolysins: the cytolytic activity of Spherechinus granularis

(Echinoidea) coelomic fluid. Comp. Biochem. Physiol. 1992, 101A, 553-556.

[8] Service, M., Wardlaw, A., Echinochrome-A as a bactericidal substance in the coelomic

fluid of Echinus esculentus (L.). Comp Biochem Physiol B Biochem Mol Biol 1984,

79, 161-165.



1998, 160, 2983-2997.

[10] Smith, L. C., Shih, C.-S., Dachenhausen, S. G., Coelomocytes express SpBf, a

homologue of factor B, the second component in the sea urchin complement

system. J Immunol 1998, 161, 6784-6793.

138



[12] Hibino, T., Loza-Coll, M., Messier, C., Majeske, A., et al., The immune gene

repertoire encoded in the purple sea urchin genome. Dev Biol 2006, 300, 349-365.




2005, 22, 33-47.



2000, 51, 1021-1033.



subpopulations of the phagocytic coelomocytes. Immunogenetics 2000, 51, 1034-

1044.



[17] Wilson, G. S., Raftos, D. A., Corrigan, S. L., Nair, S. V., Diversity and antimicrobial

activities of surface-attached marine bacteria from Sydney Harbour, Australia.

Microbiol Res 2009. In Press.







139



[20] Ball, M. S., Karuso, P., Mass spectral compatibility of four proteomics stains. J

Proteome Res 2007, 6, 4313-4320.

[21] Breci, L., Hattrup, E., Keeler, M., Letarte, J., et al., Comprehensive proteomics in


electrophoresis, and isoelectric focusing. Proteomics 2005, 5, 2018-2028.

[22] Haynes, P. A., Miller, S., Radabaugh, T., Galligan, M., et al., The wildcat toolbox: a

set of perl script utilities for use in peptide mass spectral database searching and

proteomics experiments. J Biomol Tech 2008, 17, 97-102.

[23] Rohrbough, J. G., Breci, L., Merchant, N., Miller, S., Haynes, P. A., Verification of

single-peptide protein identifications by the application of complementary database

search algorithms. J Biomol Tech 2006, 17, 327-332.

[24] Corda, D., Di Girolamo, M., Mono-ADP-Ribosylation: a tool for modulating immune

response and cell signaling. Sci. STKE 2002, 2002, 53.

[25] Roberts, M. R., de Bruxelles, G. L., Plant 14-3-3 protein families: evidence for

isoform-specific functions? Biochem. Soc. Trans. 2002, 30, 373-378.

[26] Hamm, H. E., The many faces of G protein signaling. J Biol Chem 1998, 273, 669-

672.

[27] Miller, D., Hemmrich, G., Ball, E., Hayward, D., et al., The innate immune repertoire

in cnidaria-ancestral complexity and stochastic gene loss. Genome Biology 2007, 8,

R59.

[28] Rosado, C. J., Kondos, S., Bull, T. E., Kuiper, M. J., et al., The MACPF/CDC family

of pore-forming toxins. Cell Microbiol 2008, 10, 1765-1774.

140

[29] Haag, E. S., Sly, B. J., Andrews, M. E., Raff, R. A., Apextrin, a novel extracellular

protein associated with larval ectoderm evolution in Heliocidaris erythrogramma.

Dev Biol 1999, 211, 77-87.

[30] Huang, G., Liu, H., Han, Y., Fan, L., et al., Profile of acute immune response in


infection. Dev Comp Immunol 2007, 31, 1013-1023.

[31] Gelebart, P., Opas, M., Michalak, M., Calreticulin, a Ca2+-binding chaperone of the

endoplasmic reticulum. The International Journal of Biochemistry & Cell Biology

2005, 37, 260-266.

[32] Berridge, M. J., Lipp, P., Bootman, M. D., The versatility and universality of calcium

signalling. Nat Rev Mol Cell Biol 2000, 1, 11-21.

[33] Oh-hora, M., Rao, A., Calcium signaling in lymphocytes. Current Opinion in

Immunology 2008, 20, 250-258.

[34] Arosa, F. A., de Jesus, O., Porto, G., Carmo, A. M., de Sousa, M., Calreticulin is

expressed on the cell surface of activated human peripheral blood T lymphocytes in

association with major histocompatibility complex class I molecules. J Biol Chem

1999, 274, 16917-16922.

[35] Dupuis, M., Schaerer, E., Krause, K., Tschopp, J., The calcium-binding protein

calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes. J

Exp Med 1993, 177, 1-7.

[36] Haug, T., Kjuul, A. K., Styrvold, O. B., Sandsdalen, E., et al., Antibacterial activity in

Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa

(Holothuroidea), and Asterias rubens (Asteroidea). J Invertebr Pathol 2002, 81, 94-

102.

141

Supplementary Data 3.1: Mass spectrometric (LC-MS/MS) data for protein spots

isolated from 2DE gels. The Genbank accession numbers of the identified proteins are

shown. The amino acid sequences shown are the matching peptides. Parameter definitions

are: log(e), the probability that a putative peptide sequence corresponding to a mass

spectrum arises stochastically. The lower the log(e) value, the more significant the

assignment of the peptide sequence to the mass spectrum. m + h, peptide mass in Da + 1. z,

peptide charge.

142

Ref.

Spot

GenBank

Accessio

n N

o.

Description

log(e

)m

+h

zPeptide s

equences

1gi1

15935893

PRED

ICTED

: sim

ilar

to v

acuola

r (V

-type)

H(+

)-

ATPase B

subunit isofo

rm 1

-2.7

1197

2[me 3 AAKEHTLAVTR 13 nyit

-4.3

1665

2kfpk 44 FAEIVTLTNDGSKR 58 sgqv

-1.3

1317

2kfae 47 IVTLTNDGSKR 58 sgqv

-7.4

2928

3sgtt 70 AVVQVFEGTSGIDAKNTTCEFTGDILR 96 mpvs

-6.7

1521

2sgtt 70 AVVQVFEGTSGIDAK 84 nttc

-5.4

2574

3idak 85 NTTCEFTGDILRMPVSEDMLGR 106 vfng

-4.5

1427

2idak 85 NTTCEFTGDILR 96 mpvs

-11857

3dilr 97 MPVSEDMLGRVFNGSGK 113 aidk

-4.8

1151

2dilr 97 MPVSEDMLGR 106 vfng

-14

2451

2pqsr 140 IYPEEMIQTGISAIDVMNSIAR 161 gqki

-5.3

2492

3siar 162 GQKIPIFSAA GLPHNEIAAQICR 184 qagl

-12.6

2178

2rgqk 165 IPIFSAAGLPHNEIAAQICR 184 qagl

-5.4

1728

3itpr 254 LALTAAEFLAYQCEK 268 hvlv

-1.5

1456

2ealr 286 EVSAAREEVPGRR 298 gfpg

-8.6

1897

2pgrr 299 GFPGYMYTDLATIYER 314 agrv

-4.4

1597

2ldsr 364 QIYPPINVLPSLSR 377 lmks

-17

2679

3dmtr 390 KDHADVSNQLYANYAIGKDVQAMK 413 avvg

-5.2

2007

3dmtr 390 KDHADVSNQLYANYAIGK 407 dvqa

-5.9

1356

2kfek 438 NFITQGNYENR 448 svfe

2gi1

15945943

PRED

ICTED

: sim

ilar

to E

NSAN

GP00000011972 (

rab

GD

P d

issocia

tion inhib

itor)

-2.2

1313

2smgr 70 GRDWNVDLIPK 80 flma

-1.7

1336

3ktir 207 RIKLYSESLAR 217 ygks

-1.3

1180

2tirr 208 IKLYSESLAR 217 ygks

-5.2

2523

3slar 218 YGKSPYLYPMYGLGELPQGFAR 239 lsai

-8.9

1448

2gfar 240 LSAIYGGTYMLDK 252 pide

-1.2

1748

2mldk 253 PIDEITMEDGKVTGVK 268 sgge

3gi4

7551037

cyto

skele

tal actin C

yII

b-1

1.8

1761

2[mc 3 DDDVAALVIDNGSGMVK 19 agfa

-5.2

2156

3gmvk 20 AGFAGDDAPRAVFPSIVGRPR 40 hqgv

-2.7

976.4

1gmvk 20 AGFAGDDAPR 29 acfp

-1.7

881.5

2ravf 33 PSIVGRPR 40 hqgv

-3.7

2540

3grpr 41 HQGVMVGMGQKDSYVGDEAQSKR 63 gilt

143

Ref.

Spot

GenBank

Accessio

n N

o.

Description

log(e

)m

+h

zPeptide s

equences

-92383

3grpr 41 HQGVMVGMGQKDSYVGDEAQSK 62 rgil

-3.8

1204

2grpr 41 HQGVMVGMGQK 51 dsyv

-4.3

1355

2mgqk 52 DSYCGDEAQSKR 63 gilt

-6.1

1199

2mgqk 52 DSYVGDEAQSK 62 rgil

-6.8

1954

2nelr 97 VAPEEHPVLLTEAPLNPK 114 anre

-11.2

1791

2slek 240 SYELPDGQVITIGNER 255 frap

-1.3

1575

3iadr 314 MQKEITALAPPTMK 327 ikii

4gi4

7551037

cyto

skele

tal actin C

yII

b-5

.11777

2[mc 3 DDDVAALVIDNGSGMVK 19 agfa

-5.1

2156

3gmvk 20 AGFAGDDAPRAVFPSIVGRPR 40 hqgv

-2.7

976.4

1gmvk 20 AGFAGDDAPR 29 avfp

-1.4

881.5

2ravf 33 PDIVGRPR 40 hqgv

-6.6

2540

3grpr 41 HQGVMVGMGQKDSYVGDEAQSKR 63 gilt

-8.1

2384

3grpr 41 HQGVMVGMGQKDSYVGDEAQSK 62 rgil

-2.2

1188

2grpr 41 HQGVMVGMGQK 51 dsyv

-4.5

1355

2mgqk 52 DSYVGDEAQSKR 63 gilt

-9.1

1954

2nelr 97 VAPEEHPVLLTEAPLNPK 114 anre

-9.1

1791

2slek 240 SYELPDGQVITIGNER 255 frap

-1.2

1165

2ptmk 328 IKIIAPPERK 337 ysvw

gi1

15975381

PRED

ICTED

: hypoth

etical pro

tein

(succin

ate

CoA

ligase,

AD

P f

orm

ing,

beta

subunit)

-3.4

1623

2pipr 62 AEVATTAQRAYEIAK 76 slgs

-2.3

1713

3eiak 77 SLGSGDVVVKAGVLAGGR 94 gkga

-2.1

771.4

1vvvk 87 AQVLAGGR 94 gkga

-5.7

2093

2ggvk 109 LAFSPEEVKDLASQMIGNK 127 lvtk

-4.6

1339

2yvrr 154 EYYFAITMER 163 afng

-5.2

2125

3diik 326 LHGGTPANFLDVGGGATADQVK 347 qafk

-6.4

2724

3eaka 408 IIAHSDLRILACDDLDEAAKMVVR 431 lsti

-9.1

1819

2sdlr 416 ILACDDLDEAAKMVVR 431 lsti

-5.7

1334

2sdlr 416 ILACDDLDEAAK 427 mvvr

5gi1

15956476

PRED

ICTED

: sim

ilar

to a

pextr

in-1

.42261

2fcmk 88 TQSSLDGEAWPAGSYCIYKK 107 gdcp

-1.3

1187

1geaw 98 PAGSYCIYKK 107 gdcp

-2.3

1019

2awpa 100 GSYCIYKK 107 gdcp

-1.8

2829

3ciyk 107 KGDCPSGFQSGSIRWDDEDSANINR 131 eggt

-1.3

1337

2gsir 121 WDDEDSANINR 131 eggt

144

Ref.

Spot

GenBank

Accessio

n N

o.

Description

log(e

)m

+h

zPeptide s

equences

-1.3

2145

3sipk 166 ADRFMLLSRFDSCQQVR 182 gmtv

-1.3

1109

2slpk 166 ADRFMLLSR 174 fdsc

-1.3

1803

2kadr 169 FMLLSRFDSCQQVR 182 gmtv

-5.5

1255

2qqvr 183 GMTVTKEWFR 192 wdne

-21198

2qvrg 184 MTVTKEWFR 192 wdne

-2.2

1067

2rgmt 186 TVTKEWFR 192 wdne

-1.5

965.5

2rgmt 186 VTKEWFR 192 wdne

-2.3

1357

1srad 306 WPQGNYCIYR 315 wdgs

-3.7

1171

2radw 307 PQGNYCIYR 315 wdgs

6gi4

7550939

calreticulin

-1.2

1075

2vesv 40 HKGSDAGKFK 49 wsag

-1.6

799.4

2vesv 40 HKGSDAGK 47 fkws

-7.1

2767

3sagk 55 FYGDAEQDKGIQTSQDAKFYGLSAK 79 ftdf

-12.6

2001

2sagk 55 FYGDAEQDKGIQTSQDAK 72 fygl

-3.4

1072

2sagk 55 FYGDAEQDK 63 giqt

-1.5

829.3

1sagk 55 FYGDAEQ 61 dkgi

-2.6

1957

3daeq 62 DKGIQTSQDAKFYGLSAK 79 ftdf

-3.5

1191

2daeq 62 DKGIQTSQDAK 72 fygl

-7.3

1714

2eqdk 64 GIQTSQDAKFYGLSAK 79 ftdf

-3947.5

2eqdk 64 GIQTSQDAK 72 fygl

-1.6

785.4

1qdak 73 FYGLSAK 79 ftdf

-2.4

2234

3ftvk 98 HEQKIDCGGGYAKIFPADLD 117 qedm

-1.4

3584

3gyak 111 IFPADLDQEDMHGDSPYNIMFGPDICGPGTKK 142 vhvi

-4.1

1915

3pgtk 142 KVHVIFNYKGKNLLIK 157 kdir

-3.9

1334

3pgtk 142 KVHVIFNYKGK 152 nlli

-4.9

1205

3gtkk 143 VHVIFNYKGK 152 nlli

-3.7

1319

2pmin 267 NPEYKGEWAPK 277 kien

-3.6

1205

2minn 268 PEYKGEWAPK 277 kien

-10.4

1728

2nlys 307 YPSFGAIGFDLWQVK 321 sgti

8gi1

60623362

puta

tive 1

4-3

-3 e

psilon isofo

rm-1

.71204

2dvar 16 VNEELSVEER 25 nlls

-6.6

1518

2veer 26 NLLSVAYKNVIGAR 39 rasw

-5.3

1329

2dyhr 115 YLAEFSLDSKR 125 ksas

145

Ref.

Spot

GenBank

Accessio

n N

o.

Description

log(e

)m

+h

zPeptide s

equences

11

gi1

15972829

PRED

ICTED

: sim

ilar

to g

lucose-6

-phosphate

isom

era

se

-1.4

1561

3ardr 107 MFGGEKINFTEDR 119 avlh

-2.2

878.6

2tedr 120 AVLHVALR 127 nrsn

-1.8

866.5

1crvr 154 KFTESVR 160 sgew

-3.5

1083

2esvr 161 SGEWKGTSGK 170 aitd

-2.1

1658

3kgfs 169 GKAITDAVNIGIGGSDLG 186 pvmv

-9.8

1614

2iask 234 TFTTQETITNATSAK 248 nwfl

-5.4

1218

2hvak 264 HFVALSTNAEK 274 vsaf

-1.2

1827

3nffg 355 AETHALLPYDQYLHR 369 faay

-2.5

2228

3ylhr 370 FAAYFQQGDMESNGKYVTR 388 dgrr

-7.4

2164

3mtgk 463 TKEEAEKELVASGMSAENIK 482 lilp

12

gi1

15680328

PRED

ICTED

: sim

ilar

to c

yto

solic m

ala

te

dehydro

genase

-2.4

1590

3mprr 94 EGMERADLLKANVK 107 ifes

-4.8

1761

3gmer ADLLKANVKIFESQGQ 114 ains

-4.1

971.6

2gmer ADLLKANVK 107 ifes

-81221

2dllk 104 ANVKIFESQGQ 114 ains

-3.7

1921

3ktvk 126 VLVVGNPANTNCLVCMK 142 naps

-2.7

993.5

1ikar 239 KLSSAMSSAK 248 aicd

-1.3

865.4

1kark 240 LSSAMSAAK 248 aicd

13

gi1

15936805

PRED

ICTED

: sim

ilar

to h

ete

rotr

imeric g

uanin

e

nucle

otide-b

indin

g p

rote

in b

eta

subunit isofo

rm 2

-3.2

1011

2[m 2 ATELEHLR 9 hege

-8.8

2248

3rdar 24 KAVQDTTLMQVTQNMDPVGR 43 iqmr

-8.5

2681

3dark 25 AVQDTTLMQVTQNMDPVGRIQMR 47 trrt

-6.6

2120

3dark 25 AVQDTTLMQVTQNMDPVGR 43 iqmr

-4.6

2448

3lktr 131 EGNVRVSREL PGHTGYLSCCR 151 fidd

-2.2

1892

3gnvr 136 VSRELPGHTGYLSCCR 151 fidd

-2.3

1550

2rvsr 139 ELPGHTGYLSCCR 151 fidd

-81214

2pdnr 199 TFVSGACDASAK 210 twdi

-3.3

1010

2kger 306 AGVLAGHDNR 315 vscl

16

gi1

15955510

PRED

ICTED

: sim

ilar

to J

1A c

rysta

llin

(AD

P-

ribosylg

lycohydro

lase)

-1.2

1989

3pyyk 57 LPTGKNSAYGDHLYVLLK 74 svve

-7.1

1493

2ptgk 62 NSAYGDHLYVLLK 74 svve

-2.9

1379

2tgkn 63 SAYGDHLYVLLK 74 svve

-11.2

1876

2eavk 170 VVQNTPICIKYALTGAK 186 lleg

146

Ref.

Spot

GenBank

Accessio

n N

o.

Description

log(e

)m

+h

zPeptide s

equences

-5.5

1172

2eavk 170 VVQNTPICIK 179 yalt

-1.1

723.4

1icik 180 YALTGAK 186 lleg

-8.5

2242

3llak 301 VERGQEVKALAEQLVAMRQS 320 ]

-6.2

2044

3llak 301 VERGQEVKALAEQLVAMR 318 qs]

-9.5

1858

2kver 304 GQEVKALAEQLVAMRQS 320 ]

-3.3

1659

3kver 304 GQEVKALAEQLVAMR 318 qs]

-41334

2qevk 309 ALAEQLVAMRQS 320 ]

-5.2

1118

2qevk 309 ALAEQLVAMR 318 qs]

17

gi1

15926884

PRED

ICTED

: sim

ilar

to C

G7820-P

A (

carb

onic

anhydra

se a

lpha)

-2.7

1005

1kcdk 139 GIAVLGSFIK 148 vgkp

-11402

2pclk 165 KALNKNCTAPVEG 177 gfdp

-6.4

2759

3clkk 166 ALNKNVTAPVEGGFDPSCLLPENKK 190 dywt

-3.9

1331

2clkk 166 ALNKNVTAPVEGG 178 fdps

-5.5

1274

2clkk 166 ALNKNVTAPVEG 177 gfdp

-1.9

2334

3alnk 170 NCTAPVEGGFDPSCLLPENKK 190 dywt

147

CHAPTER IV

Time course proteomic profiling of cellular responses to immunological challenge in

sea urchins (Heliocidaris erythrogramma).

Submitted to

Molecular and Cellular Proteomics


Haynes PA2 – Experimental design – Technical support

Raftos DA1 – Project supervision

Nair SV1 – Experimental design – Project supervision


2 Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109,

Australia

148

149

4.1. Preface

The 2DE gel based analysis employed in Chapter 3 revealed major modifications of

the coelomic fluid proteome resulting from injection itself, but relatively few alterations

that were specifically due to the presence of bacteria. We hypothesised that more

substantial differences in protein abundances due to the injection of pathogens might occur

over a broader time course than that assessed in Chapter 3 (20 hours p.i.). In order to better

understand protein alterations due to wounding, and identify molecules specifically

involved in anti-pathogen responses, Chapter 4 is a time course study of the coelomocyte

proteome in response to the injection of a bacterial PAMP (LPS) or saline. In this case, we

used shotgun proteomics, which was successful in identifying and quantifying a broader

range of proteins (Chapter 2) than could be identified by 2DE.

150

151

4.2. Summary

Genome sequences and high diversity cDNA arrays have provided a detailed

molecular understanding of immune responses in a number of invertebrates, including sea

urchins. However, complementary analyses have not been undertaken at the level of

proteins. Here, we use shotgun proteomics to describe changes in the abundance of

proteins from coelomocytes of sea urchins after immunological challenge and wounding.

The relative abundances of 345 reproducibly identified proteins were measured 6, 24 and

48 hours after injection. Significant changes in the relative abundance of 188 proteins were

detected. These included pathogen-binding proteins, such as the complement component

C3 and scavenger receptor cysteine rich proteins, as well as proteins responsible for

cytoskeletal remodeling, endocytosis and intracellular signaling. An initial systemic

reaction to wounding was followed by a more specific response to immunological

challenge involving proteins such as apolipophorin, dual oxidase, fibrocystin L,

aminopeptidase N, and α-2-macroglobulin.

152

4.3. Introduction

The vertebrate immune system comprises both innate and immunoglobulin-based

adaptive components. Both systems work cooperatively to recognize and kill pathogens.

Invertebrates do not express immunoglobulin antibodies or TCR, but their innate immune

systems resemble that of vertebrates with similar effectors, receptors and regulation of

gene expression. Many molecules that were first identified in invertebrates, such as

Drosophila melanogaster Toll-like receptors, have homologues in humans that are now

recognized as critical components of innate immunity [1, 2].

Sea urchins are deuterostome invertebrates and as such, they are often more similar

to vertebrates than to other invertebrates [3]. Their phylogenetic position explains why

they have been extensively used as model organisms in studies of fertilization and

developmental biology. It was also the main motivation for sequencing the genome of the

purple sea urchin, Strongylocentrotus purpuratus [4]. Sea urchins possess a broad array of

immune response genes, including components of the alternative and lectin-mediated

complement pathways, a diverse array of Toll-like receptors, NOD/NALP-like cytoplasmic

recognition proteins, and a new family of highly variable immune response proteins (the

185/333 family) [5-7]. Homologues of the vertebrate recombination activating genes, Rag

1 and Rag 2, are also present in the genome but their function in sea urchins is unknown

[8].

Despite the extensive data at the genome level, there is relatively little information

available on the involvement of many of these newly discovered molecules in the inducible

immune responses of sea urchins. Coelomocytes, which are wandering amoeboid cells

within the coelomic cavity, are the primary mediators of immunological activity in sea

urchins. They are responsible for phagocytosis and encapsulation of foreign particles, as

well as other physiological activities, such as gas exchange and transport [9]. The current

153

study uses shotgun proteomics to identify proteins involved in inducible responses to the

bacterial antigen lipopolysaccharide (LPS), as well as reactions to sterile saline injection,

in coelomocytes of sea urchins. We employed an SDS-PAGE gel slice fractionation

approach as a initial step in the shotgun proteomic analysis of coelomic fluid. Peptides, and

their corresponding proteins, were then identified using tandem mass spectrometry.

Proteins were quantified by spectral counting and normalized spectral abundance factors

(NSAFs) [10]. Using this approach, we identified significant changes in the levels of 188

coelomocyte proteins over time, of which 40 differed in relative abundance between sea

urchins injected with LPS as opposed to saline.

154

Figure 4.1: Experimental design: Twenty-one sea urchin CF samples were collected over

48 hours. 3 samples were collected from unchallenged sea urchins, one each at 6, 24 and

48 hours after the beginning of the experiment. 9 samples were collected after saline

injection: 3 after 6 hours, 3 after 24 hours and 3 after 48 hours. 9 more CF samples were

collected after the injection of LPS: 3 after 6 hours, 3 after 24 hours and 3 after 48 hours.

3 sea urchins

3 sea urchins

Saline injection

LPS injection

48 h 24 h 6 h Non injected

3 sea urchins

3 sea urchins

3 sea urchins

3 sea urchins

3 sea urchins

155

4.4. Experimental procedures

4.4.1. Sea urchins

Sea urchins (H. erythrogramma) were collected from Camp Cove in Sydney

Harbour, Australia. They were housed in a recirculating seawater facility at Macquarie

University as previously described [5, 11]. The sea urchins were left undisturbed in the

aquaria for 2 months prior to experimentation to allow them to acclimatize to aquarium

conditions [11, 12].

4.4.2. Injections and sample collections

Animals were subjected to immunological challenge by inserting 23-gauge needles

attached to 1 mL syringes through the peristomium into the coelomic cavity. Nine animals

were injected with 30 µg of LPS in 1 mL of sterile artificial coelomic fluid (aCF; 10 mM

CaCl2, 14 mM KCl, 50 mM MgCl2, 398 mM NaCl, 1.7 mM Na2SO4, 0.22 µm filtration)

[13]. This corresponds to approximately 2 µg of LPS per mL of coelomic fluid (CF) as

previously described [12, 14]. A further 9 animals were injected with an equivalent volume

of aCF without LPS (saline treatment), and three animals were left undisturbed (non-

injected controls).

Three sea urchins from each treatment (saline or LPS injections), and one animal

from the non-injected control group, were sampled 6, 24 or 48 hours after injection (Figure

4.1). Coelomic fluid was withdrawn by dissecting the Aristotle’s lantern and removing CF

directly from the coelomic cavity.

156

The CF was harvested into an equal volume of calcium-magnesium-free seawater with

EDTA and imidazole (CMFSW-EI; 10 mM KCl, 7 mM Na2SO4, 2.4 mM NaHCO3, 460

mM NaCl, 70 mM EDTA and 50 mM imidazole; pH 7.4) on ice to avoid clotting and

French pressed at 800 Pa to lyse the coelomocytes. The lysates were stored frozen at -80oC

degrees and were lyophilized overnight under vacuum immediately before protein

extraction.

4.4.3. Protein extraction

Lyophilized samples were resuspended in 3 mL of sodium dodecyl sulfate

polyacrylamide gel (SDS-PAGE) sample buffer (10% glycerol, 2% SDS, 0.1 M Tris-HCl,

pH 8.0 and 1% DTT). They were then vortexed for 10 minutes before 3 mL of phenol was

added. The samples were further vortexed for 1 minute and centrifuged at 11,000 × g for 5

minutes at -4 oC. Five volumes of ice-cold methanol containing 0.1 M ammonium acetate

were added to the phenol extracts. The tubes were then held at -20 oC for 1 hour to

precipitate the proteins. The samples were centrifuged for 5 minutes at 14,000 × g at -4 oC

and the supernatant removed. The protein pellets were then washed twice with ice-cold

methanol containing 0.1 M ammonium acetate and twice with 80% acetone. For each

wash, samples were centrifuged for 5 minutes at 14,000 × g at -4 oC and the protein pellets

were re-suspended by vortexing for 30 seconds. After the final wash, pellets were left to air

dry before being solubilized in SDS-PAGE sample buffer. The total protein content of

each sample was determined using Bradford reagent (BioRad).

157

4.4.4. One-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis (1DE

SDS-PAGE)

Coelomic fluid proteins (10 µg per well) were separated on 7.5% Bis-Tris

polyacrylamide gels at 180 V for 1 hour [15]. After electrophoresis, proteins were

visualized by blue silver staining as described previously [16]. After staining, the gels were

washed twice in sterile deionised water (10 minutes per wash), before individual lanes

were cut into 16 slices of equal sizes from top to bottom. Proteins in each slice were

reduced, alkylated and subjected to trypsin digestion as previously described [17].

4.4.5. Nanoflow liquid chromatography – tandem mass spectrometry

The tryptic digest extracts from 1DE gel slices were analyzed by nanoLC-MS/MS

using a LTQ-XL ion-trap mass spectrometer (Thermo, CA, USA) essentially according to

Breci et al. [18]. Reversed phase columns were packed in-house to approximately 7 cm

(100 mm i.d.) using 100 Å, 5 mM Zorbax C18 resin (Agilent Technologies, CA, USA) in a

fused silica capillary column with an integrated electrospray tip. A 1.8 kV electrospray

voltage was applied via a liquid junction up-stream of the C18 column. Samples (in buffer

A: 5% (v/v) ACN, 0.1% (v/v) formic acid) were injected onto the C18 column using a

Surveyor autosampler (Thermo, CA, USA). Each sample was loaded onto the C18 column

followed by an initial wash step with buffer A (5% (v/v) ACN, 0.1% (v/v) formic acid) for

10 minutes at 1 µL min-1. Peptides were subsequently eluted from the C18 column on a

gradient of 0-50% Buffer B (95% (v/v) ACN, 0.1% (v/v) formic acid) over 58 minutes at

500 nL min-1 followed by a gradient of 50-95% Buffer B over 5 minutes at 500 nL min-1.

The column eluate was directed into the nanospray ionization source of the mass

spectrometer. Spectra were scanned over the range 400–1500 amu. Automated peak

158

recognition, dynamic exclusion and tandem MS of the top six most intense precursor ions

at 35% normalization collision energy were performed using the Xcalibur software

(version 2.06) (Thermo, CA, USA).

4.4.6. Protein identification

Raw MS spectra files were converted to mzXML format and analyzed using Global

Proteome Machine (GPM) software (version 2.1.1; X!Tandem algorithm,

www.thegpm.org [19, 20]. For each experiment, the 16 SDS-PAGE fractions were

processed sequentially generating output files for each individual fraction, as well as a

merged, non-redundant output file. The merged files were used for protein identification.

MS/MS spectra were searched against a combined S. purpuratus database created with

downloaded sequences from NCBI. This FASTA format database contained 44,037 protein

sequences, which comprise all S. purpuratus predicted protein sequences and identified

proteins held by NCBI as of April 2008. Search parameters included MS and MS/MS

tolerances of ± 2 Da and ± 0.2 Da, tolerance of up to 3 missed tryptic cleavages and K/R-P

cleavages. Fixed modifications were set for carbamidomethylation of cysteine and variable

modifications were set for oxidation of methionine.

4.4.7. Data analysis

To be positively identified, proteins had to; (1) match a known sequence in the S.

purpuratus database with log (e) values of less than -1; (2) be represented by at least 10

spectra from amongst all samples, with a minimum of 2 unique peptides obtained in at

least 2 different individuals, and; (3) be represented by spectral counts from all three sea

urchins in a particular treatment group (LPS- or saline-injected) at the same time point.

159

Searches were also performed against a reversed sequence database to evaluate the false

discovery rate (FDR) [21, 22]. No protein matches were detected in the reversed database

searches, indicating a protein identification confidence level of greater than 99% [23]. A

total of 345 proteins fitted these selection criteria and were subjected to further analysis.

The normalized spectral abundance factors (NSAF) of these 345 proteins were calculated

as previously described [24] and a matrix of protein abundance was generated, where each

row corresponds to a different protein and each column corresponds to one sea urchin

(Supplementary Data 4.1).

To obtain a normal distribution of the data, we applied natural log transformations to

all NSAF values [24]. The 345 proteins were considered as 345 variables, using the log

(NSAF) data set. These data were further centered and scaled to have unit standard

deviations for both rows and columns. The 21 sea urchin CF proteomes were then

subjected to principal component analysis (PCA) using the XLstats 2009 package. The

proportion of variance for each principal component and the cumulative variance were

obtained. The three components with the highest proportion of variance were used to draw

a 3D scater plot organizing the 21 sea urchin proteomes along the principal components.

PCA confirmed that the three non-injected sea urchins proteomes could be used as a

common reference point for further analysis (see Results). So, NSAF values (NSAFt) were

normalized by dividing them by the average NSAF value of the non-injected samples

(NSAFc). A new normalized and centered dataset corresponding to the log2 of the NSAFt

divided by the NSAFc was created (log2 (NSAFt/NSAFc). The log2 (NSAFt/NSAFc) data

for individual proteins were subjected to two factor ANOVA (Factor 1 - Time: 0, 6, 24 and

48 hours; Factor 2 - Challenge: saline and LPS injection) with Tuckey’s Post Hoc test to

identify individual proteins that varied in concentration over time and/or in response to the

injection of LPS or saline solution. Differences in these analyses were considered to be

significant if p<0.05.

160

Figure 4.2: Alteration in whole proteomes in response to injections. A/ Scree plot

showing the proportion of variance for each principal component (PC; histogram) and the

cumulative proportion of variances (curve) explained by consecutive PCs. B/ 3D score plot

using the first 3 PCs shown in A. The Figure shows cumulative data for all of the proteins

identified in the CF of sea urchins; non-injected (NI), 6 hours after saline injection (W6),

24 hours after saline injection (W24), 48 hours after saline injection (W48), 6 hours after

LPS injection (LPS6), 24 hours after LPS injection (LPS24) and 48 hours after LPS

injection (LPS48).

A

0

20

40

60

80

100

0

1

2

3

4

5

6

7

8

PC

1

PC

2

PC

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PC

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PC

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PC

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PC

7

PC

8

PC

9

PC

10

PC

11

PC

12

PC

13

PC

14

PC

15

PC

16

PC

17

PC

18

PC

19

PC

20

PC

21

Cu

mu

lative

va

ria

bili

ty (

%)

Eig

en

va

lue

B PC1

PC2

PC3

NI

W6

W24

W48

LPS6

LPS24

LPS48

161

4.5. Results

4.5.1. Reproducibility and threshold levels of shotgun MS/MS analysis

Three hundred and forty five distinct coelomocyte proteins were identified by

shotgun proteomics. The false discovery rate was <1 %, suggesting that the identities of

these proteins were robust. One of the most decisive steps in protein profiling is the

reproducibility of the data among the biological replicates. Hence, coelomic fluid samples

from three individual sea urchins per treatment were collected 6, 24, and 48 hours after

saline and LPS injection. An additional three sea urchins were left uninjected and the CF

from one of these sea urchins was collected at each time point (giving a total of 21 CF

samples).

To confirm that the variations observed between samples were due to the

experimental treatments rather than chance, we undertook PCA of the 21 CF proteomes. A

first step was to determine the number of principal components (PC) to extract. A Scree

plot was created to determine if a clear breaking point was observed in variances (Figure

4.2). The Scree Plot has two forms of information: the histogram shows the proportion of

variance for each PC, while the upper line shows the cumulative variance explained by the

21 components. The PCs were sorted in decreasing order of variance, so that the most

representative PCs were listed first. In this dataset, the first three PCs explain much more

of the variance in the data (roughly 32%, 9% and 7% respectively) than do all of the

subsequent PCs, leading us to further investigate these first 3 PCs. In total, 48.7% of the

total variance was explained by these first three PCs.

Figure 4.2 shows the 3D score plot obtained using the first three PCs. The different

sea urchin proteomes were explained by different proportions of the PCs, indicating that

fundamental differences existed between samples. In this plot, sea urchin samples with

162

similar protein abundance profiles appear closer in space, whereas significantly different

samples appear more distant from each other. The three non-injected samples clustered

together with low values for PC1 and PC3, and high values for PC2. This confirmed that

the differences observed between these sea urchins and those injected with LPS or saline

were due to the experimental conditions to which the animals were subjected, and that non-

injected sea urchin samples could be used as a T=0 hour reference point. Samples collected

at 6 and 24 hours post injection (p.i.) from both treatments (LPS and saline injection)

yielded high PC1 but low PC2 and PC3 values. This suggests that a significant

modification of the total CF proteome occurs within the first 6 hours after injection.

Samples collected after 6 and 24 hours p.i. tended to cluster together regardless of

collection time or challenge conditions, indicating that significant differences did not exist

between treatments (saline vs bacteria at these time points; Figure 4.2B). However,

samples collected 48 hours p.i. formed distinct groups, with intermediate values for PC1

and PC3 but low PC2 values. Among these, samples from LPS-injected sea urchins had

lower PC1 and PC3 values than those from saline-injected animals. The significant

distinction between samples collected 48 hours after LPS and saline injections indicated

that most of the alterations in protein concentration specifically induced by LPS occurred

at 48 hours p.i.. The relative decrease along the PC1 axis of the 48 hours p.i. samples

suggests that the relative abundance of most proteins from both saline- or LPS-injected

animals began return to pre-injection levels within 48 hours, even though the 48 hours p.i.

samples from LPS- and saline-injected sea urchins remained clearly different from each

other, and from non-injected controls.

163

4.5.2. Functional classification of proteins

The 345 proteins identified in this study were grouped into 9 functional categories

based on the annotation for the corresponding genes in the S. purpuratus genome. Ten

proteins could not be assigned to any of these categories and so were grouped as ‘others’.

Hypothetical proteins in the S. purpuratus genome without a known putative function

accounted for 52 of the 345 proteins identified. To understand the potential activities of

these proteins, we undertook protein Blast searches and conserved domain searches against

the entire NCBI database. The results of these searches showed strong matches to

homologous proteins from other species in all but 6 cases. The remaining 6 proteins did

not exhibit significant similarities to proteins with predicted functions, and so were

designated as ‘unknown’ in terms of protein ontology (Figure 4.3).

Proteomes of CF samples from non-injected sea urchins were dominated by

molecules involved in cell structure shape and mobility, as well as cell signalling (Figure

4.3A). Actin, tubulin and myosin were abundant in all of these samples, followed closely

by SUArp 2 and 3 proteins, a protein similar to F actin capping proteins, alpha actinin and

gelsolin. Molecules involved in cell structure shape and mobility made up 16% of all

proteins identified (Figure 4.3A), and between 56% (non-injected) to 86% (6 hours post-

saline injection) of the total protein content of coelomocytes (NSAFs values) (Figure 4.3B-

D). Proteins involved in cell signalling were also numerous (13% of all proteins

identified), but they represented only 1.3% (24 hours post LPS injection) to 7.3% (non-

injected) of the relative CF protein content (Figure 4.3B-D). Stress response and

detoxification proteins, as well as those involved in nucleic acid/protein metabolism

represented 8% and 24% of the total number of proteins identified. Higher relative

abundance values (NSAFs) for these proteins were evident in non-injected sea urchins,

where they respectively comprised 4.8% and 10.6% of the CF protein content.

164

Number of proteins

2% 2% 3%

4%

8%

8%

12%

8%

13%

16%

24%

A

B

C

D

165

Figure 4.3: Relative abundance and functional classification of proteins. These Figures

show the functional categories of proteins identified in the CF of non-injected sea urchins

(NI), 6 hours after saline injection (W6), 24 hours after saline injection (W24), 48 hours

after saline injection (W48), 6 hours after LPS injection (LPS6), 24 hours after LPS

injection (LPS24) and 48 hours after LPS injection (LPS48). A/ Pie chart showing the

functional classification of the 345 proteins identified in all of the sea urchin CFs analysed.

Data are percentages calculated by dividing the number of proteins in a category by the

total number of proteins identified. B,C,D/ show the relative abundance of the proteins of

each functional category proportional to the sum of the NSAF values of all proteins

identified (% NSAFs).

166

Immune response proteins comprised 8% of all proteins identified and represented between

1.7% (6 hours post saline injection) and 7.6% (non-injected) of the protein content, while

those involved in energy metabolism made up to 12% of the proteins identified and

comprised between 2.7% (24 hours post LPS injection) to 7.2% (non-injected) of the

relative CF protein content (Figure 4.3B-D).

In CF samples from LPS-injected and saline-injected sea urchins, significant changes

in the relative abundance of proteins involved in the different functional categories were

observed during the course of the experiment (ANOVA p<0.01; Figure 4.3). The

abundance of proteins putatively involved in cell structure, shape and mobility increased at

6 hours after injection, comprising over 80 % of the total protein content, and decreased

over the following 42 hours to represent 60%-70% of the total protein content at 48 hours

p.i. (Figure 4.3B). Molecules involved in stress responses decreased in relative abundance

from 4.8% of the proteome at T=0 hours (non-injected sea urchins) to ~2% at 6 hours p.i.

(1.9% 6 hours after saline and 2.1% 6 hours after LPS), increasing again after 24 hours p.i.

(4% after saline and 6% after LPS) (Figure 4.3D). Proteins in all other functional

categories decreased in relative abundance over the period T=0 to T=24, and then

increased in relative concentration at 48 hours p.i. (Figure 4.3C,D). These results suggest

that the major shift in the proteome along the PC1 and PC2 axes at 6 hours p.i. was due to

a significant increase in proteins involved in cell structure, shape and mobility, resulting in

a decrease in the relative concentrations of most other proteins. Protein levels in all

categories began to return to pre-injection levels at 48 hours after challenge, confirming

that proteomes collected at 48 hours p.i. were more similar to non-injected sea urchins than

those collected at 6 or 24 hours p.i..

167

4.5.3. Identification of individual proteins affected by saline or LPS injection

Significant differences in the levels of individual proteins between LPS-and saline-

injected sea urchins during the course of the experiment were detected using two-way

ANOVA and Tuckey’s Post Hoc test on the log2 (NSAFt/NSAFc) values. The null

hypotheses tested were that there were no differences between LPS- and saline-injected sea

urchins (factor 1: challenge), and that there were no differences over time (factor 2: time).

These tests showed that the relative abundances of 188 proteins were significantly altered

by LPS and saline injections (p<0.05) (Table 4.1). Many proteins were significantly altered

for both main effects, “challenge” and “time”. Hence, ANOVA results are presented in

three categories; “time significant”, “challenge significant” and “time and challenge

significant”. Two proteins differed significantly between “challenges” only, 135 differed

significantly over time but not between challenges, and 40 differed significantly over time

and between challenges (Table 4.1).

Overall, changes in the relative abundance of individual proteins were consistent

among the three sea urchins tested at each time point for each treatment (Figure 4.4). As a

result, the sum of NSAF (NSAFs) values from each set of three individuals (same

treatment, same time point) were representative of the data and could be used to simplify

visual representation of the data (Figures 4.4, 4.5 and 4.6).

168

Figure 4.4: Consistency of the relative abundance data. The NSAF values for major

yolk protein (A) in each individual sea urchin from each treatment and (B) summed NSAF

values (NSAFs) for the three sea urchins in each treatment at each time point (LPS

injection, LPS; saline injection, W; non-injected, NI).

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169

Table 4.1: Individual proteins with significantly altered relative abundance (ANOVA

p<0.01) over time and/or between challenges (LPS- or saline-injected). Proteins that

differed significantly between challenges but not over time are classified as “challenge

significant”. Proteins that differed significantly over time but not between treatments are

classified as “time significant”. Proteins that differed significantly both over time and

between the two challenges are classified as “time and challenge significant”.

Identifier Description gi|115618052| similar to Actin, cytoskeletal IIIB, partial Challenge

Significant gi|115971469| similar to Actin, cytoplasmic gi|115945106| similar to cytosolic nonspecific dipeptidase, partial gi|115956413| similar to glutamine synthetase . gi|115976312| similar to CG8649-PC (calponin) gi|115974673| similar to LOC407663 protein (short chain dehydrogenase) gi|115753213| similar to beta COP, partial gi|115928607| hypothetical protein (flotillin) gi|115803435| hypothetical protein (similar to cleavage stimulation factor fusion) gi|115891186| hypothetical protein, partial (kynurenine aminotransferase) gi|115671024| similar to KIAA0051, partial (IQ motif containing GTPase activating protein) gi|115934826| similar to Fructose-1,6-bisphosphatase 1 (D-fructose-1,6-bisphosphate 1-phosphohydrolase 1) (FBPase 1) gi|115944296| hypothetical protein (Galectin) gi|115929526| similar to aminopeptidase N . gi|115955254| similar to alpha macroglobulin . gi|160623368| putative flotillin* gi|115968722| similar to DEAD-box RNA-dependent helicase p68 isoform 2 gi|115931135| similar to putative RNA binding protein KOC gi|115770276| similar to zonadhesin gi|115952079| hypothetical protein, partial (vacuolar protein sorting 35) gi|115924997| similar to MGC84288 protein (tissue inhibitor of metalloproteinase TIMP) gi|115950705| similar to Ribosomal protein S5 gi|115894557| similar to RACK gi|115924764| similar to formin binding protein 1 gi|115968476| similar to ENSANGP00000018350 (centaurin) gi|115940610| hypothetical protein, partial (Ribosomal protein) gi|47551023| complement component C3 gi|115926118| similar to CG6950-PC (kynurenine aminotransferase) gi|115934021| similar to Psmc6 protein (proteasome 26S subunit, ATPase) gi|115974704| similar to scavenger receptor cysteine-rich protein type 12 precursor, partial gi|115944055| similar to fibrocystin L gi|115944169| similar to dual oxidase 1 isoform 1 . gi|115933977| similar to histone H2A-3 gi|47551261| MAP kinase gi|115957127| similar to ribosomal protein L5 gi|115951428| similar to LOC615074 protein isoform 2 cAMP-dependent protein kinase) gi|115944978| similar to COP9 complex subunit 4 gi|115963308| similar to ribosomal protein L32 gi|115944173| similar to ENSANGP00000006016 isoform 1 (dual oxidase maturation factor) gi|47551251| heat shock protein gp96 gi|115940166| similar to apolipoprotein B

Time and Challenge Significant

gi|47551303| guanine nucleotide-binding protein G(q) alpha subunit gi|115953630| hypothetical protein (N-acetylneuraminic acid synthase) gi|224306| actin gi|115939823| similar to catalytic subunit of cAMP-dependent histone kinase gi|115977049| hypothetical protein (chaperonin containing TCP1) gi|115972622| similar to GA19181-PA, partial (NAD dependent epimerase/dehydratase) gi|115970015| similar to von Willebrand factor gi|115937346| similar to apolipophorin precursor protein

Time Significant

gi|115946171| similar to beta-arrestin 1, putative

170

Identifier Description gi|115975006| similar to RAB2 isoform 2 gi|115974054| similar to fibropellin Ia gi|115970592| similar to tropomyosin 1 gi|115967840| similar to 0910001A06Rik protein isoform 2 gi|115931665| similar to MGC89020 protein (mitochondrial aldehyde dehydrogenase 2) gi|115972829| similar to glucose-6-phosphate isomerase . gi|115956030| similar to chaperonin gi|115970013| similar to apolipoprotein B gi|47551123| major yolk protein gi|47551085| late histone L3 H2a gi|115697859| hypothetical protein (succinyl CoA ligase) gi|115955282| hypothetical protein (heterogenous nuclear ribonucleoprotein K) gi|115931510| hypothetical protein (sorting nexin) gi|115691123| similar to Eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa isoform 1 gi|115970523| similar to elongation factor 1 alpha isoform 2 gi|115974451| similar to GTPase SUrab10p gi|115954217| similar to hexokinase I gi|115945280| hypothetical protein, partial (ADP ribosylation factor) gi|115774726| hypothetical protein, partial (phosphatidylinositol-5-phosphate 4-kinase) gi|47825404| guanine nucleotide-binding protein G(12) alpha subunit . gi|115959822| similar to annexin gi|47551041| ER calcistorin gi|115956476| similar to apextrin gi|115943916| similar to actin gi|115956824| similar to Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 gi|73746392| major yolk protein gi|47551049| fascin gi|115974713| hypothetical protein (dysferlin C2 superfamily) gi|115968818| similar to ENSANGP00000003616 (Actin like protein) gi|115931821| hypothetical protein (fibronectin) gi|115965680| hypothetical protein (Heterogeneous nuclear ribonucleoprotein L) gi|115968684| similar to retinoblastoma binding protein 4 variant isoform 1 gi|115950487| similar to arylsulfatase isoform 2 . gi|115954976| similar to heat shock protein protein gi|115928686| similar to alpha isoform of regulatory subunit A, protein phosphatase 2, partial gi|115936805| similar to heterotrimeric guanine nucleotide-binding protein beta subunit isoform 2 . gi|47825400| guanine nucleotide-binding protein G(i) alpha subunit . gi|47825398| guanine nucleotide-binding protein G(s) alpha subunit . gi|115939698| similar to 26S proteasome subunit p44.5 gi|115944135| similar to sea urchin Arp2 (SUArp2) . gi|115975046| similar to GTP-binding nuclear protein Ran gi|115966189| similar to H(+)-transporting ATPase beta subunit gi|115925449| similar to Fructose-1,6-bisphosphatase 1, like gi|115972586| similar to 71 Kd heat shock cognate protein gi|115963192| similar to Annexin A4 (Annexin IV) (Lipocortin IV) (Endonexin I) (Chromobindin-4) (Protein II) (P32.5) gi|115966293| similar to RAS-related protein MEL . gi|115921420| hypothetical protein, partial (phosphoglycerate dehydrogenase) gi|115960968| similar to sea urchin Arp3 (SUArp3) gi|115891388| similar to heat shock 90 kDa protein, partial gi|115940258| hypothetical protein (lipid raft asssocitaed protein 2) gi|115961140| similar to gelsolin gi|115972933| similar to B-cell receptor associated protein gi|115968951| hypothetical protein (adaptor protein complex AP2) gi|115971224| hypothetical protein (aminopeptidase) gi|115937294| similar to beta-parvin gi|47551035| cytoskeletal actin CyIIIb gi|115955418| similar to 5-aminoimidazole ribonucleotide (AIR) (SAICAR) synthetase isoform 1 gi|115939485| similar to malate dehydrogenase . gi|115652043| similar to histidyl-tRNA synthetase, partial gi|115926010| similar to glutathione peroxidase, partial gi|115959476| hypothetical protein, partial (translation initiation factor eIF-2B) gi|115964543| similar to transaldolase gi|115963185| hypothetical protein isoform 2 (ribosomal protein L15) gi|115965220| similar to histone macroH2A1.1 gi|115960271| similar to protein Phosphatase 2C beta

gi|115960970| similar to proliferation-associated protein 1

171

Identifier Description gi|115958373| similar to Rab5 protein gi|115925359| similar to LZP (CCP and zona pellucida superfamily) gi|115970724| similar to Valosin containing protein, partial gi|115932417| similar to ribosomal protein S10 isoform 2 gi|115746860| similar to actin, partial gi|115929978| similar to OTTHUMP00000039401 (similar to leucine rich repeat containing 16 isoform) gi|115959301| similar to Protein phosphatase 2A, catalytic subunit, beta isoform gi|115968753| similar to scavenger receptor cysteine-rich protein type 12 precursor gi|115939093| hypothetical protein (septin 9 fusion) gi|115910910| hypothetical protein, partial (Fibronectin) gi|115961398| similar to Capzb-prov protein (F actin capping protein beta) gi|115963096| similar to CG15792-PD, partial (myosin) gi|115956638| similar to cytoskeletal actin CyIIb . gi|115744264| similar to Isocitrate dehydrogenase 2 (NADP+), mitochondrial . gi|47551147| N-ethylmaleimide-sensitive factor (NSF) gi|115973107| hypothetical protein, partial (NADH cytochrome reductase) gi|115974434| similar to adenosylhomocysteinase gi|115710920| similar to Na+/K+ ATPase alpha subunit gi|115942229| similar to integrin-linked kinase gi|115940188| similar to peptidylprolyl isomerase B isoform 2 . gi|115963700| similar to Ribosomal protein L14, partial gi|115911567| hypothetical protein, partial (aspartyl tRNA synthase) gi|115968341| similar to Ribosomal protein L3 gi|115929247| similar to heterogeneous nuclear ribonucleoprotein H . gi|115945705| similar to EH-domain-containing protein 1 (Testilin) (hPAST1) gi|115738335| similar to beta-adaptin Drosophila 1 isoform 7 gi|115943043| similar to VPS13C-1A protein (vacuolar protein sorting 13 homolog C) gi|115930179| similar to gelsolin gi|115940857| similar to aldose reductase . gi|115934007| similar to TRAF4-associated factor 2 . gi|115922509| similar to glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) isoform 2 gi|47550983| nuclear intermediate filament protein gi|115929203| similar to ENSANGP00000011796 (alpha actinin) gi|115970125| similar to MGC139263 protein (Annexin) gi|115729152| similar to triosephosphate isomerase gi|115940910| hypothetical protein (septin7) gi|115630732| similar to myosin, heavy polypeptide 10, non-muscle gi|115924280| similar to flavoprotein subunit of complex II, partial gi|115960644| similar to myosin heavy chain gi|115968074| hypothetical protein (aldehyde dehydrogenase) gi|47551307| syntaxin binding protein 1 gi|115944458| similar to calponin gi|115968724| similar to transglutaminase-like protein . gi|115905691| similar to Pkm2 protein, partial (Pyruvate kinase isoenzyme type M2) gi|115965257| similar to heterogeneous nuclear ribonucleoprotein R gi|115968696| similar to paraspeckle protein 1 isoform beta gi|115759362| similar to sulfotransferase ST1B2 . gi|47551157| scavenger receptor cysteine-rich protein type 12 . gi|115944450| similar to chaperonin subunit 8 theta gi|115973804| similar to non-receptor protein tyrosine phosphatase gi|115964994| similar to Fumarylacetoacetase (Fumarylacetoacetate hydrolase) (Beta-diketonase) (FAA), partial gi|115954867| similar to Chaperonin containing TCP1, subunit 4 (delta) gi|115673346| similar to alpha-cop protein, partial gi|115720465| similar to selectin-like protein, partial . gi|115950170| similar to long microtubule-associated protein 1A; long MAP1A gi|115971193| similar to Rac1 protein gi|115955810| hypothetical protein (ribosomal protein L4) gi|115941920| similar to ribosomal protein L7, partial gi|115972878| similar to Rps9-prov protein (ribosomal protein S9) gi|5532389| microtubule-associated protein gi|115951468| similar to coatomer protein gamma 2-subunit, partial gi|115945943| similar to ENSANGP00000011972 (GDP dissociation inhibitor) gi|115628087| similar to chaperonin containing TCP1, subunit 6A isoform 1 gi|115978426| hypothetical protein (dihydrolipoyllysin S succinyltransferase) gi|115975235| similar to signal transducer and activator of transcription 5B

gi|47550939| calreticulin

172

Identifier Description gi|115951109| similar to tensilin gi|166795321| advillin gi|115956784| similar to epsin 2 gi|115951499| hypothetical protein (septin2) gi|115940967| similar to Dynamin 2, partial

gi|115939472| similar to ENSANGP00000023777 (CDC42, cell division cycle 42)

The identity of hypothetical proteins was deduced from Blast and conserved domain

searches and is shown in brackets and Italics.

* Molecules previously characterized at the protein level are shown in bold.

173

The proteins that varied over time but not between saline- and LPS-injected sea

urchins were from all functional categories. Most of these decreased in relative abundance

at 6 and/or 24 hours after injection and returned to relative concentrations close to those of

non-injected sea urchins after 48 hours. Proteins following this pattern included myosin,

fibronectin, calreticulin, GDP dissociation inhibitor, RabGTPases, ER calcistorin and

proteins involved in glycolysis. Exceptions to this overall pattern included proteins similar

to actin (Figure 4.5), fascin (Figure 4.5), cdc42, Rac1, leucyl aminopeptidase, heat shock

protein and a nuclear intermediate filament protein. These proteins increased in relative

abundance at 6 hours after both injections. This agrees with the previous observation that

the proportional representation of proteins involved in cell structure, shape and mobility

increased at this time point. The relative increase described previously among stress

response proteins at 24 hours after injection can be explained by changes in a single

molecule in this functional category, major yolk protein (Figure 4.4). We also identified

some proteins after challenge that were not detected in non-injected samples. These

included cytoskeletal actins CyIIIb and CyIIb (Figure 4.5), a flavoprotein, an annexin A7

and a triosephosphate isomerase. Conversely, some other proteins identified in non-

injected samples were absent among LPS-injected and saline-injected sea urchins. These

included advillin, F actin capping protein, alpha actinin, tensilin, dysferlin C2, beta parvin

and apextrin.

Most of the proteins that differed significantly both over time and between

challenges increased in relative concentration in response to the injection of LPS but not

saline. The relative abundance of many of these proteins was modified 48 hours after

injection, which agrees with the previous observation that differences between LPS- and

saline-injected sea urchin proteomes were most prominent at this time point.

174

Figure 4.5: Examples of proteins that differed in relative abundance over time but not

between treatments. The sum of the NSAF values (NSAFs) for each triplicate of sea

urchins in a particular treatment/time point is shown for the cytoskeletal actins CyIIb and

CyIIIb (A), fascin (B), proteins similar to apolipoprotein B and apolipophorin precursor

(C), and for proteins similar to septins (D) (LPS injection, LPS; saline injection, W; non-

injected, NI).

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175

The LPS-specific proteins primarily fell into three categories; proteins involved in

vesicular trafficking, including a beta subunit of the coatomer protein complex, a receptor

for activated C-kinase and a vacuolar protein sorting 35; immune response proteins,

including dual oxidase 1 and dual oxidase maturation factor, an α-2-macroglobulin, a

complement component C3, a fibrocystin L, and three scavenger receptor cysteine rich

(SRCR) proteins; and proteins with suspected accessory roles in immune response, such as

two kinurenine aminotransferases and an aminopeptidase N. Other proteins that were

relatively more abundant after challenge with LPS included a guanine nucleotide binding

protein G(q) alpha subunit, a MAP kinase, an apolipoprotein B and a tissue inhibitor of

metalloproteinase (TIMP).

For some of the proteins that differed between LPS- and saline-injected animals,

differences were “all or none”, meaning that the protein was not detected in one treatment

but was relatively abundant in the other treatment. For example, fibrocystin L (Figure 4.6)

was absent directly after saline injection, but was abundant 48 hours after LPS injection. In

addition to these on/off differences, there were also proteins that were present in all

treatments, but differed substantially in relative abundance between treatments. For

instance, complement component C3 (Figure 4.6) was 3.3 fold more abundant 48 hours

post-LPS injection than 48 hours after saline injection.

176

Figure 4.6: Examples of proteins that differed in relative abundance both over time

and between treatments. The sum of the NSAF values (NSAFs) for each triplicate of sea

urchins in a particular treatment/time point is shown for a protein similar to dual oxidase

maturation factor (A), a protein similar to dual oxidase 1, isoform 1 (B), a protein similar

to aminopeptidase N (C), a protein similar to fibrocystin L (D), a protein similar to alpha 2

macroglobulin (E), and complement component C3 (F). (LPS injection, LPS; saline

injection, W).

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177

4.6. Discussion

This is the first differential proteomic analysis of sea urchin responses to

immunological challenge, paving the way for further study of the individual proteins and

protein networks affected during this process. Previous transcriptomic approaches [5, 25]

have identified differentially regulated genes. These studies measured gene expression at a

single time point after challenge. However, proteomic studies of Drosophila hemocytes

showed that immune responses are dynamic processes and that the proteome varies at

different time points after challenge [2]. In order to provide a dynamic image of the

immune response in sea urchins, we used a strategy that allowed us to monitor hundreds of

proteins over several time points.

Principal component analysis suggested that CF responses to saline and LPS

injection are similar at 6 and 24 hours after challenges, and that differences specifically

due to the presence of LPS occurred mostly 48 hours after injection. This result was

confirmed by analyzing the differential abundance of individual proteins. The

concentration of 135 proteins was significantly altered over time in both the saline and

LPS treatments. Such responses probably reflect the effects of injection, such as the

puncture wound caused by the needle and the increase in CF volume caused by the

introduction of aCF (with or without LPS). A further 40 proteins differed significantly

between saline- and LPS-injected sea urchins, suggesting that these molecules participate

in a discrete LPS-induced response.

Changes in the concentration of the 135 proteins that differed in both saline and LPS-

injected animals implies that there were 3 distinct components to wounding responses:

initial changes in coelomocyte morphology and plasticity, followed by systemic responses

primarily involving the major yolk protein, and finally the induction of wound repair

mechanisms.

178

The initial (T=6 hours) changes observed in the CF proteome primarily involve

alterations in major cytoskeletal proteins. This might reflect an increased capacity for

coelomocyte agglutination at the wound site, the restructuring of actin filaments allowing

the development of filopodia and the recruitment of coelomocytes to the site of injury. We

identified a significant increase in the levels of actin isoforms at T=6 hours suggesting

significant changes in cell shape and amoeboid activity (Figure 4.5). One of these

isoforms, actin CyIIIb, was abundant only at 6 hours p.i. (Figure 4.5). Other proteins that

were abundant during this initial phase included fascin (Figure 4.5), a protein with

similarities to lipid raft associated protein 2, Rac 1 and cdc42. In mouse fibroblasts, cdc 42

activates Rac and together they control the assembly and organization of the actin

cytoskeleton, leading to the formation of filopodia and adhesion complexes [26, 27].

Similarly, fascin is involved in elongation and bundling of filopodia. It is the only actin

cross-linker localized in filopodia [28]. The relatively increased concentration of these

proteins supports previous cytological studies, which demonstrated that phagocytic

coelomocytes grow extensive filopodia that induce the formation of clots involved in

sealing wounds [29-31]. A previous study [32] compared the intense clotting of sea urchin

coelomocytes with a palisade protecting the organism against infectious agents.

Changes in the relative abundance of other proteins classified in the cell structure,

shape and mobility category occurred later in the response, reflecting ongoing changes in

the coelomocyte cytoskeleton. For example, gelsolin, which is involved in severing actin

filaments [33], as well as septins 2, 6 and 7, increased significantly in relative

concentration (Figure 4.5). Septins are a family of GTP-binding proteins that have been

found to regulate the formation of microtubules in mammalian cells and the overall

organization of the cytoskeleton under the influence of rho and cdc42 signaling modules

[34, 35]. Septin 2 can arrange itself in filaments and is necessary for phagocytosis [36, 37],

whereas septins 2, 6, and 7 form a complex of filamentous septins [38]. Our results suggest

179

the existence of the Sept2/6/7 complex that is involved in the modulation of microtubule

stability in sea urchin coelomocytes during wound repair [34].

In addition to effects on cytoskeletal activity, wounding also seemed to result in

systemic changes in the CF. This was reflected by major yolk protein (MYP; also called

vitellogenin) [39], which increased significantly in relative abundance between 6 and 24

hours p.i., before declining in concentration after 48 hours (Figure 4.4). Major yolk protein

is a member of the large lipid transfer protein (LLTP) family [40]. It is a transferrin-like

protein used to shuttle iron, but its exact activity has not yet been defined [41]. The high

relative abundance of MYP at 24 hours after challenge suggests that it has an important

role in wounding responses. Cervello et al. [42] demonstrated that this protein is stored

within the colorless spherule cells of sea urchins and is discharged into the coelomic fluid

in response to stress. As a transferrin, MYP could be involved in iron depletion to prevent

bacterial infection in a systemic response to wounding.

Other members of the LLTP family, an apolipophorin precursor and two

apolipoprotein B, were relatively abundant 48 hours after injection (Figure 4.5). This

suggests an important role for lipid metabolism in coelomocytes that might be implicated

in the induction of clotting reactions or immunological responses [43, 44]. Avarre et al.

[45] found that among insects as well as vertebrates, members of the LLTP family with a

vWD domain tend to be involved in clotting and defense reactions. The two ApoB-like

proteins included a vWD domain suggesting their involvement in clotting reactions. In

insects, ApoLp is involved in the activation of immune responses [46, 47] and is able to

recognize and inactivate LPS by forming insoluble aggregates [48]. Our data showed that

ApoB was relatively more abundant 24 hours after LPS injection than after saline

injection, suggesting that in sea urchins, these proteins may also be involved in reaction to

LPS.

180

Other evidence for the induction of clotting and wound healing activity 48 hours after

injection included the identification of proteins similar to von Willebrand factor,

cyclophilin B, selectins, and other molecules putatively involved in protein degradation,

such as the COP9/signalosome complex, 26S proteasome, TIMP and a

fumarylacetoacetase. In vertebrates, Von willebrand factor and cyclophilin B contribute to

platelet adhesion to collagen exposed at wounded sites [49, 50]. Vertebrate selectins

mediate cell migration to sites of injury during the process of wound healing [51]. In the

context of wound healing, the proteolytic systems identified could be involved in the

degradation of superfluous or damaged proteins [52]. TIMPs in particular are involved in

the degradation of connective tissue during wound healing [53]. Interestingly, the TIMP

identified in our study was more abundant after LPS injection than after saline injection. In

insects, TIMPs can act as direct effectors of defense mechanisms, by inhibiting

metalloproteases that allow pathogens to penetrate and proliferate within the host [54]. In

the oyster, Crassostrea gigas, the number of transcripts of Cg-Timp increases significantly

in response to the injection of Vibrio [55]. A number of G proteins were also significantly

altered over time after injection (G(i), G(s), G(12)) but not between different challenges.

Overall, these data suggest that there were generalized wounding responses to

injection, involving components of coagulation cascades, cytoskeletal remodelling,

systemic responses and protein degradation. These responses occurred in both the presence

or absence of LPS. However, additional cellular processes appeared to be specifically

activated in response to the injection of LPS. These LPS specific responses involved

proteins associated with vesicular transport, immunological reactions and intracellular

signaling. Most often, the induction of these proteins occurred 48 hours after LPS

injection.

The vesicular transport proteins that differed significantly in relative abundance

between LPS- and saline-injected sea urchins included homologues of coatomer protein

181

complex I (COPI), beta subunit, which functions as a receptor of activated C kinase

(RACK) [56], and another protein similar to RACK. RACK is an adaptor molecule that

binds the activated form of protein kinase C and directs the localization of the activated

enzyme to distinct cellular compartments [56]. This protein also increases after injection of

LPS in flies [57].

Other signalling proteins that were more abundant in LPS-injected sea urchins after 6

or 24 hours included guanine nucleotide binding protein (G protein) alpha subunit G(q)

and MAP kinase. G protein coupled receptors modify the activity of numerous enzymes,

including MAP kinase [58]. MAP kinase pathways regulate a large range of biological

process in cells from yeast to mammals, including wound healing and immune responses.

In response to stimuli from upstream regulators (beta-arrestin, Ras, rac1, cdc42, TRAF, all

found in our samples and regulated over time), they phosphorylate serine and threonine

residues on numerous target proteins, which in turn induce cell proliferation, inflammation,

cell motility, differentiation or apoptosis [59]. The enhanced relative abundance of G(q)

and MAP kinase in response to LPS injection suggests that LPS specifically induces a

range of sub-cellular signaling processes that are not activated after wounding.

Most of the immune response proteins that were specifically induced by LPS

potentially contribute to the activity of phagocytes. These included proteins similar to dual

oxidase 1 and dual oxidase maturation factor, complement component C3, α-2

macroglobulin, fibrocystin L, SRCRs and aminopeptidase N (Figure 4.6). Dual oxidase 1

and dual oxidase maturation factor are involved in catalyzing the one-electron reduction of

molecular oxygen to superoxide. In vertebrates, the NADPH oxidase is primarily found in

professional phagocytic cells, such as monocytes and macrophages, where it actively

participates in the killing of invading bacteria [60]. α2-macroglobulin and complement

component C3 are members of the complement component thioester protein family [61].

α2-macroglobulin proteins are acute phase proteins in vertebrates and have also been

182

identified in a range of invertebrates. They undertake C3-like functions such as

opsonisation, whilst also acting as protease inhibitors [61]. In sea urchins, complement

component SpC3 [62] is known to function as an opsonin promoting phagocytosis [12, 63].

Previous studies of the induction of SpC3 after LPS injection are in close agreement with

our data, showing a marked increase in SpC3 expression 48 hours after injection [12, 62,

63]. These results suggest that the sea urchin complement pathway is activated in response

to LPS injection, but not by saline.

The induction of opsonins, such as SpC3, and oxidative killing systems suggest that

LPS activates the phagolysosomal system of coelomocytes. This activation may also

involve aminopeptidase N, which was specifically induced by LPS 48 hours after injection.

Even though it has a range of functions, aminopeptidase N has been implicated in

FcgammaR mediated phagocytosis in mammals [64]. It spontaneously redistributes to the

zones of phagocyte–target interaction and is internalized into phagosomes. The most

actively phagocytic cells express more than twice the amount of aminopeptidase N than

less phagocytic cells [65]. Interestingly, galectin, a carbohydrate binding C-type lectin that

we also found in abundance 48 hours after challenge, binds aminopeptidase N and

activates NADPH-oxidase [66, 67]. There are 7 complete aminopeptidase N genes in the

sea urchin genome that are sufficiently similar to human aminopeptidase N gene to suggest

that they have similar functions [68].

A number of immunological receptor proteins were also found to be relatively more

abundant 48 hours after LPS injection. These included a protein similar to fibrocystin L

(Figure 4.6) and a SRCR. In humans, fibrocystin L is a receptor protein upregulated

specifically in T lymphocytes activated by lectins [69]. The corresponding sea urchin gene

has a similar structure to its human counterparts, with ten TIG and two G8 domains

(TMEM homology) [7, 69], suggesting that it may also have a receptor function. SRCRs

constitute a family of highly variable cell surface receptors and secreted proteins involved

183

in the recognition of pathogens, the development of immune systems and the regulation of

immune responses. In the S. purpuratus genome, there are an estimated 1,200 distinct

SRCR-like domains [70], and numerous SRCR proteins with variable expression patterns

have already been characterized in sea urchin coelomocytes [70, 71].

The time course analysis reported in the current study extends previous proteomic

and transcriptomic analyses of inducible immune responses in S. purpuratus that

investigated just a single timepoint (Dheilly et al., unpublished data)[5]. In a previous

proteome analysis, a total of 323 proteins were identified in S. purpuratus CF collected 24

hours after LPS injection into immunoquiescent animals (Dheilly et al., unpublished data).

We identified 236 (73%) of these proteins in the current analysis of H. erythrogramma CF,

and an additional 109 proteins that were not identified in the previous study. The strong

similarities between our analysis of H. erythrogramma CF proteome with the one

undertaken on S. purpuratus suggest that the two species of sea urchins undertake

comparable responses to LPS. Similarly, a macroarray analysis of coelomocyte gene

expression undertaken by Nair et al. [5] revealed that the injection of LPS into

immunoquiescent S. purpuratus significantly altered the transcriptional state of

coelomocytes. A large proportion of the proteins encoded by the transcripts identified in

Nair et al.’s work were also found to be significantly up-regulated in the current study.

These included defense related proteins, such as amassin, integrin, galactin, lectins,

complement component C3, cathepsin, MAP kinase, arylsulfatase. Nair et al. [5] also

highlighted the abundance of transcripts involved in amoeboid movement, such as actin,

profilin, cofilin or gelsolin.

The combination of genomic, transcriptomic and proteomic analysis is beginning to

build a comprehensive picture of the biology of sea urchin coelomocytes. In order to better

characterize the biological pathways involved in the inducible immune response of sea

urchins, future proteomics studies could make use of cell separation and sub-cellular

184

fractionation techniques to improve the characterization of low abundance proteins and

determine their precise role in sea urchin immunity. Regardless, the current study clearly

shows that coelomocytes are professional amoebocytes and phagocytes equipped to

respond to wounding and infection. The major advance made by the current study is to

characterize distinct molecules responsible in sea urchin responses to wounding and to the

presence of pathogen-associated antigens, such as LPS. In particular, it shows that there

are discrete suites of proteins that respond specifically to immunological challenge.

185


We thank Dr. Dana Pascovici for assistance with statistical analysis. The research

has been facilitated by access to the Australian Proteome Analysis Facility established

under the Australian Government’s Major National Research Facilities Program. NM

Dheilly is the recipient of an iMURS postgraduate scholarship from Macquarie University.

This study was funded in part by an Australian Research Council Discovery grant to DA

Raftos and LC Smith (DP0880316).

186

4.8. References

[1] Rock, F. L., Hardiman, G., Timans, J. C., Kastelein, R. A., Bazan, J. F., A family of

human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A

1998, 95, 588-593.

[2] Hoffmann, J. A., The immune response of Drosophila. Nature 2003, 426, 33-38.

[3] Sodergren, E., Weinstock, G.M., Davidson, E.H., Cameron, R., A. et al. The genome of

the sea urchin Strongylocentrotus purpuratus. Science 2006, 314, 941-952.

[4] Cameron, R., Mahairas, G., Rast, J., Martinez, P., et al., A sea urchin genome project:

sequence scan, virtual map, and additional resources. Proc Natl Acad Sci U S A

2000, 97, 9514-9518.




2005, 22, 33-47.

[6] Smith, L. C., Clow, L. A., Terwilliger, D. P., The ancestral complement system in sea

urchins. Immunol Rev 2001, 180, 16-34.




evolutionary origin of the Rag1/2 gene locus. Proc Natl Acad Sci U S A 2006, 103,

3728-3733.

[9] Gross, P. S., Al-Sharif, W. Z., Clow, L. A., Smith, L. C., Echinoderm immunity and the

evolution of the complement system. Dev Comp Immunol 1999, 23, 429-442.

[10] Domon, B., Aebersold, R., Mass spectrometry and protein analysis. Science 2006,

312, 212-217.

187




1044.



2000, 51, 1021-1033.







[15] Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 1970, 227, 680-685.

[16] Candiano, G., Bruschi, M., Musante, L., Santucci, L., et al., Blue silver: a very

sensitive colloidal coomassie G-250 staining for proteome analysis. Electrophoresis

2004, 25, 1327-1333.

[17] Dheilly, N. M., Nair, S. V., Smith, L. C., Raftos, D. A., Highly variable immune-


proteomic analysis identifies diversity within and between individuals. J Immunol

2009, 182, 2203-2212.

[18] Breci, L., Hattrup, E., Keeler, M., Letarte, J., et al., Comprehensive proteomics in


electrophoresis, and isoelectric focusing. Proteomics 2005, 5, 2018-2028.

188

[19] Craig, R., Beavis, R. C., A method for reducing the time required to match protein

sequences with tandem mass spectra. Rapid Communications in Mass Spectrometry

2003, 17, 2310-2316.

[20] Craig, R., Beavis, R. C., TANDEM: matching proteins with tandem mass spectra.

Bioinformatics 2004, 20, 1466-1467.

[21] Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., Gygi, S. P., Evaluation of

multidimensional chromatography coupled with tandem mass spectrometry

(LC/LC‚ MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome

Res 2002, 2, 43-50.

[22] Haynes, P. A., Miller, S., Radabaugh, T., Galligan, M., et al., The wildcat toolbox: a

set of perl script utilities for use in peptide mass spectral database searching and

proteomics experiments. J Biomol Tech 2008, 17, 97-102.

[23] Rohrbough, J. G., Breci, L., Merchant, N., Miller, S., Haynes, P. A., Verification of

single-peptide protein identifications by the application of complementary database

search algorithms. J Biomol Tech 2006, 17, 327-332.

[24] Zybailov, B. L., Florens, L., P., W. M., Quantitative shotgun proteomics using a

protease with broad specificity and normalized spectral abundance factors. Mol

Biosyst 2007, 3, 354-360.





[26] Nobes, C. D., Hall, A., Rho, Rac, and Cdc42 GTPases regulate the assembly of

multimolecular focal complexes associated with actin stress fibers, lamellipodia,

and filopodia. Cell 1995, 81, 53-62.

[27] Hall, A., Rho GTPases and the actin cytoskeleton. Science 1998, 279, 509-514.

189

[28] Vignjevic, D., Kojima, S.-i., Aratyn, Y., Danciu, O., et al., Role of fascin in filopodial

protrusion. J Cell Biol 2006, 174, 863-875.



[30] Edds, K. T., The formation and elongation of filopodia during transformation of sea

urchin coelomocytes. Cell Motil Cytoskeleton 1980, 1, 131-140.

[31] Chien, P. K., Johnson, P. T., Holland, N. D., Chapman, F. A., The coelomic elements

of sea urchins (Strongylocentrotus). Protoplasma 1970, 71, 419-442.

[32] Johnson, P., The coelomic elements of the sea urchins (Strongulocentrotus) III. In


[33] Wang, L., Spudich, J., A 45,000-mol-wt protein from unfertilized sea urchin eggs

severs actin filaments in a calcium-dependent manner and increases the steady-state

concentration of nonfilamentous actin. J. Cell Biol. 1984, 99, 844-851.

[34] Kremer, B. E., Haystead, T., Macara, I. G., Mammalian septins regulate microtubule

stability through interaction with the microtubule-binding protein MAP4. Mol Biol

Cell 2005, 16, 4648-4659.

[35] Spiliotis, E. T., Nelson, W. J., Here come the septins: novel polymers that coordinate

intracellular functions and organization. J Cell Sci 2006, 119, 4-10.

[36] Huang, Y.-W., Yan, M., Collins, R. F., DiCiccio, J. E., et al., Mammalian septins are

required for phagosome formation. Mol Biol Cell 2008, 19, 1717-1726.

[37] Mendoza, M., Hyman, A. A., Glotzer, M., GTP binding induces filament assembly of

a recombinant septin. Curr Biol 2002, 12, 1858-1863.

[38] Low, C., Macara, I. G., Structural analysis of septin 2, 6, and 7 complexes. J Biol

Chem 2006, 281, 30697-30706.

[39] Shyu, A. B., Raff, R. A., Blumenthal, T., Expression of the vitellogenin gene in

female and male sea urchin. Proc Natl Acad Sci U S A 1986, 83, 3865-3869.

190

[40] Smolenaars, M. M. W., Madsen, O., Rodenburg, K. W., Van der Horst, D. J.,

Molecular diversity and evolution of the large lipid transfer protein superfamily. J

Lipid Res 2007, 48, 489-502.

[41] Brooks, J. M., Wessel, G. M., The major yolk protein in sea urchins is a transferrin-

like, iron binding protein. Dev Biol 2002, 245, 1-12.


vitellogenin in a subpopulation of sea urchin coelomocytes. Eur J Cell Biol 1994,

64, 314-319.

[43] Miller, Y. I., Chang, M. K., Binder, C. J., Shaw, P. X., Witztum, J. L., Oxidized low

density lipoprotein and innate immune receptors. Curr Opin Lipidol 2003, 14, 437-

445.

[44] Elzen, P. v. d., Garg, S., Leon, L., Brigl, M., et al., Apolipoprotein-mediated pathways

of lipid antigen presentation. Nature 2005, 437, 906-910.

[45] Avarre, J.-C., Lubzens, E., Babin, P., Apolipocrustacein, formerly vitellogenin, is the

major egg yolk precursor protein in decapod crustaceans and is homologous to

insect apolipophorin II/I and vertebrate apolipoprotein B. BMC Evol Biol 2007, 7,

3.

[46] Kim, H. J., Je, H. J., Park, S. Y., Lee, I. H., et al., Immune activation of

apolipophorin-III and its distribution in hemocyte from Hyphantria cunea. Insect

Biochem Mol Biol 2004, 34, 1011-1023.

[47] Cheon, H., Shin, S., Bian, G., Park, J., Raikhel, A., Regulation of lipid metabolism

genes, lipid carrier protein lipophorin, and its receptor during immune challenge in

the mosquito Aedes aegypti. J Biol Chem 2006, 281, 8426-8435.

[48] Ma, G., Hay, D., Li, D., Asgari, S., Schmidt, O., Recognition and inactivation of LPS

by lipophorin particles. Dev Comp Immunol 2006, 30, 619-626.

191

[49] Allain, F., Durieux, S., Denys, A., Carpentier, M., Spik, G., Cyclophilin B binding to

platelets supports calcium-dependent adhesion to collagen. Blood 1999, 94, 976-

983.

[50] Farndale, R. W., Sixma, J. J., Barnes, M. J., Groot, P. G. D., The role of collagen in

thrombosis and hemostasis. Journal of Thrombosis and Haemostasis 2004, 2, 561-

573.

[51] Vasta, G. R., Quesenberry, M., Ahmed, H., O'Leary, N., C-type lectins and galectins

mediate innate and adaptive immune functions: their roles in the complement

activation pathway. Dev Comp Immunol 1999, 23, 401-420.

[52] Schwechheimer, C., Deng, X.-W., COP9 signalosome revisited: a novel mediator of

protein degradation. Trends Cell Biol 2001, 11, 420-426.

[53] Nagase, H., Woessner, J. F., Jr., Matrix metalloproteinases. J Biol Chem 1999, 274,

21491-21494.

[54] Lepore, L. S., Roelvink, P. R., Granados, R. R., Enhancin, the granulosis virus protein

that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease. J


[55] Montagnani, C., Le Roux, F., Berthe, F., Escoubas, J.-M., Cg-TIMP, an inducible

tissue inhibitor of metalloproteinase from the Pacific oyster Crassostrea gigas with

a potential role in wound healing and defense mechanisms. FEBS Lett 2001, 500,

64-70.

[56] Csukai, M., Chen, C.-H., De Matteis, M. A., Mochly-Rosen, D., The coatomer protein

beta-COP, a selective binding protein (RACK) for protein kinase C epsilon. J Biol

Chem 1997, 272, 29200-29206.

[57] Loseva, O., Engstrom, Y., Analysis of signal-dependent changes in the proteome of

Drosophila blood cells during an immune response. Mol Cell Proteomics 2004, 3,

796-808.

192

[58] Eglen, R. M., Bosse, R., Reisine, T., Emerging concepts of guanine nucleotide-

binding protein-coupled receptor (GPCR) function and implications for high

throughput screening. Assay Drug Dev Technol 2007, 5, 425-452.

[59] Qi, M., Elion, E. A., MAP kinase pathways. J Cell Sci 2005, 118, 3569-3572.

[60] Rada, B. K., Geiszt, M., Kaldi, K., Timar, C., Ligeti, E., Dual role of phagocytic

NADPH oxidase in bacterial killing. Blood 2004, 104, 2947-2953.

[61] Blandin, S., Levashina, E. A., Thioester-containing proteins and insect immunity. Mol

Immunol 2004, 40, 903-908.



1998, 160, 2983-2997.



[64] Mina-Osorio, P., Ortega, E., Aminopeptidase N (CD13) functionally interacts with Fc

gamma Rs in human monocytes. J Leukoc Biol 2005, 77, 1008-1017.

[65] Tokuda, N., Levy, R. B., 1,25-dihydroxyvitamin D3 stimulates phagocytosis but

supresses HLA-DR and CD13 antigen expression in human mononuclear

phagocytes. Proceedings of the society for experimental biology and medicine

1996, 211, 244-250.

[66] Granhagen, K., Lundstron, A., Bjorstad, A., Bylund, J., et al., Galectin-3 binds CD 13,

activates the NADPH-oxidase, and augments neutrophil inactivation of the

chemotactic peptide fMLF: 69. Eur J Clin Invest 2007, 37, 23.

[67] Mina-Osorio, P., Soto-Cruz, I., Ortega, E., A role for galectin-3 in CD13-mediated

homotypic aggregation of monocytes. Biochem Biophys Res Commun 2007, 353,

605-610.

193

[68] Ingersoll, E., An analysis of aminopeptidase N genes in the sea urchin genome. Dev

Biol 2008, 319, 566-566.

[69] Hogan, M. C., Griffin, M. D., Rossetti, S., Torres, V. E., et al., PKHDL1, a homolog

of the autosomal recessive polycystic kidney disease gene, encodes a receptor with

inducible T lymphocyte expression. Hum Mol Genet 2003, 12, 685-698.

[70] Pancer, Z., Rast, J. P., Davidson, E. H., Origins of immunity: transcription factors and

homologues of effector genes of the vertebrate immune system expressed in sea

urchin coelomocytes. Immunogenetics 1999, 49, 773-786.


coelomocytes of the purple sea urchin. Proc Natl Acad Sci U S A 2000, 97, 13156-

13161.

194

195

Supplementary Data 4.1: Normalized spectral abundance factors. NSAF values for the

345 proteins identified in the CF of non-injected sea urchins (NI), 6 hours after saline

injection (W6), 24 hours after saline injection (W24), 48 hours after saline injection

(W48), 6 hours after LPS injection (LPS6), 24 hours after LPS injection (LPS24) and 48

hours after LPS injection (LPS48).

196

Identifier Description NI1 NI2 NI3 W61 W62 W63

gi|115942229| PREDICTED: similar to integrin-linked kinase 1E-09 3E-06 7E-06 3E-06 3E-06 2E-06

gi|115959822| PREDICTED: similar to annexin 2E-05 2E-05 2E-05 1E-09 1E-09 1E-05

gi|115940188| PREDICTED: similar to peptidylprolyl isomerase B isoform 2 . 6E-06 6E-06 7E-06 1E-05 1E-09 1E-09

gi|115975224| PREDICTED: similar to G-cadherin 3E-06 1E-09 1E-06 4E-07 1E-09 1E-09

gi|115910910| PREDICTED: hypothetical protein, partial (Fibronectin) 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115720465| PREDICTED: similar to selectin-like protein, partial . 1E-09 1E-09 1E-09 1E-09 1E-05 1E-09

gi|115960842| PREDICTED: similar to talin 2E-06 4E-06 3E-06 5E-07 1E-06 2E-06

gi|115970015| PREDICTED: similar to von Willebrand factor 1E-05 1E-05 8E-06 1E-09 1E-06 1E-09

gi|115963192| PREDICTED: similar to Annexin A4 (Annexin IV) (Lipocortin

IV) (Endonexin I) (Chromobindin-4) (Protein II) (P32.5)

(Placental

6E-05 9E-05 6E-05 6E-05 3E-05 5E-05

gi|115944296| PREDICTED: hypothetical protein (Galectin) 5E-05 7E-05 0.0001 6E-05 1E-09 7E-05

gi|75753591| amassin 5E-05 1E-05 5E-05 6E-05 2E-05 2E-06

gi|115951499| PREDICTED: hypothetical protein (septin2) 6E-06 3E-06 4E-06 1E-09 1E-09 1E-09

gi|115968740| PREDICTED: similar to Septin 6 3E-06 6E-06 1E-09 5E-06 3E-06 1E-05

gi|115940910| PREDICTED: hypothetical protein (septin7) 3E-06 1E-05 3E-06 1E-09 1E-09 5E-06

gi|115939093| PREDICTED: hypothetical protein (septin 9 fusion) 2E-05 2E-05 1E-05 1E-09 1E-09 8E-06

gi|115955362| PREDICTED: similar to apextrin 1E-09 7E-06 1E-05 1E-09 1E-09 1E-09

gi|115956476| PREDICTED: similar to apextrin 6E-06 9E-05 0.0001 5E-06 2E-05 1E-09

gi|115959937| PREDICTED: similar to echinonectin 6E-06 8E-06 8E-06 3E-06 1E-09 2E-06

gi|115770276| PREDICTED: similar to zonadhesin 2E-05 5E-05 4E-05 6E-06 2E-06 3E-06

gi|115899605| PREDICTED: similar to echinonectin, partial 3E-05 3E-05 2E-05 4E-05 1E-09 1E-09

gi|115945705| PREDICTED: similar to EH-domain-containing protein 1

(Testilin) (hPAST1)

9E-05 9E-05 9E-05 7E-05 7E-05 8E-05

gi|115944978| PREDICTED: similar to COP9 complex subunit 4 3E-06 4E-06 1E-09 1E-09 1E-09 1E-09

gi|34100406| calmodulin-dependent protein kinase type II 1E-09 1E-09 3E-06 1E-09 1E-09 1E-09

gi|115958659| PREDICTED: hypothetical protein (arfaptin) 1E-09 3E-06 1E-09 1E-09 1E-09 1E-09

gi|115894557| PREDICTED: similar to RACK 1E-05 1E-05 6E-06 1E-09 1E-09 1E-09

gi|115940258| PREDICTED: hypothetical protein (lipid raft asssocitaed

protein 2)

1E-09 1E-09 1E-09 1E-09 1E-09 5E-06

gi|115955418| PREDICTED: similar to 5-aminoimidazole ribonucleotide (AIR)

carboxylase-5- aminoimidazole-4-N-succinocarboxamide

ribonucleotide (SAICAR) synthetase isoform 1

3E-06 6E-06 4E-06 1E-09 1E-09 1E-09

gi|115969931| PREDICTED: similar to Creatine kinase, flagellar 3E-06 1E-09 8E-06 1E-06 1E-09 1E-09

gi|115671024| PREDICTED: similar to KIAA0051, partial (IQ motif containing

GTPase activating protein)

3E-06 1E-05 1E-05 1E-09 1E-09 1E-09

gi|47551303| guanine nucleotide-binding protein G(q) alpha subunit 1E-05 2E-05 1E-05 1E-09 1E-09 1E-09

gi|115975377| PREDICTED: similar to protein kinase C, partial 1E-09 7E-06 8E-06 1E-09 1E-09 1E-09

gi|115926118| PREDICTED: similar to CG6950-PC (kynurenine

aminotransferase)

9E-06 3E-06 7E-06 1E-05 7E-06 5E-06

gi|115973422| PREDICTED: similar to adenylate kinase 2 5E-06 2E-05 1E-05 2E-05 1E-09 1E-09

gi|115951428| PREDICTED: similar to LOC615074 protein isoform 2 cAMP-

dependent protein kinase)

1E-05 7E-06 8E-06 1E-09 1E-09 3E-06

gi|115959301| PREDICTED: similar to Protein phosphatase 2A, catalytic

subunit, beta isoform

8E-06 8E-06 9E-06 1E-09 1E-09 1E-09

gi|115960271| PREDICTED: similar to protein Phosphatase 2C beta 3E-06 7E-06 3E-05 1E-09 1E-09 1E-09

gi|115946171| PREDICTED: similar to beta-arrestin 1, putative 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115937294| PREDICTED: similar to beta-parvin 1E-05 2E-05 1E-05 1E-09 4E-06 1E-09

gi|115974058| PREDICTED: similar to GTP binding protein 3E-06 3E-06 2E-05 9E-06 4E-06 3E-06

gi|115944458| PREDICTED: similar to calponin 7E-06 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115974451| PREDICTED: similar to GTPase SUrab10p 6E-05 1E-09 6E-05 5E-06 1E-09 1E-09

gi|115965021| PREDICTED: similar to BAI1-associated protein 2 9E-07 4E-06 2E-06 2E-06 3E-06 3E-06

gi|47550939| calreticulin 6E-06 6E-06 1E-09 1E-09 1E-09 1E-09

gi|115975046| PREDICTED: similar to GTP-binding nuclear protein Ran 2E-05 2E-05 3E-05 2E-05 1E-09 1E-09

gi|115960970| PREDICTED: similar to proliferation-associated protein 1 6E-06 2E-05 4E-05 1E-09 1E-09 1E-09

gi|115975006| PREDICTED: similar to RAB2 isoform 2 3E-05 4E-05 4E-05 1E-09 1E-09 1E-09

gi|115940411| PREDICTED: similar to 14-3-3-like protein 2 3E-05 1E-09 1E-09 1E-05 1E-09 1E-09

gi|115971397| PREDICTED: similar to centaurin, alpha 1 2E-05 5E-05 1E-09 1E-09 1E-09 1E-05

gi|115945715| PREDICTED: similar to Ras-related protein ORAB-1 1E-05 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115958373| PREDICTED: similar to Rab5 protein 4E-05 2E-05 1E-05 5E-06 1E-09 1E-05

gi|115774726| PREDICTED: hypothetical protein, partial

(phosphatidylinositol-5-phosphate 4-kinase)

7E-06 7E-06 2E-05 1E-09 1E-09 1E-09

gi|115968476| PREDICTED: similar to ENSANGP00000018350 (centaurin) 3E-05 4E-05 4E-06 7E-06 1E-09 1E-09

gi|115926041| PREDICTED: similar to protein-tyrosine phosphatase 1E-09 7E-06 8E-06 1E-09 1E-09 6E-06

gi|115939472| PREDICTED: similar to ENSANGP00000023777 (CDC42, cell

division cycle 42)

1E-09 1E-09 1E-09 1E-05 1E-09 3E-05

gi|115976839| PREDICTED: similar to GDP-dissociation inhibitor 1E-09 2E-05 1E-05 1E-05 4E-06 1E-09

gi|115938855| PREDICTED: similar to Rab11b 3E-05 6E-06 3E-05 4E-05 7E-06 2E-05

gi|115939823| PREDICTED: similar to catalytic subunit of cAMP-dependent

histone kinase

2E-05 3E-05 1E-05 6E-06 4E-06 3E-06

gi|47825404| guanine nucleotide-binding protein G(12) alpha subunit . 2E-05 2E-05 4E-05 9E-06 1E-09 1E-09

gi|115953926| PREDICTED: similar to Rho1 GTPase isoform 2 3E-05 1E-09 2E-05 2E-05 1E-09 1E-09

gi|47551197| src-family protein tyrosine kinase 1E-09 7E-06 3E-06 9E-06 6E-06 1E-09

gi|47825398| guanine nucleotide-binding protein G(s) alpha subunit . 3E-05 5E-05 2E-05 2E-05 1E-05 8E-06

gi|115929326| PREDICTED: similar to KIAA1646 protein (ceramide kinase) 1E-05 2E-06 7E-06 2E-06 2E-06 2E-06

gi|115949757| PREDICTED: similar to Rap1b-prov protein 7E-06 2E-05 1E-09 6E-06 1E-09 1E-05

gi|115966293| PREDICTED: similar to RAS-related protein MEL . 7E-05 7E-05 7E-05 1E-05 1E-09 5E-06

gi|160623362| putative 14-3-3 epsilon isoform 6E-05 1E-09 1E-09 4E-05 7E-06 3E-05

NSAF

197

NSAF

W241 W242 W243 W481 W482 W483 LPS61 LPS62 LPS63 LPS241 LPS242 LPS243 LPS481 LPS482 LPS483

1E-09 1E-09 1E-09 5E-06 7E-06 1E-09 1E-09 3E-06 1E-09 1E-09 1E-09 1E-09 6E-06 9E-06 1E-05





2E-06 3E-05 4E-05 6E-05 6E-05 4E-05 1E-09 2E-05 1E-09 3E-06 3E-06 2E-05 7E-05 0.0001 0.0001




2E-05 7E-06 4E-05 0.0001 0.0002 5E-05 5E-05 0.0001 3E-05 2E-06 2E-06 1E-09 0.0001 0.0001 4E-05

2E-05 4E-05 5E-05 8E-05 5E-05 7E-05 5E-05 0.0001 1E-06 3E-05 1E-05 2E-05 6E-05 4E-05 0.0002










4E-05 3E-05 6E-05 4E-05 4E-05 5E-05 4E-05 4E-05 0.0001 9E-05 3E-05 4E-05 5E-05 7E-05 6E-05






































4E-06 1E-05 2E-05 4E-06 2E-05 2E-05 3E-05 0.0001 7E-06 5E-06 1E-09 7E-06 3E-05 7E-05 1E-09







198


gi|115891186| PREDICTED: hypothetical protein, partial (kynurenine

aminotransferase)

1E-09 1E-09 6E-06 4E-05 2E-05 2E-05

gi|47551261| MAP kinase 1E-05 7E-06 2E-05 6E-06 4E-06 6E-06

gi|115974362| PREDICTED: similar to Rab7 4E-05 6E-05 7E-05 2E-05 1E-09 1E-05

gi|115960532| PREDICTED: similar to Src-type protein tyrosine kinase

isoform 2

7E-06 1E-05 1E-05 2E-05 2E-05 1E-09

gi|115934007| PREDICTED: similar to TRAF4-associated factor 2 . 2E-05 3E-05 2E-05 2E-05 2E-05 2E-05

gi|115976312| PREDICTED: similar to CG8649-PC (calponin) 3E-05 2E-05 2E-05 1E-09 6E-06 1E-09

gi|47825400| guanine nucleotide-binding protein G(i) alpha subunit . 5E-05 5E-05 4E-05 3E-05 1E-09 3E-05

gi|115945943| PREDICTED: similar to ENSANGP00000011972 (GDP

dissociation inhibitor)

5E-05 3E-05 5E-05 2E-05 1E-05 2E-05

gi|115968621| PREDICTED: similar to calponin 0.0001 0.0001 0.0001 9E-05 2E-05 9E-05

gi|115936805| PREDICTED: similar to heterotrimeric guanine nucleotide-

binding protein beta subunit isoform 2 .

0.0002 0.0003 0.0004 2E-05 1E-09 4E-05

gi|115970013| PREDICTED: similar to apolipoprotein B 1E-05 9E-06 1E-05 7E-06 2E-06 4E-06

gi|115940166| PREDICTED: similar to apolipoprotein B 1E-05 8E-06 2E-05 1E-09 1E-09 1E-09

gi|115956784| PREDICTED: similar to epsin 2 5E-06 2E-06 3E-06 1E-09 1E-09 1E-09

gi|115964211| PREDICTED: similar to Actr1a-prov protein (actin related

protein 1 homologue A)

1E-09 1E-09 1E-09 6E-06 4E-06 6E-06

gi|115699240| PREDICTED: similar to ankyrin 2,3/unc44 1E-09 1E-09 3E-06 1E-09 2E-06 1E-09

gi|115974713| PREDICTED: hypothetical protein (dysferlin C2 superfamily) 1E-05 2E-05 1E-05 1E-09 1E-09 1E-09

gi|115951109| PREDICTED: similar to tensilin 3E-05 2E-05 2E-05 1E-09 1E-09 4E-06

gi|115974054| PREDICTED: similar to fibropellin Ia 6E-06 6E-06 7E-06 1E-09 1E-09 1E-09

gi|115975440| PREDICTED: similar to moesin 2E-06 2E-06 2E-06 1E-09 5E-06 1E-09

gi|115961148| PREDICTED: similar to gelsolin 1E-09 1E-09 2E-05 3E-06 4E-06 1E-09

gi|115974666| PREDICTED: similar to coronin 3E-06 5E-06 6E-06 1E-05 1E-09 2E-06

gi|115950170| PREDICTED: similar to long microtubule-associated protein

1A; long MAP1A

2E-06 3E-06 3E-06 1E-09 1E-09 1E-09

gi|115925414| PREDICTED: similar to KIAA0727 protein (myosin) 1E-06 4E-06 1E-06 2E-06 7E-06 1E-09

gi|115746860| PREDICTED: similar to actin, partial 1E-09 1E-09 1E-09 3E-05 2E-05 6E-06

gi|115945231| PREDICTED: similar to KIAA0587 protein (NCR associate

protein)

1E-06 3E-06 2E-05 1E-06 1E-06 1E-06

gi|115971193| PREDICTED: similar to Rac1 protein 1E-09 1E-09 1E-09 2E-05 1E-09 2E-05

gi|115970592| PREDICTED: similar to tropomyosin 1 4E-06 9E-06 3E-05 1E-05 1E-09 4E-06

gi|115945717| PREDICTED: similar to Enabled homolog (Drosophila) 1E-05 2E-05 2E-05 6E-06 7E-06 8E-06

gi|115630732| PREDICTED: similar to myosin, heavy polypeptide 10, non-

muscle

6E-06 1E-05 3E-05 1E-09 1E-09 1E-06

gi|115961398| PREDICTED: similar to Capzb-prov protein (F actin capping

protein beta)

9E-05 6E-05 5E-05 3E-05 1E-09 1E-05

gi|115955651| PREDICTED: similar to Col protein 1E-09 1E-09 1E-05 6E-05 4E-05 3E-06

gi|115944055| PREDICTED: similar to fibrocystin L 3E-06 2E-06 7E-06 1E-09 1E-09 1E-09

gi|166795321| advillin 2E-05 1E-05 1E-05 8E-06 5E-06 1E-05


gi|115931821| PREDICTED: hypothetical protein (fibronectin) 6E-06 1E-05 1E-05 1E-09 1E-09 1E-09

gi|115960968| PREDICTED: similar to sea urchin Arp3 (SUArp3) 4E-05 3E-05 5E-05 2E-05 2E-05 2E-05


gi|115960587| PREDICTED: similar to alpha tubulin 8E-05 1E-09 8E-05 1E-09 1E-09 1E-09

gi|115944135| PREDICTED: similar to sea urchin Arp2 (SUArp2) . 0.0002 0.0002 0.0002 4E-05 2E-05 6E-05

gi|115951150| PREDICTED: similar to beta tubulin 9E-05 1E-09 0.0001 1E-09 7E-05 1E-09

gi|115929203| PREDICTED: similar to ENSANGP00000011796 (alpha

actinin)

6E-05 4E-05 5E-05 1E-05 2E-05 3E-05

gi|115963096| PREDICTED: similar to CG15792-PD, partial (myosin) 6E-05 9E-05 0.0001 4E-06 2E-05 1E-05

gi|115960585| PREDICTED: similar to tubulin, alpha 2 isoform 1 1E-09 0.0001 1E-09 6E-05 1E-09 3E-05

gi|115938253| PREDICTED: similar to Tubulin, beta 2, partial 9E-05 0.0001 0.0001 5E-05 0.0001 1E-09

gi|21667225| alpha-tubulin 3 0.0001 0.0001 0.0001 6E-05 0.0001 3E-05

gi|115960583| PREDICTED: similar to tubulin, alpha 2 isoform 3 0.0001 1E-09 0.0001 1E-09 0.0001 1E-09

gi|115960644| PREDICTED: similar to myosin heavy chain 2E-05 4E-05 7E-05 1E-05 9E-06 3E-06

gi|115618052| PREDICTED: similar to Actin, cytoskeletal IIIB, partial 1E-09 0.0007 0.0007 0.0008 1E-09 1E-09

gi|47551049| fascin 0.0001 5E-05 5E-05 0.0001 8E-05 0.0002

gi|115974233| PREDICTED: similar to Actin, cytoplasmic 0.0005 0.0005 1E-09 0.0003 1E-09 0.0003

gi|115971215| PREDICTED: hypothetical protein (tubulin) 0.0002 0.0002 0.0002 0.0002 0.0002 3E-05

gi|115968818| PREDICTED: similar to ENSANGP00000003616 (Actin like

protein)

0.0001 1E-04 0.0002 3E-05 4E-05 7E-05

gi|115971469| PREDICTED: similar to Actin, cytoplasmic 0.0003 0.0004 0.0005 0.0004 0.0004 0.0006

gi|224306| actin 1E-09 1E-09 1E-09 1E-09 0.0017 1E-09

gi|115747662| PREDICTED: similar to actin 1E-09 0.0006 1E-09 0.0007 0.0006 0.0008

gi|115974235| PREDICTED: similar to Actin, cytoskeletal IIIB 1E-09 1E-09 1E-09 0.0008 0.0007 0.0008

gi|115971461| PREDICTED: similar to actin 1E-09 0.0005 1E-09 0.0006 0.0006 0.0007

gi|47551035| cytoskeletal actin CyIIIb 1E-09 1E-09 1E-09 0.0019 0.0017 0.0021

gi|115943916| PREDICTED: similar to actin 0.0004 0.0005 0.0005 0.0011 0.001 0.0012

gi|871546| actin 0.0009 0.001 1E-09 0.0012 1E-09 0.0013

gi|1703135| Actin, cytoskeletal 3A (Actin, cytoskeletal IIIA) 1E-09 0.0013 0.0014 0.0019 0.0017 0.0021

gi|81158087| actin 2 1E-09 0.0015 0.0016 0.002 0.0017 0.0023

gi|115956638| PREDICTED: similar to cytoskeletal actin CyIIb . 1E-09 1E-09 1E-09 0.0021 0.0019 1E-09

gi|115949854| PREDICTED: similar to cytoskeletal actin . 0.0012 0.0013 0.0014 0.0019 0.0017 0.0021

gi|115940604| PREDICTED: similar to CyI cytoplasmic actin 0.0013 0.0014 0.0014 1E-09 0.002 0.0025

gi|47551037| cytoskeletal actin CyIIb 0.0017 0.0018 1E-09 0.0025 0.0023 0.0029

gi|115935893| PREDICTED: similar to vacuolar (V-type) H(+)-ATPase B

subunit isoform 1 .

1E-05 3E-05 3E-05 7E-06 1E-09 1E-09

NSAF

199

NSAF










4E-05 5E-05 9E-05 0.0001 7E-05 1E-09 0.0002 0.0002 0.0001 1E-05 3E-05 5E-05 9E-05 0.0002 2E-05




























1E-05 1E-05 1E-05 0.0001 1E-04 7E-05 2E-05 7E-05 7E-06 5E-06 4E-05 5E-06 6E-05 8E-05 4E-05

1E-09 1E-09 1E-09 8E-05 7E-05 0.0001 1E-09 0.0001 0.0001 8E-05 6E-05 1E-09 7E-05 7E-05 1E-09


2E-05 1E-09 1E-09 9E-05 0.0001 7E-05 8E-05 0.0001 3E-05 7E-05 5E-05 1E-09 7E-05 6E-05 1E-09



0.0001 7E-05 0.0002 1E-09 0.0002 1E-09 0.0002 1E-09 1E-09 1E-09 0.0001 8E-05 0.0001 1E-09 0.0001

5E-05 5E-05 0.0001 9E-05 0.0001 0.0001 1E-09 0.0001 4E-05 0.0001 0.0001 7E-05 9E-05 8E-05 7E-05

0.0001 1E-09 1E-09 0.0002 0.0001 0.0002 1E-09 0.0002 0.0001 0.0001 1E-09 9E-05 0.0001 1E-09 1E-09

1E-04 1E-09 1E-09 0.0002 0.0001 0.0002 1E-09 0.0002 0.0001 0.0001 0.0001 7E-05 1E-09 0.0001 1E-09

5E-06 5E-05 1E-09 2E-05 8E-05 8E-05 3E-06 1E-05 6E-06 2E-06 7E-06 1E-05 6E-05 3E-05 0.0002

0.0015 0.0006 1E-09 0.0008 0.001 0.0011 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

0.0001 8E-05 1E-04 0.0002 0.0001 0.0002 0.0002 0.0001 0.0002 0.0002 0.0001 0.0001 0.0003 0.0002 0.0002

1E-09 0.0003 0.0004 0.0004 1E-09 0.0004 0.0004 1E-09 0.0003 0.0004 1E-09 1E-09 0.0005 0.0004 0.0003

0.0001 0.0001 0.0003 0.0003 0.0003 0.0003 0.0002 0.0004 0.0001 0.0002 0.0002 0.0001 0.0002 0.0002 0.0003

4E-05 2E-05 1E-05 0.0001 5E-05 3E-05 2E-05 3E-05 1E-05 9E-06 1E-05 2E-05 8E-05 0.0001 3E-05

0.0006 0.0004 1E-09 0.0003 0.0004 0.0005 1E-09 1E-09 0.0004 1E-09 1E-09 0.0006 1E-09 1E-09 1E-09

1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 0.0021 0.0018 0.0018 1E-09 1E-09 1E-09 1E-09 0.0016 1E-09

1E-09 0.0007 0.0011 1E-09 1E-09 1E-09 0.0007 0.0006 0.0007 1E-09 0.0006 0.0008 1E-09 0.0005 1E-09

1E-09 0.0003 0.0005 0.0007 0.0009 1E-09 1E-09 1E-09 0.0009 1E-09 1E-09 0.0012 0.0012 1E-09 0.001

0.0007 1E-09 1E-09 0.0004 0.0005 1E-09 0.0006 0.0005 0.0005 0.0004 1E-09 0.0007 1E-09 1E-09 0.0004

1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 0.0022 0.0018 0.0018 1E-09 1E-09 1E-09 1E-09 1E-09 0.0019

0.0006 0.0012 0.0017 0.0006 0.0006 0.0008 0.0011 0.0009 0.001 0.0009 0.0006 0.0005 0.0007 0.0007 0.0009

0.001 0.0009 0.0013 0.0009 1E-09 0.0009 1E-09 0.0012 0.0011 0.0009 0.0008 0.001 0.0013 1E-09 1E-09

0.0017 1E-09 1E-09 0.0012 0.0014 0.0016 1E-09 1E-09 0.0018 0.0017 1E-09 0.0015 0.0019 1E-09 1E-09

0.0018 1E-09 1E-09 0.0013 0.0015 0.0016 1E-09 0.0018 0.0019 0.0017 0.0015 1E-09 1E-09 1E-09 1E-09

0.0018 0.0019 0.0025 0.0013 0.0014 0.0016 0.0024 0.002 0.002 0.0019 0.0015 0.0017 1E-09 0.0017 1E-09

0.0017 0.0017 0.0022 0.0012 0.0014 0.0016 0.0021 1E-09 1E-09 0.0017 0.0014 0.0016 0.0019 0.0016 0.002

0.0019 0.0018 0.0026 0.0015 0.0017 0.0019 0.0024 0.0019 0.0021 0.002 0.0017 0.0017 0.0019 0.0017 1E-09

0.0024 0.0022 0.0032 0.0017 0.0021 0.0022 0.003 0.0024 0.0025 0.0025 0.002 0.002 0.0021 0.002 0.0025


200


gi|115926329| PREDICTED: hypothetical protein (solute carrier family 8

sodium calcium exchanger)

3E-06 9E-06 2E-05 1E-09 7E-06 1E-09

gi|115955930| PREDICTED: similar to CG16944-PC (solute carrier family 25) 1E-09 2E-05 1E-09 1E-09 5E-06 4E-06

gi|115940494| PREDICTED: similar to MGC69168 protein (solute carrier

family, mitochondrial)

1E-09 1E-09 1E-09 1E-09 2E-06 3E-06

gi|115956824| PREDICTED: similar to Solute carrier family 25

(mitochondrial carrier; adenine nucleotide translocator),

member 4

5E-05 4E-05 5E-05 2E-05 1E-09 1E-09

gi|115935979| PREDICTED: similar to voltage-dependent anion channel 2 . 7E-05 8E-05 9E-05 2E-05 1E-09 5E-05

gi|115940270| PREDICTED: hypothetical protein, partial (oxoglutarate) 1E-09 1E-09 3E-06 1E-09 1E-09 1E-09

gi|115940857| PREDICTED: similar to aldose reductase . 1E-05 1E-05 2E-05 1E-09 1E-09 1E-09

gi|115978426| PREDICTED: hypothetical protein (dihydrolipoyllysin S

succinyltransferase)

5E-06 1E-05 3E-06 1E-09 1E-09 1E-09

gi|115945027| PREDICTED: hypothetical protein isoform 1 (integral

membrane protein 1 oligosaccharyltransferase complex)

1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115968074| PREDICTED: hypothetical protein (aldehyde dehydrogenase) 5E-06 7E-06 8E-06 1E-09 1E-09 6E-06

gi|115929988| PREDICTED: similar to Dihydrolipoyl dehydrogenase,

mitochondrial precursor (Dihydrolipoamide dehydrogenase)

1E-09 5E-06 6E-06 1E-09 1E-09 2E-06

gi|115924009| PREDICTED: similar to Methylthioadenosine phosphorylase 1E-09 1E-09 1E-09 3E-05 1E-09 2E-05

gi|115956413| PREDICTED: similar to glutamine synthetase . 1E-05 3E-05 1E-05 7E-06 4E-06 3E-06

gi|115729152| PREDICTED: similar to triosephosphate isomerase 1E-09 1E-09 6E-06 5E-06 1E-09 5E-06

gi|115697859| PREDICTED: hypothetical protein (succinyl CoA ligase) 6E-06 1E-05 1E-05 1E-09 1E-09 1E-05

gi|115925449| PREDICTED: similar to Fructose-1,6-bisphosphatase 1, like 3E-05 2E-05 4E-05 1E-09 1E-09 1E-09

gi|115968426| PREDICTED: similar to arginine kinase 7E-06 3E-06 4E-06 6E-06 4E-06 3E-06

gi|115961332| PREDICTED: similar to glutamate dehydrogenase 1 9E-06 7E-06 1E-09 1E-09 1E-09 1E-09

gi|115931667| PREDICTED: similar to aldehyde dehydrogenase 1A2 isoform

2 .

1E-05 8E-06 9E-06 7E-06 1E-09 1E-09

gi|115922509| PREDICTED: similar to glutamic-oxaloacetic transaminase 2,

mitochondrial (aspartate aminotransferase 2) isoform 2

6E-06 1E-05 2E-05 1E-09 1E-09 1E-09

gi|115697801| PREDICTED: similar to Nicotinamide nucleotide

transhydrogenase

4E-06 1E-09 9E-06 2E-06 1E-06 1E-09

gi|115964994| PREDICTED: similar to Fumarylacetoacetase

(Fumarylacetoacetate hydrolase) (Beta-diketonase) (FAA),

partial

1E-09 1E-09 8E-06 1E-09 1E-09 3E-06

gi|115924987| PREDICTED: similar to Aldehyde dehydrogenase 9 family,

member A1 like 1

6E-06 1E-05 4E-06 6E-06 7E-06 5E-06

gi|115932265| PREDICTED: hypothetical protein (glutamic-oxaloacetic

transaminase 1 fusion)

2E-06 8E-06 1E-05 1E-09 7E-06 1E-09

gi|115934826| PREDICTED: similar to Fructose-1,6-bisphosphatase 1 (D-

fructose-1,6-bisphosphate 1-phosphohydrolase 1) (FBPase 1)

2E-05 3E-05 4E-05 3E-06 1E-09 3E-06

gi|115921420| PREDICTED: hypothetical protein, partial ((phosphoglycerate

dehydrogenase)

5E-05 0.0001 0.0001 0.0001 6E-05 0.0003

gi|115972622| PREDICTED: similar to GA19181-PA, partial (NAD dependent

epimerase/dehydratase)

2E-05 5E-05 5E-05 3E-06 4E-06 1E-05

gi|115744264| PREDICTED: similar to Isocitrate dehydrogenase 2 (NADP+),

mitochondrial .

1E-05 1E-05 2E-05 8E-06 6E-06 7E-06

gi|115954217| PREDICTED: similar to hexokinase I 4E-05 3E-05 5E-05 1E-05 1E-05 1E-05

gi|115735613| PREDICTED: similar to D-3-phosphoglycerate dehydrogenase

.

2E-05 3E-05 2E-05 1E-05 9E-06 3E-05

gi|115929815| PREDICTED: similar to Nucleoside diphosphate kinase family

protein

4E-05 4E-05 1E-09 1E-09 1E-09 1E-09

gi|115929324| PREDICTED: similar to glucose-6-phosphate 1-

dehydrogenase, partial

2E-05 2E-05 2E-05 9E-06 7E-06 1E-05

gi|115964543| PREDICTED: similar to transaldolase 7E-05 8E-05 8E-05 8E-06 1E-05 3E-05

gi|115973105| PREDICTED: hypothetical protein (pyruvate kinase) 3E-05 1E-05 3E-05 2E-05 2E-05 1E-05

gi|115944251| PREDICTED: similar to transketolase isoform 1 1E-05 3E-05 3E-05 6E-06 1E-05 1E-05

gi|115959412| PREDICTED: similar to fructose-biphosphate aldolase . 5E-05 4E-05 4E-05 5E-05 2E-05 2E-05

gi|115972829| PREDICTED: similar to glucose-6-phosphate isomerase . 4E-05 3E-05 3E-05 2E-05 2E-05 1E-05

gi|115685450| PREDICTED: similar to MGC68486 protein, partial

(phosphogluconate dehydrogenase)

0.0002 0.0001 0.0003 0.0002 0.0005 0.0006

gi|115738231| PREDICTED: similar to glyceraldehydephosphate

dehydrogenase isoform 1

8E-05 6E-05 5E-05 5E-05 3E-05 0.0001

gi|115939485| PREDICTED: similar to malate dehydrogenase . 2E-05 4E-05 5E-05 7E-06 1E-09 7E-06

gi|115929978| PREDICTED: similar to OTTHUMP00000039401 (similar to

leucine rich repeat containing 16 isoform)

7E-06 1E-06 7E-06 1E-09 1E-09 1E-09

gi|115939602| PREDICTED: similar to GRAAL2 protein (SRCR, kringle,pan

apple, trypsin domains)

3E-06 1E-09 1E-06 1E-09 1E-09 1E-09

gi|115972933| PREDICTED: similar to B-cell receptor associated protein 1E-05 4E-05 3E-05 1E-09 1E-09 1E-09

gi|115941346| PREDICTED: hypothetical protein, partial (ATPase H+

transporting)

5E-05 7E-05 2E-05 2E-05 4E-05 3E-05

gi|115939860| PREDICTED: similar to ENSANGP00000014225 (lectin,

mannose binding)

2E-06 2E-06 1E-05 1E-09 1E-09 1E-09

gi|115929116| PREDICTED: similar to aminoimidazole-4-carboxamide

ribonucleotidetransformylase/IMP cyclohydrolase

1E-05 1E-05 1E-05 2E-06 1E-09 4E-06

gi|115710920| PREDICTED: similar to Na+/K+ ATPase alpha subunit 1E-05 2E-05 1E-05 6E-06 8E-06 9E-06

gi|115944063| PREDICTED: hypothetical protein (srcr domains) 3E-05 2E-05 2E-05 7E-06 3E-05 1E-09

NSAF

201

NSAF



























3E-05 4E-05 1E-09 3E-05 1E-09 4E-05 4E-05 5E-05 0.0001 3E-05 2E-05 2E-05 0.0001 0.0001 8E-05












0.0002 0.0002 0.0003 0.0003 0.0002 0.0002 0.0001 0.0003 0.0001 0.0002 0.0002 0.0001 0.0003 0.0003 0.0003

4E-05 6E-05 7E-05 9E-05 0.0001 5E-05 0.0001 9E-05 6E-05 2E-05 3E-05 3E-05 8E-05 0.0001 0.0001










202


gi|115619038| PREDICTED: similar to arylsulfatase 1E-09 1E-05 2E-05 1E-09 1E-09 4E-06

gi|115968753| PREDICTED: similar to scavenger receptor cysteine-rich

protein type 12 precursor

0.0006 0.0003 0.0004 0.0001 1E-09 1E-09


protein type 12 precursor, partial

2E-05 2E-05 6E-06 1E-09 1E-09 1E-09



1E-09 1E-09 2E-06 1E-09 1E-09 1E-09

gi|47551157| scavenger receptor cysteine-rich protein type 12 . 5E-05 3E-05 4E-05 1E-05 1E-05 1E-09

gi|115950487| PREDICTED: similar to arylsulfatase isoform 2 . 4E-05 7E-05 8E-05 3E-05 2E-05 8E-06

gi|47551023| complement component C3 4E-05 4E-05 3E-05 3E-06 3E-06 1E-05

gi|115937346| PREDICTED: similar to apolipophorin precursor protein 8E-06 1E-05 1E-05 2E-06 3E-06 2E-06

gi|115966189| PREDICTED: similar to H(+)-transporting ATPase beta

subunit

8E-05 0.0001 0.0001 6E-05 8E-05 6E-05

gi|115970375| PREDICTED: similar to melanotransferrin/EOS47 9E-05 6E-05 3E-05 7E-05 7E-05 6E-05



0.0003 0.0002 0.0003 4E-05 0.0001 2E-05

gi|115757308| PREDICTED: hypothetical protein (clathrin coat assembly

protein)

3E-06 3E-06 1E-09 1E-09 1E-09 1E-09

gi|115924764| PREDICTED: similar to formin binding protein 1 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115951468| PREDICTED: similar to coatomer protein gamma 2-subunit,

partial

9E-06 1E-05 1E-05 1E-09 1E-09 1E-09

gi|115943043| PREDICTED: similar to VPS13C-1A protein (vacuolar protein

sorting 13 homolog C)

5E-07 3E-06 2E-06 1E-09 1E-09 1E-09

gi|47551307| syntaxin binding protein 1 4E-06 2E-06 5E-06 1E-09 1E-09 1E-09

gi|115753213| PREDICTED: similar to beta COP, partial 7E-06 2E-05 2E-05 1E-09 1E-09 1E-09

gi|115932047| PREDICTED: similar to archain 1E-09 7E-06 1E-09 1E-09 1E-09 1E-09

gi|115673346| PREDICTED: similar to alpha-cop protein, partial 1E-06 3E-06 5E-06 1E-09 2E-06 1E-09

gi|115934500| PREDICTED: similar to Adaptor protein complex AP-2, alpha

2 subunit

1E-09 1E-09 1E-06 2E-06 2E-06 1E-09

gi|47551147| N-ethylmaleimide-sensitive factor (NSF) 2E-06 2E-06 2E-06 3E-06 2E-06 1E-06

gi|115738335| PREDICTED: similar to beta-adaptin Drosophila 1 isoform 7 2E-06 5E-06 8E-06 1E-09 1E-09 1E-09

gi|115928436| PREDICTED: hypothetical protein (SH3-domain GRB2-like

endophilin B1)

1E-05 7E-06 4E-06 6E-06 1E-09 3E-06

gi|115976584| PREDICTED: similar to Coatomer protein complex, subunit

beta 2 (beta prime)

1E-09 1E-06 6E-06 1E-06 1E-06 1E-09

gi|115662788| PREDICTED: similar to ENSANGP00000019991, partial

(adaptin)

1E-09 1E-05 6E-06 1E-05 1E-05 1E-09

gi|115940967| PREDICTED: similar to Dynamin 2, partial 2E-05 2E-05 2E-05 1E-09 1E-09 1E-09

gi|115931510| PREDICTED: hypothetical protein (sorting nexin) 2E-05 1E-05 2E-05 3E-06 1E-09 1E-09

gi|115968951| PREDICTED: hypothetical protein (adaptor protein complex

AP2)

8E-06 6E-06 2E-05 1E-05 1E-09 2E-06

gi|160623368| putative flotillin 2E-05 1E-05 2E-05 3E-05 1E-09 1E-05

gi|115952079| PREDICTED: hypothetical protein, partial (vacuolar protein

sorting 35)

1E-05 2E-05 2E-05 2E-06 2E-06 1E-09

gi|115889707| PREDICTED: similar to Pdcd6ip protein (Programmed cell

death 6 interacting protein)

1E-05 1E-05 7E-06 4E-06 5E-06 1E-06

gi|115970724| PREDICTED: similar to Valosin containing protein, partial 2E-06 7E-06 1E-05 4E-06 1E-09 1E-09

gi|115928607| PREDICTED: hypothetical protein (flotillin) 1E-05 1E-05 2E-05 1E-09 1E-05 1E-09

gi|115963658| PREDICTED: major vault protein 2E-05 3E-05 2E-05 1E-05 5E-06 3E-05

gi|115936009| PREDICTED: similar to ENSANGP00000009431 (flotilin) 4E-05 7E-05 1E-04 2E-05 2E-05 3E-05

gi|115759362| PREDICTED: similar to sulfotransferase ST1B2 . 1E-05 8E-06 1E-05 1E-09 1E-09 7E-06

gi|115955254| PREDICTED: similar to alpha macroglobulin . 9E-07 1E-09 2E-06 1E-09 1E-09 1E-09

gi|115929526| PREDICTED: similar to aminopeptidase N . 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115940918| PREDICTED: hypothetical protein (aminopeptidase like 1) 3E-06 5E-06 6E-06 1E-09 6E-06 1E-09

gi|115945106| PREDICTED: similar to cytosolic nonspecific dipeptidase,

partial

1E-05 5E-06 1E-05 1E-09 1E-09 1E-09

gi|115953922| PREDICTED: similar to Peptidase (mitochondrial processing)

beta isoform 1

3E-06 5E-06 1E-05 7E-06 9E-06 9E-06

gi|115924997| PREDICTED: similar to MGC84288 protein (tissue inhibitor of

metalloproteinase TIMP)

2E-05 3E-05 1E-05 5E-06 1E-09 1E-05

gi|115971224| PREDICTED: hypothetical protein (aminopeptidase) 1E-09 1E-09 3E-06 5E-06 1E-05 1E-05

gi|115972931| PREDICTED: similar to sulfatase 1 precursor 1E-09 1E-09 3E-05 2E-06 1E-09 1E-09

gi|115968724| PREDICTED: similar to transglutaminase-like protein . 8E-06 2E-05 1E-05 5E-06 8E-06 1E-06

gi|115953033| PREDICTED: similar to Aminopeptidase puromycin sensitive 2E-06 1E-05 8E-06 1E-05 9E-06 3E-06

gi|115973804| PREDICTED: similar to non-receptor protein tyrosine

phosphatase

6E-06 6E-06 7E-06 3E-06 1E-09 1E-09

gi|115958557| PREDICTED: similar to Arginyl-tRNA synthetase 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115691123| PREDICTED: similar to Eukaryotic translation initiation factor

2, subunit 1 alpha, 35kDa isoform 1

1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115652043| PREDICTED: similar to histidyl-tRNA synthetase, partial 1E-09 1E-09 1E-09 2E-06 1E-09 1E-09

gi|115941920| PREDICTED: similar to ribosomal protein L7, partial 2E-05 5E-05 4E-05 1E-09 1E-09 1E-09

gi|115963308| PREDICTED: similar to ribosomal protein L32 1E-09 1E-09 1E-09 4E-05 1E-09 6E-05

gi|115963185| PREDICTED: hypothetical protein isoform 2 (ribosomal

protein L15)

6E-06 2E-05 2E-05 1E-09 1E-09 2E-05

gi|115970594| PREDICTED: similar to interleukin enhancer binding factor 3,

partial

1E-09 5E-06 3E-06 1E-09 1E-09 1E-09

gi|115921067| PREDICTED: similar to ribosomal protein L23a 1E-09 1E-09 8E-06 1E-09 1E-09 1E-05

gi|115972878| PREDICTED: similar to Rps9-prov protein (ribosomal protein

S9)

1E-05 2E-05 7E-06 1E-09 1E-09 1E-09

NSAF

203

NSAF



5E-05 0.0002 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09 0.0002 0.0002 7E-05 1E-09 6E-05 1E-09




2E-05 1E-09 5E-05 3E-05 8E-05 6E-05 5E-05 0.0001 3E-05 1E-05 1E-05 1E-05 4E-05 6E-05 8E-05





7E-05 8E-05 6E-05 1E-09 0.0001 7E-05 2E-05 3E-05 2E-05 9E-05 9E-05 6E-05 7E-06 5E-05 9E-05














































204


gi|115954825| PREDICTED: similar to S6 ribosomal protein isoform 1 1E-09 5E-06 1E-09 9E-06 1E-09 2E-05

gi|115957127| PREDICTED: similar to ribosomal protein L5 1E-05 2E-05 2E-05 1E-09 1E-09 1E-09

gi|5532389| microtubule-associated protein 8E-06 2E-06 1E-09 1E-09 1E-09 1E-09

gi|115936334| PREDICTED: hypothetical protein (proteasome subunit) 1E-05 2E-05 1E-09 4E-06 1E-09 1E-09

gi|115965220| PREDICTED: similar to histone macroH2A1.1 7E-06 1E-05 1E-05 1E-09 1E-09 1E-09

gi|115949865| PREDICTED: similar to putative eukaryotic petide chain

release factor subunit 1

1E-09 3E-06 7E-06 1E-09 1E-09 1E-09

gi|115935400| PREDICTED: hypothetical protein (inosine monophosphate

dehydrogenase)

6E-06 1E-09 1E-05 6E-06 3E-06 3E-06

gi|115934021| PREDICTED: similar to Psmc6 protein (proteasome 26S

subunit, ATPase)

5E-06 1E-05 3E-06 5E-06 1E-09 4E-06

gi|115968684| PREDICTED: similar to retinoblastoma binding protein 4

variant isoform 1

8E-06 3E-06 7E-06 1E-09 1E-09 1E-09

gi|115965680| PREDICTED: hypothetical protein (Heterogeneous nuclear

ribonucleoprotein L)

1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115959476| PREDICTED: hypothetical protein, partial (translation

initiation factor eIF-2B)

2E-05 4E-06 1E-05 1E-09 9E-06 1E-09

gi|115939698| PREDICTED: similar to 26S proteasome subunit p44.5 1E-09 1E-09 1E-09 1E-09 1E-09 3E-06

gi|115940610| PREDICTED: hypothetical protein, partial (Ribosomal protein) 1E-05 9E-06 2E-05 8E-06 1E-09 8E-06

gi|115932417| PREDICTED: similar to ribosomal protein S10 isoform 2 1E-09 1E-09 9E-06 1E-09 1E-09 1E-09

gi|115968696| PREDICTED: similar to paraspeckle protein 1 isoform beta 6E-06 8E-06 5E-06 1E-09 1E-09 1E-09

gi|115963700| PREDICTED: similar to Ribosomal protein L14, partial 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115968722| PREDICTED: similar to DEAD-box RNA-dependent helicase

p68 isoform 2

6E-06 9E-06 9E-06 1E-06 1E-09 1E-09

gi|115946250| PREDICTED: similar to Shmt2 protein, partial 8E-06 8E-06 1E-09 1E-05 2E-05 1E-05

gi|115970676| PREDICTED: similar to 40S ribosomal protein S13 1E-09 1E-09 3E-05 7E-06 1E-09 1E-09

gi|115840564| PREDICTED: similar to DEAD (Asp-Glu-Ala-Asp) box

polypeptide 48

3E-06 3E-06 1E-09 1E-09 1E-09 1E-09

gi|68534982| translation elongation factor 1B gamma subunit 1E-05 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115934350| PREDICTED: similar to ubiquitin-activating enzyme E1 1E-09 2E-06 6E-06 1E-09 1E-09 3E-06

gi|115975522| PREDICTED: similar to Ribophorin II 1E-09 2E-06 4E-06 1E-09 1E-09 3E-06

gi|115951495| PREDICTED: similar to ribosomal protein L6 isoform 2 5E-06 1E-09 1E-05 9E-06 1E-09 8E-06

gi|115673317| PREDICTED: similar to translation initiation factor 2 gamma

subunit

3E-06 3E-06 1E-09 3E-06 3E-06 1E-05

gi|115939546| PREDICTED: similar to Ribosomal protein S2 9E-06 1E-09 1E-05 2E-05 1E-09 2E-05

gi|148539604| polyA-binding protein 4E-06 1E-09 1E-09 2E-06 1E-09 7E-06

gi|115936872| PREDICTED: similar to 26S proteasome regulatory chain 4 8E-06 6E-06 1E-09 1E-09 1E-09 1E-09

gi|115955282| PREDICTED: hypothetical protein (heterogenous nuclear

ribonucleoprotein K)

3E-06 3E-06 4E-06 1E-09 1E-09 1E-09

gi|115945280| PREDICTED: hypothetical protein, partial (ADP ribosylation

factor)

0.0001 0.0001 7E-05 1E-09 1E-09 1E-09

gi|115953630| PREDICTED: hypothetical protein (N-acetylneuraminic acid

synthase)

4E-05 2E-05 5E-05 4E-06 1E-09 7E-06

gi|115661720| PREDICTED: hypothetical protein (sucinate semialdehyde

dehydrogenase)

1E-09 1E-09 6E-06 5E-06 6E-06 1E-05

gi|115950705| PREDICTED: similar to Ribosomal protein S5 1E-09 1E-09 1E-09 1E-05 1E-09 1E-05

gi|115936522| PREDICTED: similar to ribosomal protein S26e 1E-09 9E-06 1E-09 1E-09 1E-09 7E-06

gi|115973107| PREDICTED: hypothetical protein, partial (NADH cytochrome

reductase)

3E-05 6E-05 4E-05 1E-09 1E-09 1E-09

gi|115958412| PREDICTED: similar to 34/67 kD laminin binding protein 1E-09 8E-06 5E-06 1E-05 2E-05 2E-05

gi|115929247| PREDICTED: similar to heterogeneous nuclear

ribonucleoprotein H .

2E-05 1E-05 3E-05 1E-09 2E-05 1E-09

gi|115950168| PREDICTED: similar to ubiquitin-activating enzyme E1-like 2 2E-06 3E-06 7E-06 2E-06 1E-06 1E-09

gi|115960623| PREDICTED: similar to LOC496154 protein (poly (ADP-ribose)

polymerase family)

2E-06 5E-06 6E-06 6E-06 1E-09 1E-09

gi|115975235| PREDICTED: similar to signal transducer and activator of

transcription 5B

2E-06 6E-06 9E-06 1E-09 4E-06 1E-09

gi|158327684| cytochrome oxidase subunit 2 3E-05 3E-05 4E-05 2E-05 1E-09 1E-09

gi|115970083| PREDICTED: similar to ubiquitin/40S ribosomal protein S27a

fusion protein

1E-09 1E-09 5E-05 1E-09 1E-09 1E-09

gi|115959136| PREDICTED: similar to ribosomal protein S8e 5E-06 2E-05 1E-05 1E-09 1E-09 4E-05

gi|115928686| PREDICTED: similar to alpha isoform of regulatory subunit A,

protein phosphatase 2, partial

1E-09 1E-09 1E-09 5E-06 6E-06 1E-09

gi|115946604| PREDICTED: similar to nuclear RNA helicase Bat1 isoform 2 6E-06 6E-06 1E-05 5E-06 3E-06 1E-09

gi|115968341| PREDICTED: similar to Ribosomal protein L3 2E-05 3E-05 4E-05 3E-06 1E-09 2E-05

gi|115911567| PREDICTED: hypothetical protein, partial (aspartyl tRNA

synthase)

4E-05 4E-05 3E-05 8E-06 6E-06 7E-06

gi|115970525| PREDICTED: similar to elongation factor 1 alpha isoform 1 1E-09 1E-09 1E-09 3E-05 4E-05 4E-05

gi|115649138| PREDICTED: similar to ribosomal protein S3 4E-05 6E-05 3E-05 2E-05 2E-05 4E-05

gi|115965257| PREDICTED: similar to heterogeneous nuclear

ribonucleoprotein R

2E-05 3E-05 2E-05 5E-06 7E-06 2E-06

gi|115946394| PREDICTED: similar to RNA binding motif protein 28 2E-05 3E-05 5E-06 1E-09 1E-09 1E-09

gi|47550983| nuclear intermediate filament protein 4E-06 7E-06 1E-09 4E-06 1E-09 1E-05

gi|148539566| eukaryotic initiation factor 4a 1E-05 1E-05 2E-05 2E-05 2E-05 5E-06

gi|115955810| PREDICTED: hypothetical protein (ribosomal protein L4) 3E-05 6E-05 5E-05 1E-09 1E-05 2E-05

gi|115974148| PREDICTED: similar to Ribophorin I 3E-05 3E-05 3E-05 2E-05 1E-09 7E-06

gi|47551065| histone H3 1E-09 5E-05 4E-05 8E-05 5E-05 0.0002

gi|115926828| PREDICTED: similar to eukaryotic translation elongation

factor isoform 2

9E-06 6E-06 9E-06 8E-06 2E-06 2E-05

NSAF

205

NSAF











































5E-06 4E-05 1E-09 4E-05 3E-05 4E-05 1E-09 4E-05 4E-05 1E-09 1E-05 1E-09 1E-09 1E-09 0.0001














2E-05 0.0001 8E-05 3E-05 4E-05 2E-05 0.0002 0.0001 7E-05 1E-05 7E-06 7E-06 4E-05 1E-09 3E-05


206


gi|115933977| PREDICTED: similar to histone H2A-3 1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|47551085| late histone L3 H2a 0.0002 0.0001 0.0002 6E-05 1E-09 1E-09

gi|47551121| mitochondrial ATP synthase alpha subunit precursor 4E-05 3E-05 5E-05 2E-05 5E-05 2E-05

gi|115974434| PREDICTED: similar to adenosylhomocysteinase 6E-05 5E-05 0.0001 4E-05 4E-05 8E-05

gi|47551075| late histone L1 H2b 1E-09 0.0001 9E-05 8E-05 1E-09 0.0001

gi|115970523| PREDICTED: similar to elongation factor 1 alpha isoform 2 8E-05 8E-05 0.0001 1E-09 1E-09 1E-09

gi|47551061| H4 histone protein 0.0005 0.0006 0.0009 0.0004 0.0002 0.0002

gi|115974576| PREDICTED: similar to putative ribosomal protein S14e 1E-09 9E-06 2E-05 1E-09 1E-09 2E-05

gi|115970054| PREDICTED: similar to putative RNA helicase (DEAD box) 7E-06 1E-09 1E-09 2E-05 2E-05 2E-05

gi|115931135| PREDICTED: similar to putative RNA binding protein KOC 1E-05 1E-05 5E-06 7E-06 7E-06 7E-06

gi|115955764| PREDICTED: similar to Cry5 protein 9E-06 7E-06 1E-09 1E-09 1E-09 1E-09

gi|115976887| PREDICTED: similar to START domain containing protein 9E-05 0.0001 7E-05 6E-05 2E-05 4E-06

gi|47551041| ER calcistorin 5E-06 3E-05 3E-05 1E-09 1E-09 1E-09

gi|115937420| PREDICTED: catalase 1E-09 2E-05 2E-05 2E-06 8E-06 1E-05

gi|115924280| PREDICTED: similar to flavoprotein subunit of complex II,

partial

1E-09 1E-09 1E-09 1E-09 1E-09 1E-09

gi|115926010| PREDICTED: similar to glutathione peroxidase, partial 2E-05 1E-05 2E-05 2E-05 1E-09 1E-09

gi|115977049| PREDICTED: hypothetical protein (chaperonin containing

TCP1)

3E-06 9E-06 4E-06 8E-06 3E-06 5E-06

gi|115959568| PREDICTED: similar to glutathione reductase 2E-06 1E-09 3E-06 3E-06 1E-09 3E-06

gi|115628087| PREDICTED: similar to chaperonin containing TCP1, subunit

6A isoform 1

8E-06 1E-05 6E-06 1E-09 1E-09 5E-06

gi|115815329| PREDICTED: similar to Cct6a protein, partial (chaperonin

containing TCP1)

2E-05 1E-09 1E-09 5E-06 1E-05 1E-09

gi|115905691| PREDICTED: similar to Pkm2 protein, partial (Pyruvate kinase

isoenzyme type M2)

9E-05 5E-05 8E-05 1E-09 1E-09 1E-09

gi|115956030| REDICTED: similar to chaperonin 1E-09 1E-09 1E-09 6E-06 5E-06 6E-06

gi|115936458| PREDICTED: similar to Aconitase 2, mitochondrial isoform 1 1E-05 2E-05 1E-09 4E-06 1E-09 1E-05

gi|115924889| PREDICTED: similar to Chaperonin containing TCP1, subunit

5 (epsilon)

7E-06 1E-09 1E-09 1E-05 1E-05 4E-06

gi|115954976| PREDICTED: similar to heat shock protein protein 70kDa 1E-09 1E-09 1E-09 2E-05 1E-05 2E-05

gi|115944173| PREDICTED: similar to ENSANGP00000006016 isoform 1

(dual oxidase maturation factor)

9E-06 1E-05 1E-05 1E-09 1E-09 1E-09

gi|115956641| PREDICTED: similar to LOC495278 protein (chaperonin

containing TCP1)

5E-06 1E-05 1E-05 2E-06 1E-09 2E-06


4 (delta)

7E-06 1E-05 6E-06 1E-09 3E-06 2E-06

gi|115970125| PREDICTED: similar to MGC139263 protein (Annexin) 5E-06 5E-06 3E-06 1E-09 1E-09 6E-06

gi|115964822| PREDICTED: similar to mitochondrial chaperonin Hsp56 5E-06 5E-06 1E-05 1E-09 1E-09 6E-06

gi|115944450| PREDICTED: similar to chaperonin subunit 8 theta 1E-09 7E-06 6E-06 2E-06 1E-09 1E-09


3 (gamma)

2E-06 1E-09 3E-06 4E-06 1E-09 8E-06

gi|115956665| PREDICTED: similar to heat shock protein protein 70kDa 2E-05 3E-05 2E-05 2E-05 1E-09 3E-05

gi|115891388| PREDICTED: similar to heat shock 90 kDa protein, partial 3E-05 4E-05 6E-05 4E-06 3E-06 1E-09

gi|115931665| PREDICTED: similar to MGC89020 protein (mitochondrial

aldehyde dehydrogenase 2)

7E-06 8E-06 1E-05 2E-05 1E-05 6E-05

gi|115944169| PREDICTED: similar to dual oxidase 1 isoform 1 . 2E-05 3E-05 3E-05 7E-07 1E-05 1E-09

gi|47551251| heat shock protein gp96 5E-05 3E-05 4E-05 1E-09 1E-09 1E-09

gi|115972586| PREDICTED: similar to 71 Kd heat shock cognate protein 7E-05 7E-05 7E-05 6E-05 8E-05 7E-05

gi|115924727| PREDICTED: similar to major yolk protein precursor 1E-09 1E-09 1E-09 0.0002 1E-09 1E-09

gi|47551123| major yolk protein 0.0007 0.0004 0.0004 0.0002 0.0002 6E-05

gi|53913428| major yolk protein 0.0007 1E-09 0.0005 1E-09 0.0002 1E-09

gi|73746392| major yolk protein 0.0009 0.0005 0.0006 0.0002 0.0002 6E-05

gi|115974673| PREDICTED: similar to LOC407663 protein (shor chain

dehydrogenase)

2E-05 1E-05 1E-05 1E-09 4E-06 1E-09

gi|115803435| PREDICTED: hypothetical protein (similar to cleavage

stimulation factor fusion)

6E-06 6E-06 5E-06 2E-06 1E-09 1E-09

gi|115967840| PREDICTED: similar to 0910001A06Rik protein isoform 2 2E-05 3E-05 9E-06 1E-09 1E-09 1E-09

gi|115760426| PREDICTED: similar to conserved hypothetical protein 7E-06 2E-06 1E-09 2E-06 1E-09 1E-09

gi|115934013| PREDICTED: hypothetical protein (ANK domain and SH3

domain)

1E-09 1E-06 1E-09 1E-06 1E-09 5E-06

gi|115929076| PREDICTED: hypothetical protein 1E-09 1E-09 2E-05 1E-09 1E-09 1E-09

gi|115925359| PREDICTED: similar to LZP (CCP and zona pellucida

superfamily)

2E-06 6E-06 2E-05 2E-06 1E-09 1E-09

gi|115964419| PREDICTED: hypothetical protein . 1E-05 5E-06 1E-05 1E-06 5E-06 1E-09

gi|115963910| PREDICTED: hypothetical protein, partial . 2E-05 2E-05 1E-05 5E-05 5E-05 1E-09

gi|115621989| PREDICTED: hypothetical protein 3E-05 6E-06 9E-05 3E-06 1E-09 8E-06

NSAF

207

NSAF


1E-09 1E-09 1E-09 0.0004 1E-09 0.0001 0.0001 0.0002 5E-05 2E-05 1E-09 1E-09 0.0003 0.0004 6E-05

2E-05 1E-09 1E-09 0.0003 0.0004 1E-09 5E-05 1E-09 4E-05 2E-05 2E-05 2E-05 0.0003 0.0003 0.0001



0.0002 1E-09 3E-05 0.0002 1E-09 0.0002 0.0002 0.0003 0.0001 0.0002 0.0002 0.0001 0.0003 1E-09 1E-09


0.0003 0.0002 0.0003 0.0005 0.0005 0.0006 1E-09 0.0005 0.0004 0.0005 1E-09 0.0002 0.0009 1E-09 0.0004





2E-06 8E-05 9E-05 6E-05 4E-05 8E-05 6E-05 3E-05 0.0001 0.0001 4E-05 7E-05 0.0001 9E-05 8E-05


























3E-05 4E-05 4E-05 9E-05 6E-05 7E-05 4E-05 4E-05 7E-05 4E-05 2E-05 2E-05 6E-05 0.0001 0.0001

1E-09 0.0006 0.0004 0.0002 0.0002 0.0002 0.0002 1E-09 9E-05 0.0004 0.0005 1E-09 1E-09 1E-09 0.0003

0.0005 0.0007 0.0004 0.0002 0.0002 0.0002 0.0003 0.0002 9E-05 0.0004 0.0008 0.0007 0.0003 0.0004 0.0003

0.0005 0.0007 0.0004 0.0003 0.0003 0.0003 0.0003 0.0002 9E-05 0.0004 0.0008 0.0007 0.0003 0.0004 0.0004

0.0005 0.0009 0.0005 0.0003 0.0003 0.0003 0.0003 0.0003 0.0001 0.0005 0.0009 0.0007 0.0004 0.0004 0.0005










2E-05 3E-05 4E-06 6E-06 1E-05 3E-05 4E-05 4E-05 5E-06 9E-06 5E-06 5E-06 3E-05 2E-05 0.0004

208

209

CHAPTER V

Highly variable immune-response proteins (185/333) from the sea urchin,

Strongylocentrotus purpuratus: Proteomic analysis identifies diversity within and

between individuals.

Published in

Journal of Immunology

182(4): 2203-2212


Nair SV1 - Experimental design -Project supervision

Smith LC2 – Technical support

Raftos DA1 - Experimental design - Project supervision

1Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia.

2Department of Biological Sciences, George Washington University, Washington, DC, 20052, USA.

Dheilly N.M., Nair S.V., Smith L.C., Raftos D.A. (2009) Highly variable immune-

response (185/333) from the sea urchin Strongylocentrotus purpuratus: Proteomic analysis

identifies diversity within and between individuals. Journal of Immunology. 182(4): 2203-

12.

210

211

5.1. Preface

A major difference observed between previous macroarray analysis and the current

proteomics studies (Chapters 2 to 4) was the low abundance of 185/333 proteins identified

by 2DE and shotgun proteomics. Even though these molecules constituted up to 60% of

bacterially challenged cDNA library, only a single 185/333 protein was identified in the

coelomic fluid proteome of S. purpuratus and none were found in H. erythrogramma. The

following two Chapters of this thesis specifically investigated 185/333 proteins with the

aims of further characterizing their variability and providing new clues on their function in

the sea urchin immune system. In Chapter 5, we characterized the variability of 185/333

proteins within and between individuals and identified differences in expression in

response to different challenges using a combination of proteomics technologies.

212

213

5.2. Abstract

185/333 genes and transcripts from the purple sea urchin, Strongylocentrotus

purpuratus, predict high levels of amino acid diversity within the encoded proteins. Based

on their expression patterns, 185/333 proteins appear to be involved in immune responses.

In the present study, one- and two-dimensional Western blots show that 185/333 proteins

exhibit high levels of molecular diversity within and between individual sea urchins. The

molecular masses of 185/333-positive bands or spots range from 30 to 250 kDa with a

broad array of isoelectric points. The observed molecular masses are higher than those

predicted from mRNAs, suggesting that 185/333 proteins form strong associations with

other molecules or with each other. Some sea urchins expressed > 200 distinct 185/333

proteins, and each animal had a unique suite of the proteins that differed from all other

individuals. When sea urchins were challenged in vivo with pathogen-associated molecular

patterns (PAMPs; bacterial LPS and peptidoglycan), the expression of 185/333 proteins

increased. More importantly, different suites of 185/333 proteins were expressed in

response to different PAMPs. This suggests that the expression of 185/333 proteins can be

tailored toward different PAMPs in a form of pathogen-specific immune response.

214

5.3. Introduction

Recent studies of host defense have uncovered profound differences among animal

phyla in the molecules used to mediate immune responses [1-3]. It seems that the immune

systems of different animals have evolved a variety of solutions to meet a basic

requirement to combat pathogens. The resulting, highly diversified immune responses may

reflect the specific physiology, lifespan, habitat, and associated microbial populations of

particular animal groups, or they may have arisen by chance via evolutionary radiation. A

number of metazoan phyla have now been studied, identifying a range of alternative

immunological mechanisms that exhibit high levels of molecular diversity [2]. These

discoveries have driven a paradigm shift in our understanding of invertebrate immune

responses, from systems that are simple and static, to those that are complex and have

novel mechanisms for generating molecular hypervariability, a key requirement for

keeping pace in the “arms race” against microbial pathogens.

Some invertebrate immune systems are proving to be surprisingly complex. Recent

analyses of the purple sea urchin (Strongylocentrotus purpuratus) genome identified

several large gene families, including gene models for 222 TLRs, 203 NOD-like receptors

(NLR), 218 scavenger receptor cysteine-rich (SRCR) molecules, and 104 C-type lectins [4-

6]. The complexity and large size of these gene families suggest that the receptors they

encode may recognize individual pathogen-associated molecular patterns (PAMPs) with a

high degree of specificity. They might also act combinatorially, providing highly diverse

recognitive capabilities [5]. In addition to diversified receptors, the S. purpuratus genome

contains homologues of Rag 1 and Rag 2, the molecules responsible for the somatic

recombination of immunoglobulins in vertebrates [7]. This suggests that the molecular

tools required to generate molecular hypervariability might also exist among invertebrates.

215

185/333 genes represent another high-diversity immune response system in sea

urchins. This family was first identified during a transcriptome analysis of sea urchin

immune response genes [8]. Genes that were up-regulated in coelomocytes (immune cells)

after the injection of LPS were identified by screening high-density arrayed, conventional

cDNA libraries with probes generated by subtractive suppression hybridization.

Surprisingly, ~ 60% of the expressed sequence tags (ESTs) characterized in this

transcriptome analysis were members of a set of closely related transcripts with similarities

to two uncharacterized sequences from S. purpuratus, called DD185 (GenBank accession

AF228877 [9]) and EST333 (GenBank accession R62081 [10]), hence the designation

185/333.

Screening an arrayed cDNA library constructed from immunologically activated

coelomocytes indicated that the frequency of 185/333 mRNAs was enhanced more than

75-fold compared with a nonactivated arrayed cDNA library [8]. Northern blots also

showed striking increases in 185/333 expression in coelomocytes from bacterially

activated sea urchins compared with injury controls [9]. Based on this significant increase

in gene expression, 185/333 transcripts were investigated in more detail, revealing an

unexpected level of sequence diversity [8, 11]. To date, for 185/333 cDNAs, 689 have

been characterized from 14 sea urchins. These sequences are predicted to encode 286

different proteins [11, 12].

The diversity evident among 185/333 transcripts is generated in several ways.

185/333 mRNAs are comprised of 25 different blocks or “elements” of nucleotide

sequence, that are present or absent in numerous combinations. This results in “element

patterns” that are repeatedly identified in different mRNAs [8, 12], contributing significant

diversity to the family of 185/333 messages. Single nucleotide polymorphisms (SNPs) and

small insertions or deletions (indels) are also frequent in all 185/333 sequences, adding to

diversity. Additionally, there are surprisingly high levels of frame shifts and the insertion

216

of early stop codons in the mRNA sequences [11]. The processes that generate this

diversity among 185/333 mRNAs are not yet well defined, but they may include

differences among the estimated fifty 185/333 gene loci [11, 13, 14], high levels of allelic

polymorphism at each locus, RNA editing, and/or low-fidelity RNA polymerases [15],

followed by posttranslational modifications. All of these processes may have been driven

by positive selection associated with anti-pathogen defense [8, 11, 12, 14]. Nucleotide

sequence variability results in high levels of nonconservative amino acid substitutions

among predicted proteins of the type consistent with intense evolutionary selection

pressure [8, 12, 14, 15].

185/333 mRNAs are predicted to encode proteins with a hydrophobic leader, a

glycine-rich region with multiple endoprotease cleavage sites, an RGD motif, a histidine-

rich region, numerous N-linked and O-linked glycosylation sites, acidic patches, and

several types of tandem and interspersed repeats [11]. The deduced proteins do not contain

cysteines, transmembrane regions, GPI linkage sites, identifiable domains, or any

predictable folding patterns. The only regions with at least some similarity to other

molecules are the RGD motif and one of the histidine-rich domains, which is comparable

to histatins, a group of mammalian salivary proteins with powerful antifungal activities

[16, 17]. Brockton et al. [18] have shown that 185/333 proteins are expressed by two

subsets of coelomocytes (small phagocytes and polygonal cells), and that the number of

185/333+ cells increases in response to immunological challenge. Initial analyses of the

185/333 proteins indicated that recombinant 185/333 proteins appear as multimers and that

native proteins are present in a broad range of sizes, in agreement with the mRNA

sequences [18].

217

Here, we present the first comprehensive analysis of 185/333 proteins. It reveals a

broad diversity of these proteins within individual sea urchins. Different sea urchins

express different suites of 185/333 proteins, and expression is altered by immunological

challenge. The data indicate that differential expression or posttranslational modification of

185/333 proteins might allow S. purpuratus to tailor immune responses toward specific

pathogens.

218


5.4.1. Sea urchins

Adult S. purpuratus were purchased from Marinus Scientific after collection from

the coast of southern California. They were maintained in the laboratory as described

previously [19]. S. purpuratus becomes immunoquiescent after long-term housing of >8

months without significant disturbance [19, 20]. Immunoquiescence can easily be reversed

by injecting PAMPs, or in response to injury [19-21].

5.4.2. Immunological challenge and sample collection

Animals were challenged by injecting 2 µg of LPS or 4 µg of peptidoglycan (Sigma-

Aldrich) per milliliter of coelomic fluid (CF), as previously described [10, 19]. Control

animals were injected with an equivalent volume of artificial CF (aCF) [11]. CF (100 µl)

was withdrawn from each sea urchin immediately before injection, and then at various

times after injection. A 23-gauge needle attached to a 1-ml syringe was inserted through

the peristomium into the coelomic cavity and CF was withdrawn without anticoagulant.

The CF was immediately expelled into a 1-ml tube and mixed with 100 µl of urea sample

buffer (2.4 M Tris-HCl (pH 6.8), 0.25% SDS, 4 M urea, 20% glycerol). Samples were

stored at -70°C until used. Proteins were precipitated using 2-D Clean Up kits (GE

Healthcare) according to the manufacturer’s instructions and resuspended in urea sample

buffer (8 M urea, 4% CHAPS, 60 mM DTT). The total protein content of each sample was

determined with 2-D Quant kits (GE Healthcare).

219

5.4.3. One-dimensional electrophoresis (1DE)

CF proteins in urea sample buffer (~100 µg/well) were separated on 10% Tris-

glycine precast polyacrylamide gels (Criterion Gel System; BioRad) at 130 V for 2 hours ,

or on 7.5% bis-Tris polyacrylamide gels at 180 V for 1 hour. After electrophoresis,

proteins were visualized using Sypro Ruby (Sigma-Aldrich) following the manufacturer’s

protocol, or with Coomassie Blue using standard protocols. Alternatively, the proteins

were transferred to nitrocellulose membranes by Western blotting as described below.

5.4.4. Two-dimensional electrophoresis (2DE)

Isoelectrofocusing was performed using an IPGphor IEF system (GE Healthcare).

Immobilized pH gradient (IPG) gel strips (11 cm, pH 3-6 or pH 3-10; GE Healthcare) were

rehydrated overnight with 200 µg of CF proteins in 200 µL rehydration buffer (8 M urea,

2% CHAPS, 50 mM DTT, and 0.5% carrier ampholytes; Immobiline; GE Healthcare).

Isoelectrofocusing was undertaken at 100 V for 3 hours, 250 V for 20 minutes, and 8000 V

for 6 hours, to obtain a total of 26,000-30,000 Vh. The proteins in the IPG strips were

reduced (1% DTT, 15 minutes) and alkylated (2.5% iodoacetamide, 15 minutes) before

separation in the second dimension by SDS-PAGE using 10% Tris-glycine precast

polyacrylamide gels (Criterion Gel System, BioRad) [22].

After electrophoresis, protein spots on the gels were visualized by Coomassie blue staining

or with Sypro Ruby. Alternatively, proteins were transferred to nitrocellulose membranes,

as described see below.

220

5.4.5. Antibodies

The polyclonal antisera against 185/333 proteins used in this study were the same as

those reported by Brockton et al. [18]. Antisera were generated against synthetic peptides

corresponding to elements 1, 7 and 25a, which are present in most 185/333 cDNAs (see

Ref. 11 and Figure 5.5). The peptides were conjugated to keyhole limpet haemocyanin and

injected into two rabbits per peptide on four separate occasions (Quality Controlled

Biochemicals). Only those antisera for which the preimmunization bleeds did not cross-

react with sea urchin CF proteins by Western blot [18] were used in this study. The three

antisera used were designated anti-185-66, anti-185-68, and anti-185-71.

5.4.6. Western blotting and immunostaining

Proteins separated by 1DE or 2DE were transferred from polyacrylamide gels to

nitrocellulose membranes by electroblotting using a Criterion blotting system (Bio-Rad).

Transfers were performed at 100 V for 1 hour at room temperature using Towbin buffer

(0.25 M Tris-HCl, 1.92 M glycine (pH 8.3)) with 20% methanol. Once transfer was

complete, membranes were blocked by incubation in skim milk solution (7% skim milk

powder in TBST; 10 mM Tris-HCl, 137 mM NaCl, 0.5% Tween 20 (pH 7.5)) overnight.

Following several washes with TBST (3 times for 5 minutes), membranes were incubated

with anti-185/333 antisera (1/20,000 dilution of an equal mix of anti-185-66, -68 and -71

in TBST, or each antiserum separately) for 1 hour at room temperature. The blots were

washed with TBST (3 times for 5 minutes) and incubated for 1 hour at room temperature

with goat anti-rabbit IgG conjugated with HRP (1/30,000 in TBST; Sigma-Aldrich). After

washing in TBST (3 times for 5 minutes), 185/333+ proteins were visualized using ECL

chemiluminescence (GE Healthcare) with blue light-sensitive high-performance

221

chemiluminescence film (Hyperfilm ECL, GE Healthcare). Each blot was exposed to film

for varying lengths of time (1-15 minutes) to optimize the exposure. In some cases, image

processing (Photoshop; Adobe Systems) was used to combine autoradiographs from the

various exposures into composite images. Proteome maps for 185/333+ protein spots on

2DE Western blots were established using Progenesis software (Perkin Elmer) to plot

molecular mass and isoelectric point (pI). The relative intensities of different protein spots

were calculated using Adobe Photoshop (Adobe Systems).

5.4.7. Anti-185/333 ELISA

CF from LPS- and aCF-injected sea urchins was adjusted to 5 × 105 coelomocytes/ml

with calcium-magnesium-free seawater with EDTA and imidazole (CMFSW-EI; 10 mM

KCl, 7 mM Na2SO4, 2.4 mM NaHCO3, 460 mM NaCl, 70 mM EDTA, and 50 mM

imidazole (pH 7.4)) and was mixed with an equal volume of CMFSW-EI containing 1%

(v/v) Nonidet P-40 to lyse the cells, followed by centrifugation (12,000 × g, 10 seconds) to

remove debris. The supernatant was diluted 1/20 with TBS and aliquoted in triplicate into

96-well ELISA plates (200 µl/well; Corning). Plates were incubated overnight at 4°C so

that proteins could adhere to the wells. The plates were washed once with TBS and

blocked for 1 hour with TBS containing 4% (w/v) BSA. After blocking, 100 µl of anti-185

antisera (1/20,000 dilution of an equal mix of anti-185-66, -68 and -71 in TBST) was

added per well for 2 hours at room temperature with gentle shaking. The plates were

washed three times for five minutes each with TBST before adding 100 µl anti-rabbit IgG-

alkaline phosphatase conjugate (1/20,000 in TBST; Sigma-Aldrich) per well. The plates

were incubated with the secondary Ab for 2 hours before being washed three times with

TBST. After the final wash, 200 µl of p-nitrophenyl phosphate (2 mg/ml in 0.1 M glycine,

1 mM MgCl2, 1 mM ZnCl2 (pH 10.4); Sigma-Aldrich) was added per well and incubated

222

for 30 minutes before absorbance was read in a microplate spectrophotometer at 415 nm.

Data were corrected for the absorbance in wells prepared without sea urchin proteins.

Controls included wells in which the primary (anti-185) Abs or both the primary and

secondary Abs were omitted, and wells in which an irrelevant Ab (rabbit anti-tunicate

collectin peptide [24, 25]) was used in place of the anti-185 antisera.

5.4.8. Mass spectrometry and data analysis

Mass spectrometry (MS) was performed on CF proteins separated by either 1DE or

2DE. To extract proteins from 1DE, Coomassie blue-stained gels were washed twice in

water (10 minutes each). Individual lanes were cut into 16 slices of equal sizes so that

proteins in different molecular mass ranges could be analyzed separately by MS (see

Figure 5.8). To extract proteins after 2DE, spots from 2DE gels that corresponded to

individual 185/333+ proteins on Western blots were excised.

Gel slices or excised protein spots were washed briefly with 100 mM NH4HCO3

before being destained (3 times for 10 minutes) with 25 mM NH4HCO3/acetonitrile (ACN)

(1/1) and dehydrated in 100% ACN for 5 minutes. After dehydration, gel pieces were air-

dried, reduced with 10 mM DTT in 100 mM NH4HCO3 for 45-60 minutes at 56°C, and

alkylated with 55 mM iodoacetamide in 100 mM NH4HCO3 for 45 minutes at room

temperature. The gel pieces were washed once with 100 mM NH4HCO3 for 5 minutes and

twice with 25 mM NH4HCO3,/ACN (1/1) for 5 minutes before being dehydrated with

100% ACN. The dehydrated gel slices were air-dried and rehydrated with trypsin

(12.5ng/µl in 50 mM NH4HCO3; Promega) for 30 minutes at 4°C. An additional aliquot of

50 mM NH4HCO3 was added before the proteins were digested at 37ºC overnight. The

resulting tryptic peptides were extracted from the gel pieces by washing them twice with

223

2% formic acid in 50% ACN. Extracts were combined and concentrated to 10 µl by

vacuum centrifugation.

Mass spectrometry was performed at the Australian Proteome Analysis Facility

(Macquarie University). The tryptic digest extracts from 1DE gel slices were subjected to

data-dependent nanocapillary reversed phase liquid chromatography followed by

electrospray ionization using a Thermo LCQ Deca ion trap mass spectrometer (Thermo

Scientific; liquid chromatography (LC)-MS/MS). For LC-MS/MS, a microbore HPLC

system (TSP4000; Thermo Scientific) was modified to operate at capillary flow rates using

a simple T-piece flow-splitter. Columns (8 cm × 100 µm inside diameter) were pack with

100 Å, 5-µm Zorbax C18 resin at 500 ψ. Integrated electrospray tips for the columns were

made from fused silica, pulled to a 5-µm tip using a laser puller (Sutter Instrument). An

electrospray voltage of 1.8 kV was applied using a gold electrode via a liquid junction

upstream of the column. Samples were introduced onto the analytical column using a

Surveyor autosampler (Thermo Scientific). The HPLC column eluent was eluted directly

into the electrospray ionization source of the ion trap mass spectrometer. Peptides were

eluted with a linear gradient of buffer A (0.1% formic acid) and buffer B (ACN containing

0.1% formic acid) at a flow rate of 500 nl/min. Automated peak recognition, dynamic

exclusion, and daughter ion scanning of the top three most intense ions were performed

using the Xcalibur software as previously described [23].

GPM open source software (Global Proteome Machine Organization;

www.thegpm.org) was used to search peptide sequences against a combined

Strongylocentrotus database created with sequences downloaded from the National Center

for Biotechnology Information (NCBI). This FASTA format database contained 44,037

protein sequences comprising all S. purpuratus sequences held by NCBI as of April 2008.

This database also incorporated a list of common human and trypsin peptide contaminants.

Search parameters included MS and MS/MS tolerances of ±2 Da and ±0.2 Da, tolerance of

224

up to three missed tryptic cleavages, and K/R-P cleavages. Fixed modifications were set

for carbamidomethylation of cysteine, and variable modifications were set for oxidation of

methionine. Peptides isolated from 1DE gel slices were deemed to significantly match

corresponding peptides in the available databases if their GPM log(e) score was lower than

the statistically significant cut-off values assigned by the GPM algorithm.

Protein spots from 2DE gels were analyzed using MALDI-TOF-TOF using an LTQ

FT Ultra hybrid mass spectrometer (Thermo Scientific). MS ion searches were compared

with 185/333 sequences in a custom database containing 81 translated full-length 185/333

cDNA sequences [12] and 689 EST sequences [8] using the Mascot search engine (Matrix

Sciences; www.matrixscience.com/) set for carbomethylation (C) and oxidation (M)

variable modifications, with peptide and fragment mass tolerances of ±50 ppm and ±0.5

Da respectively, and a maximum missed cleavage of one.

225

5.5. Results

5.5.1. One-dimensional SDS-PAGE analysis of CF proteins

1DE was used to provide a preliminary assessment of the diversity of proteins in CF,

particularly those that were detected by anti-185 Abs (Figure 5.1). The molecular masses

of CF proteins from all of the sea urchins analyzed in the present study (n = 13) ranged

from 20 kDa to >193 kDa (see Figure 5.6). Sypro Ruby-stained CF proteins from the

individual sea urchins ranged from 30 kDa to > 193 kDa (Figure 5.1, lane 1). There were

substantial differences in banding patterns when electrophoretically separated CF proteins

from the same sea urchin were stained with Coomassie blue compared to Sypro Ruby

(Figure 5.1, compare lanes 1 and 2). This was probably due to different sensitivities and

physiochemical properties of the stains [26].

Immunostained 1DE Western blots of CF proteins identified numerous 185/333+

bands ranging from ~20 kDa to >193 kDa (Figure 5.1, lane 3). Controls that omitted the

primary (anti-185) and/or secondary (anti-IgG) Abs, and irrelevant controls using anti-

tunicate collectin Abs instead of anti-185 antisera, were negative (data not shown). Most

185/333+ bands from all of the sea urchin samples analyzed (n = 13) had molecular masses

ranging from 50 kDa to >193 kDa, although some were as low as 20 kDa (see Figure 5.6).

Many of the 185/333+ bands on Western blots were not visible on Sypro Ruby- and

Coomassie blue-stained 1DE gels, suggesting that the 185/333 proteins were present at

extremely low concentrations.

226

Figure 5.1: 1DE SDS-PAGE and Western blot of CF proteins from an individual sea

urchin. CF proteins were loaded at 100 µg/well onto 10% SDS-Page gels. Lane 1/ Sypro

Ruby staining. Lane 2/ Coomassie blue staining. Lane 3/ Western blot immunostained with

an equal mixture of anti-185 sera (anti-185-68, -66, and -71; 1/20,000). The positions of

molecular mass markers are shown on the left.

1 2 3

kDa

193 -

112 -

64 -

30 -

227

5.5.2. Two-dimensional Western blots of CF proteins

Given that cDNA analyses predicted a broad array of 185/333 proteins in the CF [8,

11, 12], we extended our analysis of 185/333 proteins using 2DE, which affords far greater

resolution than does 1DE. Sea urchin CF proteins were initially separated by isoelectric

focusing (pH 3-10) and then by 10% SDS-PAGE. Large numbers of 185/333+ spots were

evident after 2DE, to the extent that they often appeared as dense smears (Figure 5.2).

Most of the 185/333 proteins recognized by the antisera in all of the sea urchins analyzed

(n = 13) had pIs between 3 and 7 with apparent molecular masses of 40 kDa to >193 kDa.

To improve the resolution of individual 185/333+ proteins on 2DE Western blots,

additional isoelectric focusing separations were conducted on immobilized pH gradient

(IPG) strips with a pH range of 3-6. Composite images obtained from film exposures of

three different time intervals were used to visualize 185/333+ proteins of varying

abundance (Figure 5.3). For the individual sea urchin shown in Figure 5.3, image analysis

of the different exposures revealed that fifty-one 185/333+ spots were evident after a 1-

minute film exposure. An additional 117 spots were evident after 5 minutes, and a further

96 spots appeared after 10 minutes, making a total of 264 spots that were detected on the

composite image for this individual. The number of discrete 185/333+ spots varied

substantially among individuals. Three of the 13 sea urchins tested did not express

detectable levels of 185/333 proteins. However, the number of discrete 185/333+ spots was

often >200 in other individuals.

The enhanced protein separation capabilities of 2DE also showed that each discrete

185/333+ band evident in 1DE contained numerous variants with similar molecular masses

but different pIs. For example, at least fifteen 185/333+ spots with different pIs were

evident at ~75 kDa (Figure 5.3). A further eight 185/333+ spots appeared at ~60 kDa and at

least six different 185/333+ spots were present at ~30 kDa (Figure 5.3).

228

Figure 5.2: 2DE Western blot of 185/333+ proteins. The gel was loaded with 200 µg of

CF proteins from a single sea urchin and the blot was immunostained with an equal

mixture of anti-185 sera (anti-185-66, -68, and -71; 1/20,000). pIs are shown as pH units

on the top of the blot, and molecular masses (kDa) are indicated on the left.

3 4 5 6 7 8 9 10

30 -

64 -

193 - 112 -

pI

kDa

229

These pI variants were often regularly spaced from each other, differing by ~0.1-0.2 pH

units. There were also numerous 185/333+ spots with identical pI but different molecular

masses (Figure 5.4). For instance, three proteins in the pI 7.5-7.75 range each had three

different molecular mass forms at ~150 kDa, 193 kDa and >193 kDa (Figure 5.4).

The three different anti-185 sera, which were raised against different regions of the

most commonly predicted 185/333 polypeptide sequences, identified subsets of 185/333

proteins (Figure 5.5). Within the small region of 2DE Western blot (pI range of 4-5 and

molecular masses of 30 kDa or lower), anti-185-66 resolved 14 distinct variants, of which

7 were 30 kDa and had a pI range of 4-5 (Figure 5.5A). An additional seven anti-186-66-

positive spots were smaller than 30 kDa with a pI range of 4.5-5. In comparison, anti-185-

68 recognized five of the seven proteins at 30 kDa with a pI range of 4-5 that were present

on the anti-185-66 blot, but did not recognize the set of seven lower molecular mass spots.

Anti-185-71 recognized only two of the 30 kDa proteins that were recognized by the other

two antisera. This result is consistent with the expression of truncated 185/333 cDNAs

[11].

5.5.3. Diversity of 185/333 proteins between individuals

There were major differences in the expression profiles of 185/333 proteins among

different sea urchins. Some animals did not show 185/333 expression before LPS injection

(Figure 5.6A, lanes 3 and 11), although expression was evident after challenge (Figure

5.6B, lanes 3 and 11). In other cases, 185/333 expression, which was very low in

preinjection samples, increased significantly after LPS injection. A broad distribution of

185/333 molecular masses ranging from 20 kDa to >193 kDa was apparent among the 13

animals analyzed by 1DE Western blots (Figure 5.6).

230

Figure 5.3: Composite image of a 2DE Western blot. The gel was loaded with 200 µg of

CF proteins from sea urchin 12. The blot was immunostained with an equal mixture of the

three anti-185 antisera (anti-185-66, -68, and -71; 1/20,000) and exposed to

autoradiographic film for 1, 5, or 10 minutes. The different exposures were merged to give

a final composite image. pIs are shown on the top as pH units, and molecular masses (kDa)

are shown on the left.

Figure 5.4: Enlarged region of a 2DE Western blot of CF proteins from animal 12

immunostained with an equal mixture of the three different anti-185 sera (anti-185-66, -68,

and -71; 1/20,000). pIs are shown on the top as pH units, and molecular masses (kDa) are

shown on the left.

6 |

3 l

193 -

64 -

112 -

30 -

kDa 1 min

5 min

10 min

10 min

pI

7.5 8 8.5 9 9.5 10 ! ! ! ! ! !

112 -

64 -

193 -

pI

kDa

231

Figure 5.5: Different anti-185 sera recognize subsets of 185/333 proteins. A/ Enlarged

regions of three different 2DE Western blots loaded with 200 µg of CF proteins from the

same sea urchin and immunostained separately with anti-185-66, anti-185-68, or anti-185-

71 (1/20,000). B/ Schematic diagram of a 185/333 protein showing the peptides used to

generate the three different anti-185 sera. The rectangle represents the protein sequence,

which is composed of the leader and 25 nucleotide sequence “elements”. The positions and

sequences of the synthetic peptides against which the three anti-185 sera were raised are

indicated [15].

4

!

5

!

4

!

5

!

4

!

5

! 30 -

68 66 71

kDa

A.

B.

66 - element 1 (AHAQRDFNERRGKENDTER)

68 - element 7 (GGRRGDGEEETDAAQQIGDGLC)

71 - element 25a (GTEEGSPRRDGQRRPYGNR)

pI

232

Figure 5.6.: 1DE Western blots of CF from 13 different sea urchins sampled before

(A) and 96 hours after (B) challenge with LPS. The equivalent lane numbers in both

panels refer to the same animals from which CF samples were obtained before and after

LPS challenge. The blots were immunostained with an equal mixture of the three different

anti-185 sera (anti-185-66, -68, and -71; 1/20,000).

193 -

112 -

64 -

kDa

1 2 3 4 5 6 7 8 9 10 11 12 13

30 -

1 2 3 4 5 6 7 8 9 10 11 12 13

193 -

112 -

64 -

kDa

30 -

A

B

233

Although many individuals expressed some 185/333 proteins with identical

molecular masses (e.g., 112 kDa, Figure 5.6, lanes 1-4; 100 and 120 kDa, Figure 5.6, lanes

7 and 8), the suite of 185/333+ bands expressed by each individual sea urchin was unique.

Three animals did not express detectable 185/333 proteins, even after challenge with LPS

(data not shown). In total, nineteen 185/333+ bands with distinct molecular masses were

identified among the 13 sea urchins analyzed by 1DE. The molecular masses of these

proteins seemed to be evenly spaced at 8-10 kDa apart over the molecular mass range of

20 to >193 kDa (Figure 5.6).

5.5.4. MS analysis of 185/333+ proteins

Proteins on coomassie blue-stained 2DE gels that corresponded to 185/333+ spots on

Western blots were analyzed by MS. This analysis failed to identify 185/333 proteins

unambiguously using the default criteria on the Mascot search engine. Although MS of

each of the protein successfully identified ion fragments with mass/charge properties that

were similar to known 185/333 sequences, these matches did not yield sufficiently high

statistical probabilities to confirm the identity of any of the proteins (data not shown).

In contrast, MS analysis of CF proteins separated by 1DE unequivocally identified

185/333 proteins. A total of 41 peptides isolated from 1DE gel slices matched with known

185/333 sequences from the NCBI database (Table 5.1). The same 185/333 peptide was

often identified in more than one gel slice. For example, the peptide

FDGPESGAPQMEGR appeared in gel slices 2-4 from animal 17. It is unlikely that the

occurrence of the same peptide in multiple fractions was due to contamination of the

different fractions with exactly the same 185/333 protein. If the same protein was present

in more than one fraction, we would expect to find exactly the same combination of

peptides from that protein in more than one fraction.

234

Figure 5.7: 1DE Western blots of CF proteins from three different sea urchins

(animals 6, 17 and 22) immunostained with an equal mixture of anti-185 sera (anti-185-66,

-68, and -71; 1/20,000). The numbers on the right show the approximate positions of slices

cut from 1DE gels for analysis by mass spectrometry (see Table 5.1). In total, gels were cut

into 16 slices. The positions of only eight of those slices containing the highest molecular

mass proteins are shown here.

8

7

6

5

4

3

2

1 104 -

59 -

27 -

kDa gel slice

number

6 17 22

animal number

235

Table 5.1: Mass spectrometric (LC-MS/MS) data for peptides isolated from 1DE gels

of S. purpuratus CF that match known 185/333 sequencesa

Animal Gel Slice Log(e) m + h z Sequence Genbank Accession No.

6 6 -1.30 2594.5 3 MAVLTLATMAATTSIIIATTQKVTK1 gb|ABK88425.1|

-3.20 1735.8 3 GQGGFGGRPGGMQMGGPR gb|ABR22418.1|

-3.20 1735.8 3 GQGGFGGRPGGMQTGSPR gb|ABZ10666.1

-3.30 1493.6 2 FDGPESGAPQMEGR gb|ABZ10664.1|

-9.80 2886.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGPGR gb|ABZ10668.1|

-2.40 1735.8 3 GQGGFGGRPGGMQMGGLR gb|ABK88417.1|

-3.30 2131.9 3 RGDGEEETDAAQQIGDGLGGR gb|ABZ10666.1|

-1.70 1506.7 2 FDGPGFGAPQMGGPR gb|ABR22467.1|

-4.60 1157.6 2 KPFGDHPFGR gb|ABR22410.1|

2

-2.00 2819.2 3 GDGEEETDAAQQIGDGLGGSGQFDGPRR gb|ABR22477.1|

-2.40 1735.8 3 GQGGFGGRPGGMQMGGPR gb|ABR22418.1|

-2.40 1735.8 3 GQGGFGGRPGGMQTGSPR gb|ABZ10666.1|

-4.20 1649.7 3 RFDGPESGAPQMEGR gb|ABZ10664.1|

-5.20 1493.6 2 FDGPESGAPQMEGR gb|ABZ10664.1|

-3.00 2131.9 3 RGDGEEETDAAQQIGDGLGGR gb|ABZ10666.1|

-1.50 2320.2 2 PQTDQRNNRLVSATKAAMRM1 gb|ABK88425.1|


-1.40 1735.8 3 GQGGFGGRPGGMQMGGLR gb|ABK88417.1|

-2.60 1979.9 3 MGGRNSTNPEFGGSRPDGAG1 gb|ABR22330.1|

-1.70 2744.3 3 RNSTNPEFGGSRPDGAGGRPLFGQGGR1 gb|ABR22330.1|

3

-4.60 2927.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGHGR gb|ABZ10669.1|

-2.40 1479.6 2 FDGPESGAPQMDGR gb|ABZ10666.1|

-4.00 1980.0 3 FGGSRPDGAGGRPFFGQGGR gb|ABZ10669.1|


-2.00 1157.6 2 KPFGDHPFGR gb|ABR22410.1|


-0.17 2564.1 3 FDGPESGAPQMEGRRQNGVPMGGR gb|ABK88329.1|

-3.10 2132.0 3 RGDGKEETDAAQQIGDGLGGR gb|ABR22436.1|

4


-1.7 3398.5 3 DFNERREKENDTERGQGGFGGRPGGMQMGGP gb|ABK88476.1|


5

-1.5 1831.9 3 RFDGPEPGAPQMEGRR gb|ABK88803.1|

-2.7 2131.0 3 RGDGEEETDAAQQIGDGLGGR gb|ABZ10666.1| 6


-1.6 1515.7 2 ADVVEIAVNEEDVN1 gb|ABA19607.1|


-4 1493.6 2 FDGPESGAPQMEGR gb|ABZ10664.1|

17

7


-2.50 1033.5 2 FGAPQMGGPR gb|ABR22467.1|

-1.40 2814.3 3 RGRGQGRFGGRPGGMQMGGPRQDGGPMG gb|ABK88373.1|

22 2


a The data are from 1DE gel slices of CF from three different sea urchins. The GenBank accession numbers of the 185/333 sequences that matched peptides from 1DE gels are also shown. The amino acid sequences shown are the matching 185/333 peptides from the NCBI database. Parameter definitions are: log(e), values indicate the probability that a putative peptide sequence corresponding to a mass spectrum arises stochastically. The lower the log(e) value, the more significant the assignment of the peptide sequence to the mass spectrum. The log(e) values listed in this table are all lower than the significance cutoff values assigned by the GPM algorithm, and so they are deemed to be statistically significant (43); m + h, peptide mass in Da + 1; z, peptide charge. b Peptides that matched sequences for previous analysis of nucleotide sequences that might represent frameshift mutations [12, 13].

236

However, this was not the case. Only single peptides were found in each fraction. Given

this repetitive identification of the same peptide in different gel slices, a total of 23 unique

185/333 peptides were identified among all of the gel slices. In some cases, the GPM

algorithm allocated slightly different predicted amino acid sequences to the same MS

spectrum. For instance, GPM identified two peptides (GQGGFGGRPGGMQMGGPR and

GQGGFGGRPGGMQTGSPR; underlining identifies amino acids that differ) from the

same MS spectrum. This occurred because the amino acid differences between these two

peptides were predicted to yield peptides with indistinguishable mass/charge ratios.

Matches to both of these peptides were present in the NCBI database. In some cases, the

subtle differences between peptides involved in the inclusion of an arginine resulting in

closely related peptides of different lengths, such as RGDGEEETDAAQQIGDGLGGR

and RGDGEEETDAAQQIGDGLGGPGQFDGHGR. It is noteworthy that of the 41

peptides identified, 36 were located in the glycine-rich (N-terminal) and central regions of

the predicted proteins. This is consistent with frameshifts and early stop codons that have

been identified in about half of the cDNAs [11]. In many cases, the introduction of these

frameshifts or early stop codons would have resulted in the loss of epitopes for the

antiserum anti-185-68, which was raised against the C-terminal region of full-length

185/333 proteins, and to a lesser extent, the epitope for anti-185-71, which is in the central

region of predicted full-length 185/333 proteins. As a result, these antisera would not have

identified many of the 185/333 proteins encoded by truncated mRNAs in the Western blot

shown in Figure 5.5A. Some of the peptides shown in Table 5.1 matched sequences that

previous analyses have suggested are derived from frameshift mutations [12, 13].

237

All of the peptides that matched 185/333 sequences came from gel slices 2-7, which were

also shown by Western blot to contain the vast majority of 185/333 proteins (Figures 5.6

and 5.7). Of the three sea urchins analyzed by 1DE and LC-MS/MS, animal 17 had the

highest overall expression levels for 185/333 proteins (Figure 5.7) and yielded 37 of the 41

peptides that matched 185/333 sequences.

5.5.5. 185/333 protein expression increases after LPS challenge

1DE Western blots of CF from five out of the six animals injected with LPS showed

increases in 185/333+ protein expression between 24 and 192 hours (representative results

are shown in Figure 5.8A). Repeated injections with LPS performed 14 days after the

primary challenge increased 185/333 protein expression to levels that were higher than

those evident after the first injection (Figure 5.8A). Sea urchins injected with aCF either

did not respond to the injection or showed weak responses in terms of 185/333 protein

expression (data not shown).

These Western blot data were confirmed by ELISA (Figure 5.8B). Injecting LPS into

sea urchins significantly (p < 0.05) increased the titer of 185/333 proteins detected in CF

by ELISA. By 48 hours after LPS injection, anti-185 reactivity had increased 2.4-fold

compared with the 0 hour time point (p < 0.05). It returned to levels that were

indistinguishable (p > 0.05) from those before LPS challenge by 96 h. In response to aCF

(control) injections, the titer of 185/333 proteins in CF also increased up to 48 hours

postinjection, but only to a level that was 1.4-fold greater than the preinjection time point.

This increase was significantly less than the response to LPS (p < 0.05). Negative controls

confirmed the specificity of the ELISA.

238

Figure 5.8: The titer of 185/333 proteins in CF increases after immune challenge. A/

1DE Western blots of CF from a single sea urchin collected at various times after the

animal was injected twice with LPS. The second LPS injection was administered 360

hours after the first. B/ 185/333 protein expression levels determined by ELISA (ΔODU415)

at various times after naive sea urchins (n = 5) were injected with LPS or aCF (controls).

Bars indicate SEM. Asteriks denote time points that differed significantly (p <0.05)

between LPS-injected sea urchins and controls.

0.05 -

0.10 -

0.15 -

0.20 -

0.25 -

0 20 40 60 80 100

LPS

control

time after injection (hours)

!O

DU

415

B

193 -

A first LPS injection second LPS injection

0 24 48 192 24 48 192

kDa

time after injection (hours)

* *

239

Wells in which the primary (anti-185 antisera) or secondary (anti-rabbit IgG) Abs

were omitted, or when the primary Ab was replaced by an irrelevant control (anti-tunicate

collectin), did not yield absorbance readings that were significantly greater than

background levels (p > 0.05, data not shown).

5.5.6. Diversity of 185/333 protein expression after immunological challenge

1DE analysis did not detect changes in the types of 185/333 proteins expressed by

individual sea urchins in response to LPS or PG injections, even though densitometry (data

not shown) indicated there was an increase in the relative quantity of 185/333 proteins

after repeated challenge (Figure 5.9). The molecular masses of the predominant 185/333

proteins in CF from animals 6 and 25 did not change in response to an initial injection of

LPS, or after a second LPS injection 2 weeks later. A similar result was apparent for

animals 22 and 31, which were challenged with LPS followed by PG 2 weeks later. The

185/333+ bands of these two animals did not show any major changes in molecular mass

when the challenge was switched from LPS to PG.

In contrast, 2DE revealed clearly discernable changes in the pIs of 185/333 proteins

when sea urchins were challenged with PG after initially responding to LPS, even though

the molecular masses of the predominant forms remained the same (Figure 5.10). This is

best exemplified in the small region of the 2DE Western blots of CF from animal 31 (pIs

of 4-6, molecular masses of 40-65 kDa) shown in Figure 5.10B. CF from this sea urchin

was collected after an initial challenge with LPS (Figure 5.10B, upper panel), and then

again after a subsequent injection of PG (Figure 5.10B, lower panel). Of the thirty

185/333+ spots in this region, seven were found only after LPS challenge (e.g., 63 kDa, pI

4.8), and six were found only after challenge with PG (e.g. 42 kDa, pI 5.5). The remaining

17 proteins were present after both the LPS and PG challenges.

240

Figure 5.9: 1DE Western blots of CF collected after sea urchins had been injected with

LPS or PG. Animals 6 and 25 were injected with LPS twice at 360-hours interval, while

animals 22 and 31 were injected first with LPS and then with PG 360 hours later. CF was

collected 96 hours after each injection, and the Western blots were immunostained with an

equal mixture of the three different anti-185 sera (anti-185-66, -68, and -71; 1/20,000).

193 -

112 -

64 -

LPS ! LPS LPS ! PG

6 25 22 31

animal number

kDa

241

The relative expression levels of some of these shared proteins also differed between

challenges. For instance, one of the proteins (63 kDa, pI 5.2) had a densitometry value of

5.1 after LPS injection and 1224 after PG challenge, representing a 240-fold increase in

expression intensity in response to PG.

242

Figure 5.10: 2DE Western blots of CF collected from a single sea urchin (animal 31)

that had been injected first with LPS and then 360 hours later with PG. CF was

withdrawn at 96 hours after each injection. Western blots were immunostained with an

equal mixture of the three different anti-185 sera (anti-185-66, -68, and -71; 1/20,000). A/

Full proteome maps (pI of 3-6; 40-150 kDa; 30-seconds film exposure) of the CF samples

after LPS and PG challenges. B/ Enlargements (pI of 4.0-6.3; 40-85 kDa; 5-minutes film

exposure) of the boxed area in A. The numbers shown in each panel are the relative

expression intensities of five 185/333+ spots that differed substantially in expression after

LPS compared with PG injections.

3 4 5 6

64 -

64 -

kDa

LPS

PG

A

B

pI

LPS

PG

2.5

0

4.1

0 1224

261

0

5.1

1.5

0

64 -

64 -

kDa

4 6 pI

243

5.6. Discussion

This study has identified substantial diversity among 185/333 proteins, as reflected

by their broad range of molecular masses and pIs. Most importantly, there are obvious

differences in the suites of 185/333 proteins expressed by different sea urchins, and these

suites of proteins undergo subtle but extensive changes in response to different types of

immune challenge. The diversity of 185/333 proteins and the changes in their expression

detected by the current proteomic analysis correspond with previous studies of mRNAs

from sea urchins responding to different PAMPs [11]. All of these data suggest that sea

urchins may be capable of altering the expression of 185/333 proteins to tailor specific

responses against different PAMPs.

Despite the overall agreement between our current observations of 185/333 proteins

and predictions based on the prior analyses of 185/333 nucleotide sequences [11, 12], there

are some discrepancies. The predicted sizes of 185/333 proteins based on cDNA sequences

range from 4 to 55.3 kDa [11, 12], with predicted pIs ranging from 5.42 to 11.54.

However, the present study shows that native 185/333 proteins have a far wider range of

molecular masses (20 to >193 kDa using 1DE analysis), with predominant bands often

being at the high end of this range. They also have far more acidic pIs than expected,

mainly between 3 and 7. It is unlikely that the discrepancy between predicted and observed

molecular masses is due to disulphide-bonded oligomerization because none of the

predicted 185/333 amino acid sequences identified to date contains cysteines [8, 11]. Even

if cysteines were present in missense sequences of frame-shifted proteins, the strong

reducing conditions used to prepare samples would have disrupted any oligomers held

together by disulphide bonds. Another explanation for the discrepancy in molecular masses

is that 185/333 proteins are glycosylated and form large complexes covalently linked to

carbohydrates. There are numerous conserved sites for N-linked glycosylation within the

244

histidine-rich region of the 185/333 proteins (elements 11-25), and there are conserved

sites for O-linked glycosylation in the carboxyl-terminal region [8, 11]. However,

deglycosylation of N-linked oligosaccharides failed to decrease the molecular masses of

185/333 proteins to the size of predicted monomers (Smith LC, unpublished data). Given

these results, it seems likely that the disparity between predicted and observed molecular

masses reflects oligomerization based on mechanisms other than disulphide bond

formation that are resistant to the reducing treatments used in 1DE and 2DE. This

conclusion is supported by studies of recombinant 185/333 proteins, in which the

expression of a single form of 185/333 protein yields a range of expressed proteins with

molecular masses corresponding to monomers, homodimers and higher order oligomers

[18].

Other data also suggest that the diversity of molecular masses evident among

185/333 proteins is increased by the expression of truncated molecules. Many of the SNPs

and indels found previously in 185/333 transcripts are predicted to result in frameshifts,

and the encoded proteins may be either truncated and/or have missense sequences [11, 13].

No such frameshifts have been identified in the 185/333 genes. However, comparisons of

gene and mRNA sequences from individual animals suggest that posttranscriptional

modification may be responsible for these missense proteins [15]. If these messages are

translated, it would explain why the three different anti-185 antisera used in the present

study detected different subsets of 185/333 proteins. The antisera were raised against

amino acid sequences in N-terminus (element 1), the middle (element 7) and the C-

terminus (element 25a) of the predicted proteins. The antiserum targeted to the N-terminus

(anti-185-66) identified the largest number of 185/333 proteins on 2DE Western blots,

presumably because its epitope is most likely to be present in every protein, including the

shortest truncated forms. Of the 689 translated cDNA sequences identified to date, 676

contain the epitope within element 1 that is recognized by the anti-66 antiserum [11].

245

Antisera directed toward more C-terminal regions recognize decreasing numbers of

185/333+ spots, probably because these epitopes are not present on truncated proteins or

those with missense sequence at the C-terminus. Current cDNA sequence data from

Terwilliger et al. [11] show that 660 of 689 cDNA sequences contain the anti-68 epitope in

element 7, while only 375 cDNA sequences contain the anti-71 epitope in element 25a.

The variability evident in the molecular masses of 185/333 proteins, was matched by

substantial diversity in their pIs. One source for this diversity is found in mRNAs where

sequence variability results in changes in charged amino acids at particular positions

leading to predicted proteins with very similar molecular masses but different pIs [8, 11,

12]. Terwilliger et al. (see supplemental Figures S1 and S2 in Refs. 11, 12) identified

numerous sequence positions in 185/333 mRNAs that encode two to four different amino

acids, many with different charges. In the present study, similar subtle differences were

detected by MS, which identified a number of 185/333 peptides that differ by just a single

amino acid. 2DE Western blots also showed a variety of pIs for 185/333 proteins that had

very similar molecular masses. In many cases, the charge variants making up a single

molecular mass class of 185/333 proteins had a pI spanning the full pH range from 3 to 10,

although most variants had a pI within the range of 3-6. The different pI forms were often

equally spaced, suggesting that adjacent protein spots represent variants that differ by just

a single charged residue.

Although the variability in pI detected in the current study agrees with the diversity

seen among cDNA sequences, our proteomic data also provide evidence for additional

posttranslational modifications. Some 185/333 proteins have identical isoelectric points but

significantly different molecular masses. This suggests that individual 185/333 proteins

may be conjugated with some other molecule(s) that alters their molecular mass but not

their isoelectric point. Such conjugation would provide another explanation for why many

185/333+ bands are much larger in molecular mass than predicted. However, the cDNA

246

data suggest that it is also feasible for 185/333 proteins with significantly different

sequences and different pIs to have the same molecular mass, resulting in a vertical ladder

of spots on 2DE Western blots.

The diversity of 185/333 proteins explains our inability to unequivocally identify

185/333 proteins by MS of individual proteins isolated by 2DE. MS analysis of the

185/333+ spots from 2DE gels did not identify any statistically significant matches to

proteins in a custom database of 185/333 sequences. However, all the 185/333 proteins

isolated by 2DE yielded ion fragments that possessed mass/charge characteristics similar to

those of known 185/333 protein sequences. The available evidence suggests that the

existing database of 185/333 sequences may represent only a small fraction of the 185/333

variants present in sea urchin populations. Consequently, the chance of finding a

statistically significant match in this restricted data set to a single 185/333 protein isolated

from the CF of an individual sea urchin may be extremely low, even though the purity of

the proteins isolated by 2DE would have been high. In other words, we may not have

identified matches because the variability of 185/333 sequences means that many 185/333

proteins will not yet be in our database of 185/333 sequences and so cannot be identified

by MS. Our difficulties in matching isolated proteins to the existing 185/333 database

highlight the problems of employing MS techniques, which search for precise similarities

between peptides to characterize hypervariable proteins. It is interesting that even though

members of the Down syndrome cell adhesion molecule (Dscam) family are important

proteins in the immunological responses of insects, proteomics analysis of hemolymph

extracts from immunologically challenged Drosophila have not yet been able to identify

Dscams by MS.

To circumvent the problem of matching individual 185/333+ proteins to our

restricted sequence data set, we employed “shotgun” MS (LC-MS/MS), in which all of the

CF proteins within a particular molecular mass range from 1DE gels were analyzed

247

simultaneously by MS. Unlike 2DE, in which the individual protein spots analyzed by MS

presumably contain just a single 185/333 isotype, the 1DE gel slices subjected to shotgun

MS were likely to contain numerous different 185/333 isotypes. This greatly increased the

chance that at least one of these isotypes would match a previously identified sequence in

our 185/333 database. 1DE also has the advantage that it is not limited by the complex

separation characteristics of 2DE, which, for instance, is relatively inefficient in separating

hydrophobic proteins. Additionally, we used an alternative search engine (GPM as

opposed to Mascot) with slightly altered search stringencies (three missed tryptic

cleavages) to increase our chances of identifying matching 185/333 peptides in the

available databases. This process successfully identified numerous peptides that matched

precisely to sequences in the NCBI database without affecting the robustness of the

sequence matches. The gel slices in which matching peptides were identified corresponded

to regions on 1DE and 2DE Western blots that contained high concentrations of 185/333

proteins, which confirms the specificities of the anti-185 sera used in this study. However,

shotgun MS probably still identified just a small fraction of the 185/333 peptides present

within the peptide mixture, because of the limited numbers of potentially matching

peptides in the currently available dataset. The limited sensitivity of MS in identifying

highly variable proteins in the absence of comprehensive sequence databases also explains

why our MS analysis identified multiple forms of 185/333 proteins in one of the sea

urchins analyzed, even though both 1DE and 2DE Western blotting revealed substantial

intraindividual diversity in all sea urchins.

The difficulties associated with analyzing the diversity of 185/333 proteins were

compounded by the substantial variation evident in the suites of 185/333 proteins

expressed by different sea urchins. None of the 13 sea urchins analyzed by 1DE Western

blotting expressed the same pattern of 185/333+ bands, even though there were a number

of molecular mass forms that were shared by some individuals. On average, two to three

248

major bands were evident in each sea urchin, along with numerous minor bands. The

differences between individuals implie that there may be a variety of mechanisms acting in

concert to produce novel arrays of 185/333 proteins in individual sea urchins. These

factors could include the presence or absence of different 185/333 genes in different sea

urchins, the presence of different alleles at a given 185/333 gene locus, differential gene

expression of 185/333 family members, posttranscriptional processing or editing of the

transcripts that insert frameshifts and SNPs [15], and posttranslational processing of the

proteins [13].

Our data suggest that these potential mechanisms for molecular diversification may

allow sea urchins to vary the suites of 185/333 proteins that they express in response to

different types of immunological challenge. ELISA provided direct evidence that 185/333

expression increases after the injection of PAMPs, while 2DE analysis of 185/333 proteins

from individual sea urchins identified many subtle changes in the patterns of 185/333

proteins responding to different PAMPs. There were clear differences in the pIs and

molecular masses of the proteins expressed in response to LPS compared with those

synthesized by the same sea urchin in response to PG. These results agree with changes

that are apparent in the cDNAs from animals responding to different PAMPs, including

LPS, β-1,3-glucan, or dsRNA [11]. Terwilliger et al. [11] showed that a diverse size range

of 185/333 messages was present in the CF of immunoquiescent sea urchins, but this broad

expression profile changed to one single, major mRNA size after immune challenge.

Sequence analysis of mRNAs from animals responding to immune challenge also indicated

that the predominant 185/333 variants in CF are different before and after challenge. Such

alterations in 185/333 transcription explain the changes detected during the present study

in the suites of 185/333 proteins found in the CF of the same sea urchin responding to

different PAMPs.

249

The identification of PAMP-specific responses suggests that 185/333 protein

expression can be tailored to meet different forms of immunological challenge. The

obvious implication is that 185/333 proteins are involved in some form of pathogen-

specific immunological response. There is growing evidence for this level of

immunological specificity among a variety of invertebrates [1, 2, 5, 27-29]. For instance,

bacterial immunization in bumble bees can induce acquired immune responses that are

highly specific to the original, inoculating species of bacteria [29], although the

mechanisms underlying this response are not knwon. In addition to 185/333 proteins, other

large gene families may be capable of fine-scale discrimination between pathogens. These

gene families encode variable lymphocyte receptors from agnathans [30-34], V-region-

containing chitin-binding proteins (VCBPs) from cephalochordates and tunicates [3, 35,

36], Dscams in insects [37, 38] and fibrinogen-related proteins (FREPs) from molluscs

[39-42]. A repertoire of >19,000 different Dscams is expressed in hemocytes of

Drosophila and mosquitoes [37, 38], and thousands of FREPs are present in freshwater

snails [39-42]. None of these gene families is closely related to one another, suggesting

that mechanisms for immune diversification have evolved many times and that additional

unique systems are likely to be identified among other organisms in the future. In this

context, our continuing investigations of 185/333 proteins will provide insights into how

animals other than mammals adapt in the “arms race” against the microbes.

250


We thank Dr. Virginia Brockton for assistance in processing and shipping protein

samples and Dr. Katherine Buckley for suggesting improvements to the manuscript. The

research has been facilitated by access to the Australian Proteome Analysis Facility

established under the Australian Government’s Major National Research Facilities

Program.

5.8. Disclosures

The authors have no financial conflicts of interest.

251

5.9. References

[1] Flajnik, M. F., Immunology: another manifestation of GOD. Nature 2004, 430, 157–

158.

[2] Flajnik, M. F., Du Pasquier, L., Evolution of innate and adaptive immunity: can we

draw a line? Trends Immunol. 2004, 25, 640–644.

[3] Litman, G. W., Cannon, J. P., Dishaw, L. J., Reconstructing immune phylogeny: new

perspectives. Nat. Rev. Immunol. 2005, 5, 866–879.

[4] Hibino, T., Loza-Coll, M., Messier, C., Majeske, A. J., et al., The immune gene

repertoire encoded in the purple sea urchin genome. Dev. Biol. 2006, 300, 349–365.


into the immune system of the sea urchin. Science 2006, 314, 952–956.

[6] Sodergren, E., Weinstock, G. M., Davidson, E. H., Cameron, R. A., et al., The genome

of the sea urchin Strongylocentrotus purpuratus. Science 2006, 314, 941–952.


evolutionary origin of the RAG1/2 gene locus. Proc. Natl. Acad. Sci. USA 2006,

103, 3728–3733.


analysis of coelomocyte gene expression in response to LPS in the sea urchin:

identification of unexpected immune diversity in an invertebrate. Physiol.

Genomics 2005, 22, 33–47.

[9] Rast, J. P., Pancer, Z., Davidson, E. H., New approaches towards an understanding of

deuterostome immunity. Curr. Top. Microbiol. Immunol. 2000, 248, 3–16.

[10] Smith, L. C., Chang, L., Britten, R. J., Davidson, E. H., Sea urchin genes expressed in

activated coelomocytes are identified by expressed sequence tags: complement

252


homology within the deuterostomes. J. Immunol. 1996, 156, 593–602.

[11] Terwilliger, D. P., Buckley, K. M., Brockton, V., Ritter, N. J., Smith, L. C.,

Distinctive expression patterns of 185/333 genes in the purple sea urchin,

Strongylocentrotus purpuratus: an unexpectedly diverse family of transcripts in

response to LPS, _-1,3-glucan, and dsRNA. BMC Mol. Biol. 2007, 8, 16.

[12] Terwilliger, D. P., Buckley, K. M., Mehta, D., Moorjani, P. G., Smith, L. C.,

Unexpected diversity displayed in cDNAs expressed by the immune cells of the

purple sea urchin, Strongylocentrotus purpuratus. Physiol. Genomics 2006, 26,

134–144.

[13] Buckley, K. M., Smith, L. C., Extraordinary diversity among members of the large

gene family, 185/333, from the purple sea urchin, Strongylocentrotus purpuratus.

BMC Mol. Biol. 2007, 8, 68.

[14] Buckley, K. M., Munshaw, S., Kepler, T. B., Smith, L. C., The 185/333 gene family is

a rapidly diversifying host-defense gene cluster in the purple sea urchin,

Strongylocentrotus purpuratus. J.Mol. Biol. 2008, 379, 912–928.

[15] Buckley, K. M., Terwilliger, D. P., Smith, L. C., Sequence variations in 185/ 333


increase diversity. J. Immunol. 2007, 181, 8585–8594.

[16] Tsai, H., Bobek, L. A., Human salivary histatins: promising anti-fungal therapeutic

agents. Crit. Rev. Oral Biol. Med. 1998, 9, 480–497.

[17] Xu, T., Telser, E., Troxler, R. F., Oppenheim, F. G., Primary structure and

anticandidal activity of the major histatin from parotid secretion of the subhuman

primate, Macaca fascicularis. J. Dent. Res. 1990, 69, 1717–1723.


diversity of 185/333 proteins from the purple sea urchin – unexpected protein-size

253

range and protein expression in a new coelomocyte type. J. Cell Sci. 2008, 121,

339–348.

[19] Clow, L. A., Gross, P. S., Shih, C. S., Smith, L. C., Expression of SpC3, the sea


2000, 51, 1021–1033.


the evolution of the complement system. Dev. Comp. Immunol. 1999, 23, 429–442.


homologue, SpC3, functions as an opsonin. J. Exp. Biol. 2004, 207, 2147–2155.

[22] Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 1970, 227, 680–685.

[23] Andon, N. L., Hollingworth, S., Koller, A., Greenland, A. J., et al., Proteomic

characterization of wheat amyloplasts using identification of proteins by tandem

mass spectrometry. Proteomics 2002, 2, 1156–1168.

[24] Green, P., Luty, A., Nair, S., Radford, J., Raftos, D., A second form of collagenous

lectin from the tunicate, Styela plicata. Comp. Biochem. Physiol. B Biochem. Mol.

Biol. 2006, 144, 343–350.

[25] Green, P. L., Nair, S. V., Raftos, D. A., Secretion of a collectin-like protein in

tunicates is enhanced during inflammatory responses. Dev. Comp. Immunol. 2003,

27, 3–9.

[26] Miller, I., Crawford, J., Gianazza, E., Protein stains for proteomic applications: which,

when, why? Proteomics 2006, 6, 5385–5408.

[27] Kurtz, J., Franz, K., Innate defence: evidence for memory in invertebrate immunity.

Nature 2003, 425, 37–38.

[28] Kurtz, J., Armitage, S. A., Alternative adaptive immunity in invertebrates. Trends

Immunol. 2006, 27, 493–496.

254

[29] Sadd, B. M., Schmid-Hempel, P., Insect immunity shows specificity in protection

upon secondary pathogen exposure. Curr. Biol. 2006, 16, 1206–1210.

[30] Litman, G. W., Cannon, J. P., Rast, J. P., New insights into alternative mechanisms of

immune receptor diversification. Adv. Immunol. 2005, 87, 209–236.

[31] Litman, G. W., Hawke, N. A., Yoder, J. A., Novel immune-type receptor genes.

Immunol. Rev. 2001, 181, 250–259.

[32] Pancer, Z., Amemiya, C. T., Ehrhardt, G. R., Ceitlin, J., et al., Somatic diversification

of variable lymphocyte receptors in the agnathan sea lamprey. Nature 2004, 430,

174–180.

[33] Pancer, Z., Mayer, W. E., Klein, J., Cooper, M. D., Prototypic T cell receptor and

CD4-like coreceptor are expressed by lymphocytes in the agnathan sea lamprey.

Proc. Natl. Acad. Sci. USA 2004, 101, 13273–13278.

[34] Pancer, Z., Saha, N. R., Kasamatsu, J., Suzuki, T., et al., Variable lymphocyte

receptors in hagfish. Proc. Natl. Acad. Sci. USA 2005, 102, 9224–9229.

[35] Cannon, J. P., Haire, R. N., Litman, G. W., Identification of diversified genes that

contain immunoglobulin-like variable regions in a protochordate. Nat. Immunol.

2002, 3, 1200–1207

[36] Cannon, J. P., Haire, R. N., Rast, J. P., Litman, G. W., The phylogenetic origins of the

antigen-binding receptors and somatic diversification mechanisms. Immunol. Rev.

2004, 200, 12–22.

[37] Dong, Y., Taylor, H. E., Dimopoulos, G., AgDscam, a hypervariable immunoglobulin

domain-containing receptor of the Anopheles gambiae innate immune system.

PLoS Biol. 2006, 4, e229.

[38] Watson, F. L., Puttmann-Holgado, R., Thomas, F., Lamar, D. L., et al., Extensive

diversity of Ig superfamily proteins in the immune system of insects. Science 2005,

309, 1874–1878.

255

[39] Adema, C. M., Hertel, L. A., Miller, R. D., Loker, E. S., A family of fibrinogen-

related proteins that precipitates parasite-derived molecules is produced by an

invertebrate after infection. Proc. Natl. Acad. Sci. USA 1997, 94, 8691–8696.

[40] Leonard, P. M., Adema, C. M., Zhang, S. M., Loker, E. S., Structure of two FREP

genes that combine IgSF and fibrinogen domains, with comments on diversity of

the FREP gene family in the snail Biomphalaria glabrata. Gene 2001, 269, 155–

165.

[41] Zhang, S. M., Loker, E. S., Representation of an immune responsive gene family

encoding fibrinogen-related proteins in the freshwater mollusc Biomphalaria

glabrata, an intermediate host for Schistosoma mansoni. Gene 2004, 341, 255–266.

[42] Zhang, S. M., Loker,E. S., The FREP gene family in the snail Biomphalaria glabrata:

additional members, and evidence consistent with alternative splicing and FREP

retrosequences: fibrinogen-related proteins. Dev. Comp. Immunol. 2003, 27, 175–

187.

[43] Fenyo, D., Beavis, R. C., A method for assessing the statistical significance of

mass spectrometry-based protein identifications using general scoring schemes.

Anal. Chem. 2003, 75, 768–774.

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257

CHAPTER VI

Ultrastructural localization of a highly variable immune response protein (185/333)

within coelomocytes and the gut of sea urchins.

In preparation for submission to

Journal of Cell Sciences


Birch D1 – Technical support

Nair SV1 – Project supervision

Raftos DA1 – Experimental design – Project supervision


258

259

6.1. Preface

Chapter 5 revealed the variability of 185/333 proteins both within and between

individual sea urchins. The proteins were more abundant after the injection of PAMPs and

it appeared that different 185/333 proteins are expressed in response to the injection of

different PAMPs. Based on these data, and the transcriptomic and genomic analyses

described in Chapter 1, it has been postulated that 185/333 proteins are involved in

pathogen recognition, specifically in the encapsulation of pathogens or phagocytosis. In the

current Chapter, we further investigated the potential functions of 185/333 proteins by

studying their ultrastructural localization in coelomocytes and in the gut of sea urchins.

260

261

6.2. Summary

There is renewed interest in the immune reponses of sea urchins. This has been

heightened by the discovery of 185/333 proteins, a family of highly variable immune

response molecules with no homology to any known proteins. In this study, we show that

185/333 proteins are expressed by filopodial amoebocytes and colorless spherule cells,

which have been shown previously to cooperate in immune responses. Filopodial

amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the

trans-Golgi and later fuse with the plasma membrane of the cell. Colorless spherule cells

contain 185/333 proteins within large spherules. In the presence of bacteria and yeast, the

ultrastucture of colourless spherule cells changes, as does the profile of expression of

185/333 proteins in those cells. However, 185/333 proteins were not found in phagosomes

of coelomocytes. Another previously uncharacterized amoebocyte type, which is localized

within the connective tissue of the gut, also expressed 185/333 proteins at high

concentrations. As in filopodial amoebocytes, these gut-associated amoebocytes express

185/333 proteins on the membrane of intracellular vesicles and on the cell surface. The

abundance of 185/333 proteins within gut amoebocytes and their ultrastructural similarities

with filopodial amoebocytes suggest that these two cell types may be related. The 185/333-

positive gut amoebocytes were often associated with anuclear bodies that appeared to

incorporate material of microbial origin that was also surrounded by 185/333 proteins.

The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggest

that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

262

6.3. Introduction

The immune systems of invertebrates lack antibody-based adaptive immune

responses. However, there are still strong similarities between other components of

invertebrate and vertebrate immune systems. The sea urchin genome revealed broad

homologies between the immune response genes of these deuterostome invertebrates and

those of vertebrates [1]. Many of the sea urchin gene families that are putatively involved

in pathogen recognition are highly diversified compared to their vertebrate homologues

[2]. Two hundred and three NOD/NALP-like cytoplasmic recognition receptors (NLR)

were identified in the S. purpuratus genome, whilst humans possess only about 20 of these

genes. There are also 222 Toll-like receptors (TLR) and 218 scavenger receptor cysteine

rich (SRCR) genes present in the sea urchin genome, but the human genome encodes only

16 SRCR proteins and 13 TLRs [2]. It has been suggested that gut-associated pathogenesis

and regulation of normal gut flora might have driven the expansion of these immune

response gene families in sea urchins [3]. Recently, sequencing of Amphoxius genome has

revealed a similar expansion of pathogen recognition genes in this early chordate [4].

The 185/333 gene family of sea urchin represents an exception to the homologies

that exist between echinoderms and vertebrates because they lack homology to any known

vertebrate gene and seem to be absent from the Amphoxius genome. Nair et al. [5] first

revealed the importance of 185/333 molecules in sea urchin immune responses. They

showed that 185/333 mRNA expression increased up to 75 fold in immunologically

activated coelomocytes so that over 60% of sequences in a subtracted cDNA library of

immune response ESTs were 185/333 mRNAs. Further studies revealed high levels of

variability within this new gene family [5, 6]. A total of 689 full length 185/333 cDNAs

have so far been sequenced from S. purpuratus [6, 7], and 171 genes have been cloned and

sequenced [8]. Alignment of these sequences necessitated the insertion of large gaps that

263

identified 25 blocks of shared sequences designated “elements”. Based on the presence or

absence of different elements, over 30 distinct element patterns have so far been described

[6, 8]. In addition to these element patterns, which appear to be generated by

recombination/duplication events [9], another level of variability is generated by single

nucleotide substitutions. A bias towards cytidine to uridine transitions was observed, which

is consistent with post-transcriptional cytidine deaminase activity [10]. Transcriptomic

analysis also showed that different 185/333 mRNAs are expressed in response to different

pathogen-associated molecules [7].

The absence of cysteines and discernable secondary structure has limited structural

predictions for 185/333 proteins [6]. It is therefore difficult to hypothesise about the

function of these proteins. However, it is known that the expression of 185/333 proteins is

induced in response to the injection of different pathogen-associated molecules [5, 11].

When sea urchins are injected with LPS or peptidoglycans, the expression of 185/333

proteins increases by up to 2.4-fold [11].

The variability among 185/333 genes and transcripts is matched by differences

among the transcribed proteins, both within and between individual sea urchins [11]. Over

two hundred 185/333 proteins can be detected by two-dimensional electrophoresis (2DE)

of coelomic fluid proteins from a single individual, and the pattern of proteins expressed

by each individual sea urchin is unique. Again, the suite of 185/333 proteins expressed by

coelomocytes changes when sea urchins are challenged with different types of pathogen-

associated molecules, such as LPS and peptidoglycans.

Previous studies have identified 4 major types of coelomocytes in sea urchins:

amoebocytes, colorless spherule cells, red spherule cells, and vibratile cells [12-14]. In

addition, 3 subtypes of amoebocytes are present in S. purpuratus: polygonal (or filopodial)

amoebocytes, and discoidal (or petaloide) amoebocytes, and small filopodial amoebocytes

[15]. These amoebocytes can be differentiated by their cytoskeletal architecture. Discoidal

264

amoebocytes are believed to be primarily phagocytic, whereas polygonal amoebocytes

play a major role in wound healing and clotting [16]. Arizza et al. [17] showed that

amoebocytes and colorless spherule cells are also cytotoxic and function co-operatively.

Amoebocytes appear to release factors into the coelomic fluid that activate colorless

spherule cells, resulting in cytolysis of rabbit erythrocytes and K562 tumor cells. Lin et al.

[18] used depletion studies to demonstrate that cytotoxic molecules in the coelomic fluid

originate from both amoebocytes and colourless spherule cells. In addition to the coelomic

fluid, amoebocytes have been observed within gonads and the axial organ of sea urchins,

but little is known about their presence in the gut and other tissues [2].

Immunofluorescence microscopy has revealed that 185/333 proteins are localized

on the surface of small amoebocytes and within perinuclear vesicles of both small

amoebocytes and polygonal amoebocytes [15]. A significant increase in the percentage of

small amoebocytes expressing 185/333 proteins was observed in response to the injection

of LPS, further supporting the idea that these molecules are involved in immune responses.

Brockton et al. [15] speculated that 185/333 proteins are secreted from coelomocytes and

subsequently associate with the cell surface, forming aggregates between cells that lead to

the encapsulation of pathogens. This was supported by the observation that 185/333-

positive amoebocytes often form aggregates or syncitia.

The current study describes the ultrastructural localization of 185/333 proteins

within coelomocytes and in the gut of sea urchins. It focuses on the pathways by which

185/333 proteins are localized on the cell surface of filopodial amoebocytes, and their role

in antimicrobial defense.

265


6.4.1. Sea urchins

Heliocidaris erythrogramma were collected from Camp Cove in Sydney Harbour,

Australia. They were housed in a recirculating sea water facility at Macquarie University

using methods previously described [5, 19]. The sea urchins were left undisturbed in the

aquaria for 2 months prior to experimentation to allow them to acclimatize to aquarium

conditions [19, 20]. Some animals were then injected with bacteria or yeast by inserting a

23-gauge needle attached to a 1 mL syringe through the peristomium into the coelomic

cavity. Yeast (Saccharomices cerevisciae, 0.25 g) were re-suspended in 0.8% of congo red

in artificial coelomic fluid [aCF; 7], heated to 95ºC for one hour and washed 3 times in

aCF. The cell pellet was re-suspended in 10 mL of aCF and 100 µL of the suspension

were injected per sea urchin. Congo red was omitted for electron microscopy observations.

Late logarithmic phase Vibrio sp. (VPSYS2, accession number EF584094; [21])

were centrifuged at 8,000 rpm for 2 minutes, and re-suspended to 1/10 of their original

volume in 0.2 M NaCl. Bacteria were then inactivated with 10% acetic acid (room

temperature, 10 minutes). The solution was neutralized with five volumes of 1 M Tris HCl

(pH 8.0) before the bacteria were washed three times with phosphate buffer saline (PBS)

and re-suspended in aCF. 100 µL of this suspension were injected per sea urchin.

Coelomic fluid (CF, 1 mL) was harvested into syringes using 23-gauge needles one

hour after injection. CF from non-injected sea urchins was also collected. An aliquot of

live coelomocytes was viewed using an Olympus BH2 optical microscope (Olympus,

Japan), and images were captured with a CCD camera (SCION and Visicapture).

Alternatively, coelomocytes were processed for immunofluorescence microscopy or

transmission electron microscopy as described below.

266

6.4.2. Electron microscopy

Coelomic fluid (750 µL) was mixed with an equal volume of fixative solution (5%

paraformaldehyde and 2% glutaraldehyde in 0.2 M PIPES buffer). Cells were fixed

overnight, washed in 0.2 M PIPES buffer (3 times for 10 minutes), and then embedded in

melted 1% agarose. Cells in 1mm agarose blocks were dehydrated in a graded series of

ethanol (70% - 100%) for 10 minutes in each solution and infiltrated with LR White Resin

for 24 hours. Samples were then embedded in gelatine capsules and polymerised at 60° C

for 48 hours. Samples of gut tissue were also extracted using syringes and 23-gauge

needles, and processed in the same way as CF.

Semi-thin resin sections (1 µm) for light microscopy were cut using glass knives

with an ultramicrotome (Ultracut S , Leica), and stained with methylene blue (1%

methylene blue, 0.6% sodium hydroxide, 40% glycerol) for 10 seconds. Sections were

viewed using an Olympus BX2 microscope (Olympus, Japan), and images were captured

with a CCD camera (SCION and Visicapture). Ultrathin sections (70 nm) were cut with a

diamond knife (Drukker,) using an Ultracut –S ultramicrotome (Leica) and mounted on

300 mesh nickel grids. Ultrathin sections were immunogold labelled (see section 6.4.3.4)

or stained with uranyl acetate (saturated aqueous) for 30 minutes and lead citrate

(Reynold’s) for 4 minutes. They were viewed using a Philips CM10 Transmission Electron

microscope (Philips, Eindhoven) at an operating voltage of 100 kV

267

6.4.3. Localization of 185/333 proteins

6.4.3.1. Anti-185/333 antisera

The polyclonal antisera used to localize 185/333 proteins have been described

previously by Brockton et al. [15]. They were prepared by injecting rabbits with three

different synthetic 185/333 peptides designated anti-185-66, anti-185-68 and anti-185-71

(Quality Controlled Biochemicals, Hopkinton MA) corresponding to three highly

conserved regions of 185/333 cDNAs (Table 1). The three different antisera were mixed in

equal proportions before use, as previously described [11, 15].

6.4.3.2. Immunofluorescence microscopy of whole cell

CF was mixed with an equal volume of chilled calcium and magnesium free sea

water (CMFSW; 460 mM NaCl, 10.73 mM KCl, 7.04 mM Na2SO4, 2.38 mM NaHCO3 in

deionised H2O) and 30 µL of the cell suspension was loaded onto microscope slides. The

cells were kept at room temperature for 30 minutes before the CMFSW was removed.

They were overlaid with cell free CF and held at room temperature for 20 minutes until

they developed filopodia. The cells were then fixed in filtered seawater (FSW) containing

4% w/v paraformaldehyde for 20 minutes. After washing (3 times for 5 minutes in FSW),

the cells were overlaid with cytology blocking buffer (10% w/v bovine serum albumin,

BSA, 10% v/v normal goat serum in FSW) for 60 minutes at 4°C. Excess buffer was

removed before adding the anti-185/333 antisera (1:250 in PBS) for 60 minutes at 4°C.

One set of controls used serum collected from rabbits before they were immunized with

the 185/333 peptides (pre-immune serum; 1:250 in PBS). Other control slides (no primary

268

antibody) were overlaid with blocking buffer rather than antisera. Following incubation,

the antisera were removed, and the slides were washed with PBS (3 times for 5 minutes)

and then overlaid with anti-rabbit Ig- Alexa Fluor® 488 (1:400 in PBS) for 60 minutes at 4

°C in the dark. To counter-stain nuclei, slides were incubated with TO-PRO® 3-iodide

(1:500 in PBS) for 5 minutes (4°C; in dark). The slides were then washed in PBS (3 times

for 5 minutes) before being mounted in GelMount™ aqueous mounting media. Cells were

visualized using a laser scanning confocal microscope (Olympus Fluoview 300 equipped

with an inverted microscope, IX70, Olympus, Japan) with a 100 oil immersion objective

(N.A 1.4). Images were sequentially scanned with an argon 488 nm laser for Alexafluor®

488, and a helium neon 633 nm laser for TO-PRO® 3-iodide. The images were captured

and analyzed using Fluoview version 4.3 software (Olympus, Japan).

6.4.3.3. Immunofluorescence staining of semi-thin resin sections

Semi-thin sections (see above) were mounted onto glass slides. Immunolabelling

was conducted at room temperature. All sections were incubated in blocking solution

(10% fetal bovine serum, FBS, in PBS) for 30 minutes. Excess buffer was then drawn off

before adding the anti-185/333 antisera (1:250 in PBS) for 1 hour. Control sections were

overlaid with blocking buffer only or pre-immune sera (1:250 in PBS). Following

incubation, the solutions were removed and the sections were washed with PBS (5 times

for 5 minutes). All sections were then incubated with anti-rabbit Ig- Alexa Fluor® 488

(1:400 in PBS) for 1 hour in the dark. Nuclei were counterstained with propidium iodide

(1:1000 in PBS) for 5 minutes in the dark. The sections were washed in PBS, and mounted

in GelMount™ (Pro Sci Tech). Cells were visualized using a laser scanning confocal

microscope as described above (helium neon 543 nm laser for propidium iodide).

269

6.4.3.4. Immuno-gold labelling for transmission electron microscopy

Ultrathin sections on 300 mesh nickel grids were incubated with 0.05 M glycine in

PBS for 15 minutes and blocking solution (5% FBS, 5% BSA in 1 PBS) for 1 hour

before being washed (3 times for 5 minutes) in incubation buffer (0.1% BSA in PBS).

Sections were then incubated in anti-185/333 antisera (1:250 in incubation buffer) for 1

hour. Controls were incubated in blocking buffer or pre-immune serum (1:250) for 1 hour.

Following five washes in incubation buffer (3 minutes each), grids were incubated in gold-

conjugated secondary antibody (1/150 in incubation buffer, BBI goat anti-rabbit IgG

F(ab’)2, EM grade, 5 nm gold, Pro Sci Tech) for 1 hour. Sections were then washed in

incubation buffer (5 times for 3 minutes), post-fixed with 2% glutaraldehyde in PBS for 5

minutes, and washed in PBS (2 times for 5 minutes) and Milli-Q water (2 times for 5

minutes). All sections were counter stained in uranyl acetate (saturated, aqueous) for 30

minutes and lead citrate for 4 minutes. Sections were analyzed using a Philips CM10

transmission electron microscope (TEM; Philips, Eindhoven) at an operating voltage of

100 kV.

270

Figure 6.1: Coelomocyte types in live cells preparation of H. erythrogramma CF. A/

filopodial amoebocyte; B/ petaloide amoebocyte; C/ colorless spherule cell; D/ red

spherule cell; E/ fusiform cell; F/ vibratile cell.

5 µm 5 µm 5 µm

5 µm 5 µm 5 µm

A B C

D E F

271

6.5. Results

6.5.1. Characterization of coelomocyte types

Analysis of live cells identified the four major types of coelomocytes that had

previously been described in S. purpuratus; amoebocytes, colorless spherule cells, red

spherule cells and vibratile cells [11, 15]. An additional cell type, designated fusiform

cells, was also observed in H. erythrogramma, (Figure 6.1E). This cell type was

characterized by the presence of two flagella on opposite poles of the cell. A comparable

cell type has previously been described in holothurians [12-14, 22, 23]. Only two

amoebocyte types could readily be identified in live cell samples of H. erythrogramma;

filopodial and petaloide amoebocytes (Figure 6.1A,B). Discoidal and polygonal

amoebocytes, as described by Brockton et al. [15], could not be differentiated because the

cells were not allowed to adhere on slides before observation. Colorless spherule cells

were seen as large amoeboid cells packed with large clear spherules (Figure 6.1C), which

contrasted with the bright red bodies of red spherule cells (Figure 6.1D). Vibratile cells, as

previously described by Johnson et al. [13], had scant cytoplasm and a single long

flagellum (Figure 6.1F).

272

Figure 6.2: Confocal images of 185/333-positive coelomocytes. Adherent filopodial

coelomocyte (A) and semi-thin sections (1 µm) of coelomocytes (B,C,D,E) stained with

anti-185/333 antisera and Alexa fluor® 488 (green) (A,B,C) or methylene blue (D,E). In

immunofluorescence micrographs, nuclei were counter stained with TO-PRO 3™ (blue) or

with propidium iodide (red). 185/333 proteins were observed in both filopodial

amoebocytes (A,B,D) and colorless spherule cells (C,E). Arrows show intense “knobs” of

185/333 staining on filopodia.

A B C

E D

2µm

Dheilly et al., Fig 2

2µm

2µm 2µm 5µm

273

6.5.2. Immunofluorescence identification of 185/333 proteins in filopodial amoebocytes

and colorless spherule cells

Immunofluorescence 185/333 staining among coelomocytes adhered to slides was

observed only on the surface of filopodial amoebocytes (Figure 6.2A). These cells had

long filopodia with discrete packets (previously called knobs; [15]) of 185/333 staining

(Figure 6.2A). However, immunofluorescence microscopy of semi-thin sections also

revealed the presence of 185/333 proteins in colourless spherule cells (Figure 6.2B,C).

In sectioned filopodial amoebocytes, 185/333 proteins were localized on the cell

membrane and within small vesicles scattered throughout the cytoplasm (Figure 6.2B,D).

In colorless spherule cells, 185/333 proteins were abundant within the large spherules that

fill the cytoplasm of these cells (Figure 6.2C,E).

6.5.3. Ultrastructure and 185/333 localization of coelomocytes

On examination by TEM, colorless spherule cells were found to possess numerous

large membrane bound vesicles (spherules) and an irregularly shaped hyperchromatic

nucleus (Figure 6.3). Free cytoplasm was scant, since most of the cell was filled with

spherules. The spherules of any one cell were uniform in size. The electron density of the

spherules was extremely variable (Figure 6.3). Gold immunolabelling confirmed that

185/333 proteins were localized within the spherules. Some spherules appeared more

185/333 positive than others (Figure 6.3). However, this variability in the concentration of

185/333 proteins seemed unrelated to variability in the electron density of the spherules.

274


colorless spherule cell. Colorless spherules fill the cytoplasm of colorless spherule cells

(A). A highly 185/333+ spherule (arrows show gold particles) is shown adjacent to a less

intensely stained spherule (B).

B

sp

sp

0.1 µm

A

sp

sp

sp

n

2 µm

Dheilly et al., Fig. 3

275

Filopodial amoebocytes could readily be discerned in methylene blue stained semi-

thin sections. Their cytoplasm appeared blue with dark granules, and filopodial formations

could be observed. The ultrathin sections used for electron microscopy often did not allow

the visualisation of filopodia. As a result, filopodial amoebocytes were difficult to

characterize by TEM apart from the high electron density of their cytoplasm, which

contrasted with the low-density cytoplasm of petaloide amoebocytes (Figure 6.4). In

addition, the nuclei of filopodial amoebocytes under TEM were often rounded with dense

heterochromatin beneath the nuclear envelop. The larger cells in this class contained

numerous membrane bound vesicles. Small round mitochondria were often observed and

Golgi apparati were frequent.

185/333 labelling was evident in the golgi network of filopodial amoebocytes and

was more intense on the trans-Golgi network than on the cis-face (Figure 6.5). Additional

gold labelling appeared on the extremities of the Golgi, within what seem to be transport

vesicles (Figure 6.5).

Different types of membrane bound 185/333 positive vesicles were observed in

filopodial amoebocytes. Some were electron lucent (Figure 6.4 and 6.6A), others had

electron dense material (Figure 6.4 and 6.6B), and a third type showed structured

concentric lamellae and a granular center (Figure 6.6C). The lumen of the vesicle

generally did not contain 185/333 proteins. However, the membranes of these three types

of vesicles were highly positive when stained with anti-185/333 antisera (Figure 6.6A-C).

A fourth type of membrane bound vesicle was sometimes observed. No 185/333 proteins

appeared on the membrane of these vesicles, but the granular content had anti-185/333

gold labelling throughout (Figure 6.6D).

276


filopodial amoebocyte. A/ whole cell. B,C/ Insets showing details of 185/333 localization.

185/333 proteins are highly expressed on the plasma membrane (arrowhead in B) and

within small vesicles (arrows in B and C). n: nucleus, m: mitochondria, pm: plasma

membrane, v: vesicle.

A

C

B

n

v

pm B

!"

!"

C

!"v

277


Golgi apparatus in a filopodial amoebocyte. Panel B is an enlargement of the boxed

inset in panel A. 185/333 proteins are found on the trans face of the Golgi apparatus,

within small vesicles and on the cell surface. Arrows point to gold particles. G: Golgi, n:

nucleus, v: vesicle, -cis: cis face of the Golgi, -trans: trans face of the Golgi

n

G

-trans

-cis

- trans

- cis

v

v

v

!"#$%&$

! "#


278

Figure 6.6: Transmission electron micrograph of 185/333-positive vesicles in filopodial

amoebocytes. Vesicles appeared empty (A), contained an electron dense material (B), had

structured concentric lamellae (C) or diffuse granular material (D). 185/333 proteins were

usually membrane bound (A,B,C), but were sometimes seen within the vesicles that

contain diffuse granular material (D).

0.1 µm 0.1 µm

A B

0.1 µm 0.2 µm

C D


279


filopodial amoebocyte cell surface. Panels B and D are boxed inset of panels A and C.

A,B/ bundles of 185/333 proteins on the cell surface are shown by arrows. C,D/ membrane

bound 185/333-positive discs (arrow heads) dissociated from a filopodial amoebocyte.

Arrows point to gold particles.

A

0.5 µm

C D

BB

D

0.5 µm

0.2 µm

0.2 µm

Dheilly et al., Fig.7

280


gut-associated amoebocytes. Panels B, C, and D are enlargements of the boxed insets in

panels A, B and C. Gut-associated amoebocytes are found adjacent to anuclear bodies (A).

Arrows point to gold particles. ab: anuclear bodies, am: amoebocyte, en: enterocytes, ER:

endoplasmic reticulum, G: Golgi apparatus, m: mitochondria, n: nucleus, v: vesicles.

am ab

en en

en

en

en

n

m

m

ER

G

en

!"#$%&$

m

A B

!"'$%&$

v v

v

v

G

ab

ER

D

B

!"

D C


281

185/333 proteins were also commonly found on the surface of filopodial

amoebocytes. When filopodia could be observed under TEM, gold particles were

extremely abundant on their surface (Figure 6.7A). 185/333 proteins often appeared

concentrated in bundles on the filopodia. These may correspond to “knobs” of 185/333

proteins reported by Brockton et al. [15] and under immunofluorescence microscopy in the

current study. Positively stained, round formations were also seen adjacent to 185/333

positive filopodial amoebocytes (Figure 6.7B). We could not determine whether these were

excreted granules or cross sections of filopodia. However, previous immunofluorescence

analysis identified dissociated discs (or “frizbees”) of 185/333 material floating free in the

coelomic fluid (Raftos DA, unpublished data)

6.5.4. 185/333 proteins in the gut

185/333 expression was also identified in gut amoebocytes, which were located on

the basal coelomic fluid face of the gut endothelium (Figure 6.8). The cytoplasm of these

amoebocytes was granular and their nuclei had dense heterochromatin beneath the nuclear

envelop. (Figure 6.8A). Mitochondria were oblong. Adjacent to the nucleus, we often

observed faint membrane bound lamellae that resemble Golgi apparati (Figure 6.8B). As in

filopodial amoebocytes from coelomic fluid samples, 185/333 proteins were abundant on

the cell surface of gut amoebocytes, on the trans face of the putative Golgi apparatus and

membrane bound within the numerous small vesicles that filled the cytoplasm (Figure

6.8D).

282


vesicles in anuclear bodies in gut tissue. Panels B, D and F are enlargements of boxed

insets in panels A, C and E. The vesicles in anuclear bodies had various types of content

(A,C,E) and their membranes were highly 185/333+ (B,D,F, arrows). Structures

ressembling bacteria were sometimes seen (E,F). Arrows point to gold particles. b:

bacteria.

1 µm

1 µm

1 µm

0.1 µm

0.1 µm

0.1 µm

b b

b

b

A B

C D

E F

Dheilly et al., Fig 9

283

Numerous anuclear bodies were observed adjacent to these 185/333 positive

amoebocytes (Figure 6.9). The anuclear bodies appeared homogeneously light blue in

methylene blue stained sections. TEM revealed the abundance of 185/333 proteins on the

surface and on the membrane of vesicles within these anuclear bodies (Figure 6.9).

Structures within these vesicles were mostly unrecognizable. However, bacteria-like

structures were observed within some vesicles (Figure 6.9E,F). 185/333 staining was also

apparent on the surface of these bacteria-like structures.

6.5.5. Phagocytosis by coelomocytes

TEM examination of CF from sea urchins injected with yeast and bacteria showed

that none of the phagocytic cells that ingested these particles were 185/333-positive

(Figure 6.10). Only 185-333-negative petaloide amoebocytes were seen phagocytosing

yeast, bacteria and other coelomocytes. Within these cells, no compartmentalisation of the

cytoplasm was observed and the nucleus had a dense chromatin and was rarely rounded

(Figure 6.10). The numerous lamellipodia protruding in all directions had the appearance

of extremely large empty vesicles (Figure 6.10C).

Even though they were not phagocytic, colorless spherule cells were also affected

by the injection of bacteria. After challenge, the density of the spherules changed (Figure

6.11). They developed an inner core that was less electron-dense than the surrounding area

(Figure 6.11B,C). Eventually, they had a central electron dense core in a loose fibrillar

shell (Figure 6.11D). During this last stage, 185/333 proteins were rarely found within the

spherules.

284

Figure 6.10: Phagocytosis by petaloide amoebocytes. Live cell preparation (A), and

transmission electron micrographs (B-F) of petaloide amoebocytes phagocytosing bacteria

(C-F) and yeast cells (B). No 185/333+ staining was observed on bacteria free in the

coelomic fluid (D), undergoing phagocytosis (E) or within phagosomes (F). y : yeast cells.

b: bacteria, n:nucleus.

5µm

y A

n

b b

b

C

bbbD E F

0.5 µm 0.5 µm 0.5 µm

2µm

B y

n

Dheilly et al., Fig.10

285


colorless spherule cells. A/ Spherules had an homogenous electron density before the

injection of bacteria or yeast. B,C,D/ Electron density changed after microbial injection.

No 185/333 proteins were observed in empty spherules with electron dense cores (D).

0.5 µm

0.5 µm 0.5 µm

A

C D

0.5 µm

B

286

6.6. Discussion

Coelomocytes are the immune effector cells of sea urchins [24]. They express highly

variable family of 185/333 proteins, the concentration of which increases after immune

challenge [5, 11, 15]. In the current study, we localized 185/333 proteins within

coelomocytes and gut amoebocytes at the ultrastructural level and investigated their role in

phagocytosis. As a prerequisite for this analysis, we first characterized coelomocyte types

by light microscopy. Six different cell types were found in the coelomic fluid: filopodial

amoebocytes, petaloide amoebocytes, vibratile cells, fusiform cells, colorless spherule cells

and red spherule cells. All these cell types have been observed previously in sea urchins,

except for the fusiform cells [14]. However, fusiform cells have been found in other

echinoderms, such as the star fish, Asterias rubens [25], and the sea cucumber,

Apostichopus japonicus [26]. They are rare in coelomic fluid and their function is

unknown.

The expression of 185/333 proteins in coelomocytes has previously been studied in

S. purpuratus by Brockton et al. [15]. They used anti-185/333 antisera and

immunofluorescence microscopy to identify intense 185/333 staining on the surface of

small amoebocytes, and within perinuclear vesicles of both small amoebocytes and

polygonal amoebocytes. However, the expression of 185/333 proteins was extremely

variable between cells and some seemed to express 185/333 proteins throughout the

cytoplasm [15]. Immunofluorescence analysis was repeated in the current study with H.

erythrogramma coelomocytes using adherent coelomocytes fixed to microscopy slides. We

could not discriminate between the two forms of filopodial amoebocyte (polygonal and

small amoebocytes) previously described [15, 19], but observed intense anti-185/333

immunostaining of filopodial amoebocytes, while all other coelomocyte types remained

unstained (Figure 6.2A). Anti-185/333 reactivity on filopodia was localized into discrete

287

packets (“knobs”) of intense staining similar to that previously observed on the small

amoebocytes of S. purpuratus (Figure 6.2A).

We also repeated the immunofluorescence experiments using semi-thin sections of

fixed cells (Figure 6.2B,C). In this case, immunofluorescence microscopy revealed the

presence of 185/333 proteins within both filopodial amoebocytes and colorless spherule

cells. The expression of 185/333 proteins in colorless spherule cells may not have been

detected previously because this cell type often does not adhere to glass slides (Smith LC,

personal communication).

Immunogold TEM analysis demonstrated that filopodial amoebocytes had well-

developed Golgi apparati and that 185/333 proteins were found in abundance within the

trans-Golgi network. They were also found on the internal membrane surfaces of vesicles

within the cytoplasm, and in abundance on the cell surface, especially along filopodial

formations. Brockton et al. [15] had suggested that, since predicted 185/333 proteins have

no obvious membrane anchoring motifs, such as transmembrane domains or GPI anchors,

185/333 proteins are secreted from coelomocytes and then re-attached to surface

membranes. However, our ultrastructural observations suggest a more conventional

pathway, with 185/333 proteins transported to the cell surface via the Golgi apparatus and

membrane transport vesicles. This leaves open the question of how 185/333 proteins are

anchored to the membrane. It may be that they are oligomerized with other membrane-

anchored proteins. Oligomerization is supported by the previously reported disparity in

molecular weights between predicted 185/333 proteins and those observed on one- and

two-dimensional Western blots [11].

Bulbous protuberances on the filopodia of amoebocytes, which have previously been

observed by Edds [27] and Brockton et al. [15], appeared to contain more 185/333 proteins

than other areas of the membrane. We also observed membranous discs of 185/333

positive material that appeared to be dissociated from the surface of filopodial cells. Whilst

288

these may simply be filopodia caught in cross-section, they might also equate to the discs

(“frizbees”) of 185/333 positive material previously found free in the coelomic fluid

(Raftos DA, unpublished data). If this was the case, it would suggest that dicrete

membranous discs of 185/333 rich material can detach from the cell surface.

In addition to filopodial amoebocytes, 185/333 proteins were detected within gut-

associated amoebocytes. As with filopodial amoebocytes in coelomic fluid, 185/333

proteins in these cells were membrane bound within cytoplasmic vesicles and on the

external face of the cell membrane. Interestingly, the 185/333 positive staining of the gut-

associated amoebocytes appeared more intense than that within filopodial amoebocytes of

the coelomic fluid. However, the ultrastructure of both gut-associated amoebocytes and

filopodial amoebocytes was similar, suggesting that the two cell types may be related.

Most significantly, we observed gut-associated amoebocytes adjacent to anuclear

bodies, located mostly in the basal region of the intestinal endothelium. The content of the

anuclear bodies was mostly uncharacterized. They seemed to contain cellular debris or

bacteria within large vesicles. Briot [28] and Arvy [29] showed that similar “brown

bodies” in the intestine of holothurians contain debris of parasitic origin. Hence, it is

possible that the anuclear bodies in H. erythrogramma are encapsulated microbial material

or degenerative amoebocytes containing phagosomes. 185/333 proteins were extremely

abundant on the membranes of anuclear bodies. This suggests that in the gut, 185/333

proteins may be involved in phagocytosis or encapsulation of microbial pathogens. Such a

hypothesis fits with previous speculation that the sea urchin immune system focuses on

control of intestinal infections or regulation of gut microflora [3].

Even though 185/333 proteins may play a role in phagocytosis in gut, we found no

evidence that they are involved in this process in the coelomic fluid. After the injection of

bacteria and yeast into the coelomic cavity, only petaloide amoebocytes, which do not

express 185/333 proteins, appeared phagocytic. 185/333 proteins were not found bound to

289

injected bacteria or yeast, and 185/333-positive coelomocytes were not phagocytic. This

agrees with previous studies, which showed that petaloide amoebocytes are involved in

phagocytosing foreign particles in the coelomic fluid, whereas filopodial amoebocytes

have roles in wound healing and clotting [19]. This includes the formation of syncitia by

185/333 positive amoebocytes, forming aggregates between cells that might lead to the

encapsulation of pathogens [15].

In addition to filopodial cells, 185/333 proteins were identified in colorless spherule

cells in the coelomic fluid. 185/333 proteins were not associated with membranes within

this cell types, as they were in amoebocytes, but were enclosed within the large membrane

bound spherules that fill the cytoplasm. These spherules had a homogeneous electron

dense content that was modified after the injection of yeast and bacteria. After injection,

the electron density of the spherules changed leaving some cells with empty spherules, or

spherules with an electron dense core but a loose outer cortex. A corresponding decrease in

the concentration of 185/333 proteins was observed with the disappearance of the content

of the spherules. Similar alterations of electron density in spherules have been observed in

holothurians [30, 31]. The changes suggest that the contents of spherules are secreted in

response to the presence of bacteria or yeast.

The nature of the contents within spherules is a long standing question that has not

yet been resolved. The general view is that spherules contain mucopolysaccharides, which

are responsible for their tinctorial properties [31-33]. In holothurians, similar spherules

contain proteins, neutral polysaccharides and sometimes lipid, as well as

mucopolysaccharides [34]. Byrne [35] suggested that spherules contain molecules that

participate in fibrogenesis (ECM deposition). Indeed, colorless spherule cells release their

vesicular content within the regenerating intestine [36] and have been associated with

wound healing. In sea urchins, Menton and Heisen [30] showed an accumulation of

colorless spherule cells within minutes after wounding, whilst Cervello et al. [37]

290

demonstrated that the spherules of colorless spherule cells contain vitellogenin, a large

glycoprotein, that is discharged in the coelomic fluid in response to stress. Because the

protein is highly adhesive, they suggested that it is involved in the clotting reaction. In

Amphoxius, vitellogenin has both hemaglutinating and antibacterial activities [38]. Thus,

colorless spherule cells might not only be involved in wound healing, but could also

contribute to the first line of immune defense. This suggestion is supported by Arizza et al.

[17] who used a plaque-forming assay on whole coelomic fluid preparations and density

gradient separated coelomocytes to show that colorless spherule cells are cytotoxic and

that this toxicity is activated by soluble factors released from amoebocytes. Monoclonal

antibodies were found to inhibit the cytotoxicity of both colorless spherule cells and

amoebocytes by interacting with unidentified 45 kDa and 100 kDa proteins [18]. In this

context, the modification of spherules in response to microbial injection and the

subsequent disappearance of 185/333 proteins may be due to either wounding or the

presence of bacteria and yeast cells.

The different cellular localization of 185/333 proteins in filopodial amoebocytes

and colorless spherule cells suggests that these proteins may have different biological

activities in the different cell types. This may fit with the observation that there are number

of discrete groups of 185/333 molecules that could be cell specific. The cellular

localization of 185/333 proteins revealed by ultrastructural analysis in the current study

provides the first definitive clues to the function of the 185/333 family. In gut-associated

amoebocytes, they may be involved in the phagocytosis of microbial material, whilst they

might be associated with wound healing and cytotoxicity in the coelomic fluid.

291

6.7. References

[1] Sodergren, E., Weinstock, G., Davidson, E., Cameron, R., et al., The genome of the sea

urchin Strongylocentrotus purpuratus. Science 2006, 314, 941-952.



[3] Pancer, Z., Cooper, M. D., The evolution of adaptive immunity. Annual Review of

Immunology 2006, 24, 497-518.

[4] Holland, L. Z., Albalat, R., Azumi, K., Benito-Gutiérrez, E., et al., The amphioxus

genome illuminates vertebrate origins and cephalochordate biology. Genome Res

2008, 18, 1100-1111.




2005, 22, 33-47.

[6] Terwilliger, D., Buckley, K., Mehta, D., Moorjani, P., Smith, L., Unexpected diversity

displayed in cDNAs expressed by the immune cells of the purple sea urchin,

Strongylocentrotus purpuratus. Physiol Genomics 2006, 26, 134-144.





[8] Buckley, K., Smith, L., Extraordinary diversity among members of the large gene

family, 185/333, from the purple sea urchin, Strongylocentrotus purpuratus. BMC

Mol Biol 2007, 8, 68.

292

[9] Buckley, K. M., Munshaw, S., Kepler, T. B., Smith, L. C., The 185/333 gene family is

a rapidly diversifying host-defense gene cluster in the purple sea urchin

Strongylocentrotus purpuratus. J Mol Biol 2008, 379, 912-928.

[10] Buckley, K. M., Terwilliger, D. P., Smith, L. C., Sequence variations in 185/333


increase immune diversity. J Immunol 2008, 181, 8585-8594.

[11] Dheilly, N. M., Nair, S. V., Smith, L. C., Raftos, D. A., Highly variable immune-


proteomic analysis identifies diversity within and between individuals. J Immunol

2009, 182, 2203-2212.



[13] Johnson, P., The coelomic elements of the sea urchins (Strongylocentrotus) III. In


[14] Smith, L., Rast, J., Brockton, V., Terwilliger, D., et al., The sea urchin immune

system. Invertebrate Survival Journal 2006, 3, 25-39.


diversity of 185/333 proteins from the purple sea urchin - unexpected protein-size

range and protein expression in a new coelomocyte type. J Cell Sci 2008, 121, 339-

348.





293



Comp Immunol 2007, 31, 465-475.




1044.



2000, 51, 1021-1033.

[21] Wilson, G. S., Raftos, D. A., Corrigan, S. L., Nair, S. V., Diversity and antimicrobial

activities of surface-attached marine bacteria from Sydney Harbour, Australia.

Microbiol Res 2009. In Press.

[22] Holland, N. D., Giese, A. C., An autoradiographic investigation of the gonads of the

purple sea urhcin (Strongylocentrotus purpuratus). Biol Bull 1965, 128, 241-258.


the evolution of the complement system. Dev Comp Immunol 1999, 23, 429-442.

[24] Smith, L. C., Davidson, E. H., The echinoderm immune system. Characters shared

with vertebrate immune systems and characters arising later in deuterostome

phylogeny. Ann. NY Acad. Sci. 1994, 712, 213-226.

[25] Kozlova, A. B., Petukhova, O. A., Pinaev, G. P., The analysis of cellular elements in

coelomic fluid of the starfish Asterias rubens L. in early regeneration time.

tsitologiia 2006, 48, 175-183.

[26] Xing, J., Chia, F. S., Phagocytosis of sea cucumber amoebocytes: a flow cytometric

study. Invertebr Biol 1998, 117, 67-74.

294



[28] Briot, A., Sur les corps bruns des holothuries. C R Seances Soc Biol Fil 1906, 60,

1156-1157.

[29] Arvy, L., Contribution a la connaissance des 'corps bruns' des Holothuridae. C R Hebd

Seances Acad Sci 1957, 245, 2543-2545.

[30] Menton, D. N., Eisen, A. Z., The structure of the integument of the sea cucumber

Thyone briareus. J. Morphol. 1970, 131, 17-36.

[31] Hetzel, H. R., Studies on holthurian coelomocytes. I. A survey of coelomocyte types

Biol Bull 1963, 125, 289-301.

[32] Endean, R., Physiology of echinodermata, Intersciences Publishers, London 1966.

[33] Cowden, R. R., Cytological and histochemical observations on connective tissue cells

and cutaneous wound healing in the sea cucumber Stichopus badionotus. J


[34] Eliseikina, M. G., Magarlamov, T. Y., Coelomocyte morphology in the holothurians

Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica

(Dendrochirota: Cucumariidae). Russ J Mar Biol 2002, 28, 197-202.

[35] Byrne, M., the ultrastructure of the morula cells of Eupencttacta quinquesemita

(Echinodermata: Holothuroidea) and their role in the maintenance of the

extracellular matrix. J. Morphol. 1986, 188, 179-189.

[36] Garcia-Arrares, J. E., Schenk, C., Rodrigues-Ramirez, R., Torres, I. I., et al.,

Spherulocytes in the echinoderm Holothuria glaberrima and their involvement in

intestinal regeneration. Dev Dyn 2006, 235, 3259-3267.



64, 314-319.

295

[38] Zhang, S., Sun, Y., Pang, Q., Shi, X., Hemagglutinating and antibacterial activities of

vitellogenin. Fish Shellfish Immunol 2005, 19, 93-95.

296

297

CHAPTER VII

General discussion

298

299

7.1. General discussion

The experiments described in this thesis have provided fundamental information on

the immune responses of sea urchins. The thesis tested whether the complex immune

system predicted by genomic and transcriptomic data is evident at the level of expressed

proteins [1-3]. As such, this work provides essential information on the proteins expressed

in response to wounding and immune challenge, with particular emphasis on 185/333

proteins within the second half of this thesis.

The aim of the initial investigation (Chapter 2) was to establish the first proteome

map for the CF of sea urchins. This allowed comparison with proteomes of immune

responsive cells from vertebrates and other invertebrate species. A total of 323 proteins

were identified in S. purpuratus CF, and homologues of 236 of these were later identified

in H. erythrogramma CF (Chapter 4). This confirmed that most protein sequences are

conserved between these two species, which validates our approach of using the genome of

S. purpuratus for the identification of proteins in H. erythrogramma.

Proteins associated with cell shape and mobility were the most abundant in both

proteomes confirming the preponderance of amoeboid coelomocytes within the CF. These

cells have previously been implicated in clotting reactions and phagocytosis, and have

been shown to express complement component C3 [4, 5]. It is interesting that a large panel

of proteins involved in clotting and the complement system in vertebrates were among the

most abundant proteins in our samples. These data suggest that similarities with the

corresponding vertebrate systems are more extensive than previously thought.

Investigation of the CF proteome also revealed the abundance of a variety of homologues

of iron-binding proteins. This indicates that iron metabolism has an important role in the

function of coelomocytes, either in cell proliferation, activation of nitric oxide responses or

depletion of iron [6-16].

300

Even though most proteins identified within CF had homologues in vertebrate

immune systems and could ultimately be related to immune responses, the precise

functions of most proteins have not been tested experimentally in sea urchins. Chapter 3

and Chapter 4 used comparative proteomic techniques to determine the role of CF proteins

in anti-pathogen responses. In the experiments, sea urchins were injected with microbes,

PAMPs, or aCF as controls. In Chapter 3, PCA identified a significant modification of the

proteome 24 hours after saline injection, with few additional differences due to the

injection of bacteria. This modification of the proteome most likely reflects responses to

the wound created by the needle used for injection or the increased volume of CF due to

the injection of aCF. Sea urchins have open circulatory system, which makes coelomocyte

coagulation in response to wounding one of the most important defense reactions to

prevent the lost of CF when injured, and to limit the spread of microbes in the coelom.

In Chapter 4, we further investigated this wounding response over time. In the first

instance, a major modification of the proteome due to injection was observed after 6 and

24 hours p.i.. The first phase of the response involved major cytoskeletal proteins,

including actin, fascin, Rac1 and cdc42. These proteins probably lead to the formation of

filopodia and adhesion complexes necessary for the formation of clots involved in sealing

wounds [17-20]. This was quickly followed by a humoral response, mostly involving an

iron-binding protein of the LLTP family, major yolk protein [12, 21]. A second discrete

phase occurred 48 hours p.i.. At this time-point, the induction of wound repair mechanisms

was evident. This included the increased abundance of proteins such as Von willebrand

factor, cyclophilin B, and selectins, which are involved in cell migration to sites of injury

and adhesion to collagen [22-24].

In addition to wounding responses, Chapters 3 and 4 also identified proteins whose

relative abundance was substantially altered specifically in response to the injection of

bacteria or PAMPs. In Chapter 3, we employed a classical 2DE based comparative

301

proteomics analysis in which the major shift in protein abundances due to the wound

response mostly masked other more specific responses to the injection of bacteria. In

addition, many significantly altered proteins isolated from 2DE gels were often not

abundant enough to be identified by mass spectrometry. However, two of the proteins

(apextrin and calreticulin) that could be identified were significantly more abundant in

response to the injection of bacteria, as opposed to saline, suggesting that they are both

involved in immunological reactions or pathogen sequestration. These results are

supported by their involvement in immunological reactions in other species [25-27].

In Chapter 4, we turned to a more sensitive shotgun proteomic method to investigate

the response to LPS injection over time. It was obvious from PCA that the alteration in

protein concentrations due to the presence of LPS occurred mostly 48 hours p.i.. Vesicular

transport proteins, such a coatomer proteins and RACK, and cellular signalling molecules,

such as G(q) and MAP kinase, increased in relative abundance after LPS injection. Many

immune response molecules such as SpC3, dual oxidase maturation factor, dual oxidase 1,

α-2 macroglobulin, SRCRs, fibrocystin L and aminopeptidase N were also more abundant

48 hours after the injection of LPS as opposed to aCF. These results suggest their

involvement in inducible immune responses in sea urchins. More specifically, fibrocystin

L and aminopeptidase N were only found 48 hours after LPS injection, indicating that

these proteins are expressed only during PAMP-induced immune responses. Both show

strong sequence similarities with their vertebrate homologues, suggesting that they could

perform similar functions in sea urchins [2, 28, 29]. In vertebrates, they are receptor

proteins expressed on immune responsive cells that bind lectins and participate in

regulating phagocytosis [29-34].

Comparisons of the results presented within Chapters 3 and 4 also revealed

differences in responses to whole microbes and PAMPs. No differences in the relative

abundance of calreticulin and apextrin were observed between saline- and LPS-injected sea

302

urchins. This contrasted with the significant increase in the abundance of these two

proteins in Chapter 3, in which sea urchins were injected with whole bacteria. This

inconsistency indicates that coelomocytes respond differently to LPS than to whole

microorganisms. Similar observations have been made in Drosophila hemocytes, which

activate different cellular pathways in response to purified PAMPs compared to live

bacteria [35]. Perhaps, the injection of whole organisms favours increased phagocytosis,

clotting and encapsulation, whilst PAMPs activate different cellular processes via distinct

pattern recognition receptors.

The comparative proteomic analyses reported in this thesis suggest that a complex

array of proteins respond to wounding and immunological challenge. A biphasic response

has been described. There is an early generalized response that shows many similarities to

reactions to sterile wounding followed by a delayed response that appeared to be specific

towards the PAMPs/microbes. Another reason to believe that the immune system of sea

urchins is extremely complex comes from the identification in its genome sequence of an

expanded array of pathogen recognition gene families when compared with humans and

insects [1]. For instance, 218 different SRCRs genes can be found in S. purpuratus

genome. In Chapter 2, we identified 16 SRCRs, of which 8 had not been characterised

previously. This confirms that a number of the available SRCRs are expressed in

coelomocytes, and agrees with previous work, which has shown that coelomocytes use

exon swapping to generate different patterns of SRCR expression [36].

Two other families of diversified proteins were also expected to be abundant in

coelomocytes based on genomic and transcriptomic data. Transcripts of 185/333 proteins

represented over 70% of novel mRNAs in an immunologically-activated coelomocyte

transcriptome, and as many as 222 distinct TLRs genes have been identified in the S.

purpuratus genome sequence [1, 2]. However, only a single member of the 185/333 family

and no TLRs were found in coelomocytes studied by proteomic analysis. The low

303

abundance of these two protein families in coelomocytes is difficult to explain. It may be

that individual 185/333 proteins and TLRs are not expressed at sufficiently high densities

in coelomocytes to enable identification by mass spectrometry. Hence, Chapters 5 and 6 of

the thesis, used more targeted methods to investigate the variability of 185/333 proteins

and study their potential role in immune defense.

Chapter 5 investigated the pattern of 185/333 protein expression in sea urchins

challenged with different PAMPs. Over 200 185/333+ protein spots could be identified on

2DE Western blots of CF from a single individual. Even though 185/333 proteins had

predicted molecular masses ranging from 27 to 56 kDa, their observed molecular masses

ranged from 20 to >193 kDa. Moreover, based on their amino acid sequences, 185/333

proteins were predicted to have pIs ranging from 6 to 11, due to single nucleotide

substitutions that are often conservative and result in amino acid substitutions of highly

charged amino acids. However, the transcribed 185/333 proteins identified on 2DE

Western blots had a range of pIs from 3 to 12 with most proteins having pIs between 3 and

6. This inconsistency between predicted and observed results suggests that 185/333

proteins interact with other molecules that have lower pIs. The fact that 185/333 proteins

have no transmembrane region nor a GPI anchor motif but are still expressed on the

surface of both filopodial amoebocytes and gut associated amoebocytes (Chapter 6) also

suggests that they may bind to other molecules that anchor them to cell membranes. Thus,

we expect future studies to demonstrate that 185/333 proteins form complexes with

membrane bound proteins.

In addition to the variability among 185/333 proteins within a single individual, we

found that the pattern of 185/333 proteins differed between individuals (Chapter 5). The

differences observed between individuals suggest variability in the expression of the

different 185/333 protein sub-families or the presence/absence of loci or alleles. However,

it was not possible to characterize unique protein spots from 2DE gels using mass

304

spectrometry to determine if single protein spots contained proteins from the same 185/333

sub-family. Instead, 185/333 proteins separated on 1DE gels were successfully identified

by shotgun mass spectrometry. Different combinations of peptide sequences were obtained

from each gel slice, suggesting that different combination of 185/333 proteins were present

within each MW range.

We also found evidence that 185/333 proteins contribute to immune responses [37].

The previously described transcription of 185/333 proteins in response to the injection of

LPS [1] was confirmed at the protein level by ELISA and Western blotting (Chapter 6).

Some modifications of the pattern of 185/333 proteins on Western blots of CF from sea

urchins stimulated with different PAMPs were also identified. Again, this corresponds with

previous transcriptomic analysis, which suggested that different groups of 185/333 proteins

are transcribed in response to different PAMPs [38].

Based on their responses to immunological challenge and their cytological

distribution, it had been assumed that 185/333 proteins are involved in pathogen

recognition, specifically in the encapsulation of pathogens or phagocytosis. In Chapter 6,

185/333 proteins were found within filopodial amoebocytes and colorless spherule cells in

the coelomic fluid. However, after the injection of yeast or bacteria into the coelomic fluid,

no 185/333+ filopodial amoebocytes or colorless spherule cells were seen undertaking

phagocytosis. This result is in accordance with previous observations that filopodial

amoebocytes are mostly involved in clotting and wound repair, while petaloide

amoebocytes are highly phagocytic [39]. However, colorless spherule cells, which

expressed 185/333 proteins within their spherules, appeared to release the content of these

spherules in response to injection. Previous studies suggested that colorless spherule cells

are involved in wounding responses and are cytotoxic [21, 40-42].

Investigation of 185/333 protein expression within gut tissue revealed that they are

also highly expressed within amoebocytes but absent in enterocytes (Chapter 6). The

305

ultrastructural localisation of 185/333 proteins in these gut-associated amoebocytes was

similar to that of filopodial amoebocytes and these two cell types shared numerous other

ultrastructural similarities, suggesting that they are closely related. Gut-associated

amoebocytes were always found adjacent to large anuclear bodies. 185/333 proteins were

also abundant on the membrane of these bodies, which appeared to be packed with

heterogeneous granular contents resembling cell debris and bacteria. The expression of

185/333 proteins in gut-associated amoebocytes and on the cell membrane of anuclear

bodies constitute the first indication that 185/333 proteins are specifically expressed by

immune cells that are involved in the ingestion of non-self.

306

7.2. Caveats and future directions

The logical extension of the proteomic characterization of coelomocytes would be to

analyse purified sub-populations of coelomocytes. This would allow us (1) to resolve the

proteome of each cell type and predict their functions and (2) to determine whether

different groups of 185/333 proteins are expressed in different cell types. Testing whether

different sub-families of 185/333 proteins are found in amoebocytes and colorless spherule

cell spherules, and defining the exact function of these different coelomocyte types, will

allow new hypotheses on the function of this highly variable family of proteins to be

developed. It will also be of interest to continue investigations on the ability of sea urchin

immune systems to discriminate between different pathogens. Sea urchins can live up to

100 years [43] and they succeed in the continuous arms race with pathogens suggesting

that the existence of specificity and memory in their immune responses might be an

evolutionary advantage. Sea urchins also constitute ideal model organisms for such studies

because they are abundant in aquatic environment, have large number of blood cells, and

can survive multiple injections and collection of coelomic fluid, permitting long-term

primary and secondary challenge experiments. Resolving whether memory and specificity

are characteristics of invertebrate immune responses is the major outstanding question in

comparative immunology.

307

7.3. Concluding remarks

The proteomic analysis of sea urchin immune responses presented in this thesis is a

first step in determining how the immune system of sea urchins works, and identifying the

actors of this system. This thesis showed that 185/333 proteins are just one component of a

complex protein system involved in host defense. Proteomic analysis paints a picture of

coelomocytes as being primarily amoeboid cells that express a large range of immune

response genes and proteins that could contribute to host defence. Many CF proteins

appear to be involved in wounding responses. However, there is also a substantial suite of

proteins that respond specifically to the presence of bacteria or PAMPs. Different sub-

populations of coelomocytes appear to be involved in the different components of this

immune response. This supports the growing realization that sea urchin immune responses

are complex and highly directed. The abundance of highly variable immune response

proteins (185/333) within gut-associated amoebocytes indicates a link between gut-

associated pathogenesis and the sea urchin immune system. Most significantly, 185/333

proteins do appear to be involved in the ingestion of degenerative material in the gut,

including bacteria. This supports their role in active host defense responses and leaves

open the possibility that the high variability of immune response protein families in sea

urchins is associated with regulation of gut flora.

308

7.4. References




2005, 22, 33-47.



[3] Smith, L., Chang, L., Britten, R., Davidson, E., Sea urchin genes expressed in activated

coelomocytes are identified by expressed sequence tags. Complement homologues

and other putative immune response genes suggest immune system homology

within the deuterostomes. J Immunol 1996, 156, 593-602.

[4] Smith, L., Rast, J., Brockton, V., Terwilliger, D., et al., The sea urchin immune system.

Invertebrate Survival Journal 2006, 3, 25-39.




1044.

[6] Baker, M., Lindley, P., New perspectives on the structure and function of transferrins. J

Inorg Biochem 1992, 47, 147-160.

[7] Brock, J., de Sousa, M., Immunoregulation by iron-binding proteins. Immunol today

1986, 7, 30-31.

309

[8] Stafford, J., M, B., Transferrin and the inante immune response of fish: identification of

a novel mechanism of macrophage activation. Dev Comp Immunol 2003, 27.

[9] Sekyere, E., Richardson, D., The membrane-bound transferrin homologue

melanotrasnferrin: roles other than iron transport? FEBS Lett 2000, 48, 11-16.

[10] Dunn, L., Sekyere, E., Rahmanto YS, Richardson, D., The function of

melanotransferrin: a role in melanoma cell proliferation and tumorigenesis.

Carcinogenesis 2006, 27, 2157-2169.

[11] Yokota, Y., Unuma, T., Moriyama, A., Yamano, K., Cleavage site of a major yolk

protein (MYP) determined by cDNA isolation and amino acid sequencing in sea

urchin, Hemicentrotus pulcherrimus. Comp Biochem Physiol B Biochem Mol Biol

2003, 135, 71-81.

[12] Brooks, J. M., Wessel, G. M., The major yolk protein in sea urchins is a transferrin-

like, iron binding protein. Dev Biol 2002, 245, 1-12.

[13] Beck, G., Ellis, T., Habicht, G. S., Schluter, S., Marchalonis, J., Evolution of the acute

phase response: iron release by echinoderms (Asterias forbesi) coelomocytes, and

cloning of an echinoderm ferritin molecule. Dev Comp Immunol 2002, 26, 11-26.

[14] Brock, J., Mulero, V., Cellular and molecular aspects of iron and immune function.

Proc Natl Acad Sci USA 2000, 59, 537-540.

[15] Ganz, T., Iron in innate immunity: starve the invaders. Current Opinion in

Immunology 2009, 21, 63-67.

[16] Fox, P., The copper-iron chronicles: the story of an intimate relationship. Biometals

2003, 16, 9-40.

310

[17] Vignevic, D., Kojima, S.-i., Aratyn, Y., Danciu, O., et al., Role of fascin in filopodial

protrusion. J Cell Biol 2006, 174, 863-875.









64, 314-319.

[22] Allain, F., Durieux, S., Denys, A., Carpentier, M., Spik, G., Cyclophilin B binding to

platelets supports calcium-dependent adhesion to collagen. Blood 1999, 94, 976-

983.

[23] Farndale, R. W., Sixma, J. J., Barnes, M. J., Groot, P. G. D., The role of collagen in

thrombosis and hemostasis. Journal of Thrombosis and Haemostasis 2004, 2, 561-

573.

[24] Vasta, G. R., Quesenberry, M., Ahmed, H., O'Leary, N., C-type lectins and galectins

mediate innate and adaptive immune functions: their roles in the complement

activation pathway. Dev Comp Immunol 1999, 23, 401-420.

[25] Arosa, F. A., de Jesus, O., Porto, G., Carmo, A. M., de Sousa, M., Calreticulin is

expressed on the cell surface of activated human peripheral blood T lymphocytes in

311

association with major histocompatibility complex class I molecules. J Biol Chem

1999, 274, 16917-16922.

[26] Dupuis, M., Schaerer, E., Krause, K., Tschopp, J., The calcium-binding protein

calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes. J

Exp Med 1993, 177, 1-7.

[27] Huang, G., Liu, H., Han, Y., Fan, L., et al., Profile of acute immune response in


infection. Dev Comp Immunol 2007, 31, 1013-1023.

[28] Ingersoll, E., An analysis of aminopeptidase N genes in the sea urchin genome. Dev

Biol 2008, 319, 566-566.

[29] Hogan, M. C., Griffin, M. D., Rossetti, S., Torres, V. E., et al., PKHDL1, a homolog

of the autosomal recessive polycystic kidney disease gene, encodes a receptor with

inducible T lymphocyte expression. Hum Mol Genet 2003, 12, 685-698.

[30] Mina-Osorio, P., Ortega, E., Aminopeptidase N (CD13) functionally interacts with

Fc{gamma}Rs in human monocytes. J Leukoc Biol 2005, 77, 1008-1017.

[31] Tokuda, N., Levy, R. B., 1,25-dihydroxyvitamin D3 stimulates phagocytosis but

supresses HLA-DR and CD13 antigen expression in human mononuclear

phagocytes. Proc Soc Exp Biol Med 1996, 211, 244-250.

[32] Granhagen, K., Lundstron, A., Bjorstad, A., Bylund, J., et al., Galectin-3 binds CD 13,

activates the NADPH-oxidase, and augments neutrophil inactivation of the

chemotactic peptide fMLF: 69. Eur J Clin Invest 2007, 37, 23.

312

[33] Mina-Osorio, P., Soto-Cruz, I., Ortega, E., A role for galectin-3 in CD13-mediated

homotypic aggregation of monocytes. Biochem Biophys Res Commun 2007, 353,

605-610.

[34] Mina-Osorio, P., The moonlighting enzyme CD13: old and new functions to target.

Trends Mol Med 2008, 14, 361-371.

[35] Johansson, K. C., Metzendorf, C., Soderholl, K., Microarray analysis of immune

challenged Drosophila hemocytes. Exp Cell Res 2005, 305, 145-155.


coelomocytes of the purple sea urchin. Proc Natl Acad Sci U S A 2000, 97, 13156-

13161.

[37] Kurtz, J., Memory in the innate and adaptive immune systems. Microbes and Infection

2004, 6, 1410-1417.









313



Comp Immunol 2007, 31, 465-475.

[42] GarcÌa-Arrares, J. E., Schenk, C., RodrÌgues-RamÌrez, R., Torres, I. I., et al.,

Spherulocytes in the echinoderm Holothuria glaberrima and their involvement in

intestinal regeneration. Dev Dyn 2006, 235, 3259-3267.

[43] Ebert, T. A., Southon, J. R., Red sea urchins (Strongylocentrotus franciscanus) can

live over 100 years: confirmation with A-bomb 14 carbon. Fish bulletin

(Sacramento, Calif.) 2003, 101, 915.

314

Highly Variable Immune-Response Proteins (185/333) from theSea Urchin, Strongylocentrotus purpuratus: Proteomic AnalysisIdentifies Diversity within and between Individuals1

Nolwenn M. Dheilly,* Sham V. Nair,2* L. Courtney Smith,† and David A. Raftos*

185/333 genes and transcripts from the purple sea urchin, Strongylocentrotus purpuratus, predict high levels of amino acid diversitywithin the encoded proteins. Based on their expression patterns, 185/333 proteins appear to be involved in immune responses. Inthe present study, one- and two-dimensional Western blots show that 185/333 proteins exhibit high levels of molecular diversitywithin and between individual sea urchins. The molecular masses of 185/333-positive bands or spots range from 30 to 250 kDa witha broad array of isoelectric points. The observed molecular masses are higher than those predicted from mRNAs, suggesting that185/333 proteins form strong associations with other molecules or with each other. Some sea urchins expressed >200 distinct185/333 proteins, and each animal had a unique suite of the proteins that differed from all other individuals. When sea urchinswere challenged in vivo with pathogen-associated molecular patterns (PAMPs; bacterial LPS and peptidoglycan), the expressionof 185/333 proteins increased. More importantly, different suites of 185/333 proteins were expressed in response to differentPAMPs. This suggests that the expression of 185/333 proteins can be tailored toward different PAMPs in a form of pathogen-specific immune response. The Journal of Immunology, 2009, 182: 2203–2212.

R ecent studies of host defense have uncovered profounddifferences among animal phyla in the molecules used tomediate immune responses (1–3). It seems that the im-

mune systems of different animals have evolved a variety of so-lutions to meet a basic requirement to combat pathogens. The re-sulting, highly diversified immune responses may reflect thespecific physiology, lifespan, habitat, and associated microbialpopulations of particular animal groups, or they may have arisenby chance via evolutionary radiation. A number of metazoan phylahave now been studied, identifying a range of alternative immu-nological mechanisms that exhibit high levels of molecular diver-sity (2). These discoveries have driven a paradigm shift in ourunderstanding of invertebrate immune responses, from systemsthat are simple and static, to those that are complex and have novelmechanisms for generating molecular hypervariability, a key re-quirement for keeping pace in the “arms race” against microbialpathogens.

Some invertebrate immune systems are proving to be surpris-ingly complex. Recent analyses of the purple sea urchin (Strongy-locentrotus purpuratus) genome identified several large gene fam-ilies, including gene models for 222 TLRs, 203 NOD-likereceptors (NLR), 218 scavenger receptor cysteine-rich (SRCR)molecules, and 104 C-type lectins (4–6). The complexity and

large size of these gene families suggest that the receptors theyencode may recognize individual pathogen-associated molecularpatterns (PAMPs)3 with a high degree of specificity. They mightalso act combinatorially, providing highly diverse recognitive ca-pabilities (5). In addition to diversified receptors, the S. purpuratusgenome contains homologs of RAG1 and RAG2, the moleculesresponsible for the somatic recombination of immunoglobulins invertebrates (7). This suggests that the molecular tools required togenerate molecular hypervariability might also exist amonginvertebrates.

185/333 genes represent another high-diversity immune re-sponse system in sea urchins. This family was first identified dur-ing a transcriptome analysis of sea urchin immune response genes(8). Genes that were up-regulated in coelomocytes (immune cells)after the injection of LPS were identified by screening high-densityarrayed, conventional cDNA libraries with probes generated bysubtractive suppression hybridization. Surprisingly, �60% of theexpressed sequence tags (ESTs) characterized in this transcriptomeanalysis were members of a set of closely related transcripts withsimilarities to two uncharacterized sequences from S. purpuratus,called DD185 (GenBank accession AF228877 (9)) and EST333(GenBank accession R62081 (10)), hence the designation 185/333.

Screening an arrayed cDNA library constructed from immuno-logically activated coelomocytes indicated that the frequency of185/333 mRNAs was enhanced more than 75-fold compared witha nonactivated arrayed cDNA library (8). Northern blots alsoshowed striking increases in 185/333 expression in coelomocytesfrom bacterially activated sea urchins compared with injury con-trols (9). Based on this significant increase in gene expression,185/333 transcripts were investigated in more detail, revealing an

*Department of Biological Sciences, Macquarie University, North Ryde, New SouthWales, Australia; and †Department of Biological Sciences, George Washington Uni-versity, Washington, DC 20052

Received for publication September 18, 2007. Accepted for publication December 3,2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was funded in part by an Australian Research Council Discovery grantto D.A.R. (DP0880316) and by a United States National Science Foundation grant toL.C.S. (MCB 04-24235). N.D. is the recipient of an International Macquarie Univer-sity Research Scholarship postgraduate scholarship.2 Address correspondence and reprint requests to Dr. Sham V. Nair Department ofBiological Sciences, Macquarie University, North Ryde, NSW 2109, Australia.E-mail address: [email protected]

3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern;aCF, artificial coelomic fluid; CF, coelomic fluid; EST, expressed sequence tag; IPG,immobilized pH gradient; LC, liquid chromatography; MS, mass spectrometry; 1DE,one-dimensional electrophoresis; pI, isoelectric point; SNP, single nucleotide poly-morphism; 2DE, two-dimensional electrophoresis.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.07012766

unexpected level of sequence diversity (8, 11). To date, for 185/333 cDNAs, 689 have been characterized from 14 sea urchins.These sequences are predicted to encode 286 different proteins(11, 12).

The diversity evident among 185/333 transcripts is generated inseveral ways. 185/333 mRNAs are comprised of 25 differentblocks or “elements” of nucleotide sequence that are present orabsent in numerous combinations. This results in “element pat-terns” that are repeatedly identified in different mRNAs (8, 12),contributing significant diversity to the family of 185/333 mes-sages. Single nucleotide polymorphisms (SNPs) and small inser-tions or deletions (indels) are also frequent in all 185/333 se-quences, adding to diversity. Additionally, there are surprisinglyhigh levels of frame shifts and the insertion of early stop codons inthe mRNA sequences (11). The processes that generate this diver-sity among 185/333 mRNAs are not yet well defined, but they mayinclude differences among the estimated fifty 185/333 gene loci(11, 13, 14), high levels of allelic polymorphism at each locus,RNA editing, and/or low-fidelity RNA polymerases (15), followedby posttranslational modifications. All of these processes may havebeen driven by positive selection associated with anti-pathogendefense (8, 11, 12, 14). Nucleotide sequence variability results inhigh levels of nonconservative amino acid substitutions amongpredicted proteins of the type consistent with intense evolutionaryselection pressure (8, 12, 14, 15).

185/333 mRNAs are predicted to encode proteins with a hydro-phobic leader, a glycine-rich region with multiple endoproteasecleavage sites, an RGD motif, a histidine-rich region, numerousN-linked and O-linked glycosylation sites, acidic patches, and sev-eral types of tandem and interspersed repeats (11). The deducedproteins do not contain cysteines, transmembrane regions, GPIlinkage sites, identifiable domains, or any predictable folding pat-terns. The only regions with at least some similarity to other mol-ecules are the RGD motif and one of the histidine-rich domains,which is comparable to histatins, a group of mammalian salivaryproteins with powerful antifungal activities (16, 17). Brockton etal. (18) have shown that 185/333 proteins are expressed by twosubsets of coelomocytes (small phagocytes and polygonal cells),and that the number of 185/333� cells increases in response toimmunological challenge. Initial analyses of 185/333 proteins in-dicated that recombinant 185/333 proteins appear as multimers andthat native proteins are present in a broad range of sizes in agree-ment with the mRNA sequences (18).

Here, we present the first comprehensive analysis of 185/333proteins. It reveals a broad diversity of these proteins within indi-vidual sea urchins. Different sea urchins express different suites of185/333 proteins, and expression is altered by immunological chal-lenge. The data indicate that differential expression or posttrans-lational modification of 185/333 proteins might allow S. purpura-tus to tailor immune responses toward specific pathogens.

Materials and MethodsSea urchins

Adult S. purpuratus were purchased from Marinus Scientific after collec-tion from the coast of southern California. They were maintained in thelaboratory as described previously (19). S. purpuratus becomes immuno-quiescent after long-term housing of �8 mo without significant disturbance(19, 20). Immunoquiescence can easily be reversed by injecting PAMPs, orin response to injury (19–21).

Immunological challenge and sample collection

Animals were challenged by injecting 2 �g of LPS or 4 �g of peptidogly-can (Sigma-Aldrich) per milliliter of coelomic fluid (CF), as previouslydescribed (10, 19). Control animals were injected with an equivalent vol-ume of artificial CF (aCF) (11). CF (100 �l) was withdrawn from each sea

urchin immediately before injection, and then at various times after injec-tion. A 23-gauge needle attached to a 1-ml syringe was inserted through theperistomium into the coelomic cavity and CF was withdrawn without an-ticoagulant. The CF was immediately expelled into a 1-ml tube and mixedwith 100 �l of urea sample buffer (2.4 M Tris-HCl (pH 6.8), 0.25% SDS,4 M urea, 20% glycerol). Samples were stored at �70°C until used. Pro-teins were precipitated using 2-D Clean-Up kits (GE Healthcare) accordingto the manufacturer’s instructions and resuspended in urea sample buffer (8M urea, 4% CHAPS, 60 mM DTT). The total protein content of eachsample was determined with 2-D Quant kits (GE Healthcare).

One-dimensional electrophoresis (1DE)

CF proteins in urea sample buffer (�100 �g/well) were separated on 10%Tris-glycine precast polyacrylamide gels (Criterion gel system; Bio-Rad) at130 V for 2 h, or on 7.5% bis-Tris polyacrylamide gels at 180 V for 1 h.After electrophoresis, proteins were visualized using Sypro Ruby (Sigma-Aldrich) following the manufacturer’s protocol, or with Coomassie blueusing standard protocols. Alternatively, the proteins were transferred tonitrocellulose membranes by Western blotting as described below.

Two-dimensional electrophoresis (2DE)

Isoelectrofocusing was performed using an IPGphor IEF system (GEHealthcare). Immobilized pH gradient (IPG) gel strips (11 cm, pH 3–6 orpH 3–10; GE Healthcare) were rehydrated overnight with 200 �g of CFproteins in 200 �l of rehydration buffer (8 M urea, 2% CHAPS, 50 mMDTT. and 0.5% carrier ampholytes; Immobiline; GE Healthcare). Isoelec-trofocusing was undertaken at 100 V for 3 h, 250 V for 20 min, and 8000V for 6 h, to obtain a total of 26,000–30,000 Vh. The proteins in the IPGstrips were reduced (1% DTT, 15 min) and alkylated (2.5% iodoacetamide,15 min) before separation in the second dimension by SDS-PAGE using10% Tris-glycine precast polyacrylamide gels (Criterion gel system; Bio-Rad) (22). After electrophoresis, protein spots on the gels were visualizedby Coomassie blue staining or with Sypro Ruby. Alternatively, proteinswere transferred to nitrocellulose membranes, as described see below.

Antibodies

The polyclonal antisera against 185/333 proteins used in this study werethe same as those reported by Brockton et al. (18). Antisera were generatedagainst synthetic peptides corresponding to elements 1, 7, and 25a, whichare present in most 185/333 cDNAs (see Ref. 11 and Fig. 5). The peptideswere conjugated to keyhole limpet hemocyanin and injected into two rab-bits per peptide on four separate occasions (Quality Controlled Biochemi-cals). Only those antisera for which the preimmunization bleeds did notcross-react with sea urchin CF proteins by Western blot (18) were used inthis study. The three antisera used were designated anti-185-66, anti-185-68, and anti-185-71.

Western blotting and immunostaining

Proteins separated by 1DE or 2DE were transferred from polyacrylamidegels to nitrocellulose membranes by electroblotting using a Criterion blot-ting system (Bio-Rad). Transfers were performed at 100 V for 1 h at roomtemperature using Towbin buffer (0.25 M Tris-HCl, 1.92 M glycine (pH8.3)) with 20% methanol. Once transfer was complete, membranes wereblocked by incubation in skim milk solution (7% skim milk powder inTBST; 10 mM Tris-HCl, 137 mM NaCl, 0.5% Tween 20 (pH 7.5)) over-night. Following several washes with TBST (three times for 5 min), mem-branes were incubated with anti-185/333 antisera (1/20,000 dilution of anequal mix of anti-185-66, -68, and -71 in TBST, or each antiserum sepa-rately) for 1 h at room temperature. The blots were washed with TBST(three times for 5 min) and incubated for 1 h at room temperature with goatanti-rabbit IgG conjugated with HRP (1/30,000 in TBST; Sigma-Aldrich).After washing in TBST (three times for 5 min), 185/333� proteins werevisualized using ECL chemiluminescence (GE Healthcare) with blue light-sensitive high-performance chemiluminescence film (Hyperfilm ECL, GEHealthcare). Each blot was exposed to film for varying lengths of time(1–15 min) to optimize the exposure. In some cases, image processing(Photoshop; Adobe Systems) was used to combine autoradiographs fromthe various exposures into composite images. Proteome maps for 185/333�

protein spots on 2DE Western blots were established using Progenesissoftware (PerkinElmer) to plot molecular mass and isoelectric point (pI).The relative intensities of different protein spots were calculated usingAdobe Photoshop (Adobe Systems).

Anti-185/333 ELISA

CF from LPS- and aCF-injected sea urchins was adjusted to 5 � 105

coelomocytes/ml with calcium-magnesium-free seawater with EDTA and

2204 185/333 PROTEIN DIVERSITY IN SEA URCHINS

imidazole (CMFSW-EI; 10 mM KCl, 7 mM Na2SO4, 2.4 mM NaHCO3,460 mM NaCl, 70 mM EDTA, and 50 mM imidazole (pH 7.4)) and wasmixed with an equal volume of CMFSW-EI containing 1% (v/v) NonidetP-40 to lyse the cells, followed by centrifugation (12,000 � g, 10 s) toremove debris. The supernatant was diluted 1/20 with TBS and aliquotedin triplicate into 96-well ELISA plates (200 �l/well; Corning). Plates wereincubated overnight at 4°C so that proteins could adhere to the wells. Theplates were washed once with TBS and blocked for 1 h with TBS con-taining 4% (w/v) BSA. After blocking, 100 �l of anti-185 antisera (1/20,000 dilution of an equal mix of anti-185-66, -68, and -71 in TBST) wasadded per well for 2 h at room temperature with gentle shaking. The plateswere washed three times for five minutes each with TBST before adding100 �l of anti-rabbit IgG-alkaline phosphatase conjugate (1/20,000 inTBST; Sigma-Aldrich) per well. The plates were incubated with the sec-ondary Ab for 2 h before being washed three times with TBST. After thefinal wash, 200 �l of p-nitrophenyl phosphate (2 mg/ml in 0.1 M glycine,1 mM MgCl2, 1 mM ZnCl2 (pH 10.4); Sigma-Aldrich) was added per welland incubated for 30 min before absorbance was read in a microplate spec-trophotometer at 415 nm. Data were corrected for the absorbance in wellsprepared without sea urchin proteins. Controls included wells in which theprimary (anti-185) Abs or both the primary and secondary Abs were omit-ted, and wells in which an irrelevant Ab (rabbit anti-tunicate collectinpeptide (24, 25)) was used in place of the anti-185 antisera.

Mass spectrometry and data analysis

Mass spectrometry (MS) was performed on CF proteins separated by either1DE or 2DE. To extract proteins from 1DE, Coomasssie blue-stained gelswere washed twice in water (10 min each). Individual lanes were cut into16 slices of equal sizes so that proteins in different molecular mass rangescould be analyzed separately by MS (see Fig. 8). To extract proteins after2DE, spots from 2DE gels that corresponded to individual 185/333� pro-teins on Western blots were excised.

Gel slices or excised protein spots were washed briefly with 100 mMNH4HCO3 before being destained (3 � 10 min) with 25 mM NH4HCO3/acetonitrile (1/1) and dehydrated in 100% acetonitrile for 5 min. Afterdehydration, gel pieces were air-dried, reduced with 10 mM DTT in 100mM NH4HCO3 for 45–60 min at 56°C, and alkylated with 55 mM iodoac-etamide in 100 mM NH4HCO3 for 45 min at room temperature. The gelpieces were washed once with 100 mM NH4HCO3 for 5 min and twicewith 25 mM NH4HCO3/acetonitrile (1/1) for 5 min before being dehy-drated with 100% acetonitrile. The dehydrated gel slices were air-dried andrehydrated with trypsin (12.5 ng/�l in 50 mM NH4HCO3; Promega) for 30min at 4°C. An additional aliquot of 50 mM NH4HCO3 was added beforethe proteins were digested at 37°C overnight. The resulting tryptic peptideswere extracted from the gel pieces by washing twice with 2% formic acidin 50% acetonitrile. Extracts were combined and concentrated to 10 �l byvacuum centrifugation.

Mass spectrometry was performed at the Australian Proteome AnalysisFacility (Macquarie University). The tryptic digest extracts from 1DE gelslices were subjected to data-dependent nanocapillary reversed phase liq-uid chromatography followed by electrospray ionization using a ThermoLCQ Deca ion trap mass spectrometer (Thermo Scientific; liquid chroma-tography (LC)-MS/MS). For LC-MS/MS, a microbore HPLC system(TSP4000; Thermo Scientific) was modified to operate at capillary flowrates using a simple T-piece flow-splitter. Columns (8 cm � 100 �m insidediameter) were packed with 100 Å, 5-�m Zorbax C18 resin at 500 �.Integrated electrospray tips for the columns were made from fused silica,pulled to a 5-�m tip using a laser puller (Sutter Instrument). An electros-pray voltage of 1.8 kV was applied using a gold electrode via a liquidjunction upstream of the column. Samples were introduced onto the ana-lytical column using a Surveyor autosampler (Thermo Scientific). TheHPLC column eluent was eluted directly into the electrospray ionizationsource of the ion trap mass spectrometer. Peptides were eluted with a lineargradient of buffer A (0.1% formic acid) and buffer B (acetonitrile contain-ing 0.1% formic acid) at a flow rate of 500 nl/min. Automated peak rec-ognition, dynamic exclusion, and daughter ion scanning of the top threemost intense ions were performed using the Xcalibur software as previ-ously described (23).

GPM open source software (Global Proteome Machine Organization;www.thegpm.org) was used to search peptide sequences against a com-bined S. purpuratus database created with sequences downloaded from theNational Center for Biotechnology Information (NCBI). This FASTA for-mat database contained 44,037 protein sequences comprising all S. purpu-ratus sequences held by NCBI as of April 2008. This database also incor-porated a list of common human and trypsin peptide contaminants. Searchparameters included MS and MS/MS tolerances of �2 Da and �0.2 Da,tolerance of up to three missed tryptic cleavages, and K/R-P cleavages.

Fixed modifications were set for carbamidomethylation of cysteine, andvariable modifications were set for oxidation of methionine. Peptides iso-lated from 1DE gel slices were deemed to significantly match correspond-ing peptides in the available databases if their GPM loge score was lowerthan the statistically significant cutoff values assigned by the GPMalgorithm.

Protein spots from 2DE gels were analyzed using MALDI-TOF-TOFusing an LTQ FT Ultra hybrid mass spectrometer (Thermo Scientific). MSion searches were compared with 185/333 sequences in a custom databasecontaining 81 translated full-length 185/333 cDNA sequences (12) and 689EST sequences (8) using the Mascot search engine (Matrix Sciences; www.matrixscience.com/) set for carbomethylation (C) and oxidation (M) vari-able modifications, with peptide and fragment mass tolerances of �50 ppmand �0.5 Da respectively, and a maximum missed cleavage of one.

ResultsOne-dimensional SDS-PAGE analysis of CF proteins

1DE was used to provide a preliminary assessment of the diversityof proteins in CF, particularly those that were detected by anti-185Abs (Fig. 1). The molecular masses of CF proteins from all of thesea urchins analyzed in the present study (n � 13) ranged from 20kDa to �193 kDa (see Fig. 6). Sypro Ruby-stained CF proteinsfrom the individual sea urchin ranged from 30 kDa to �193 kDa(Fig. 1, lane 1). There were substantial differences in banding pat-terns when electrophoretically separated CF proteins from thesame sea urchin were stained with Coomassie blue compared withSypro Ruby (Fig. 1, compare lanes 1 and 2). This was probablydue to different sensitivities and physiochemical properties of thestains (26).

Immunostained 1DE Western blots of CF proteins identified nu-merous 185/333� bands ranging from �20 kDa to �193 kDa (Fig.1, lane 3). Controls that omitted the primary (anti-185) and/orsecondary (anti-IgG) Abs, and irrelevant controls using anti-tunicate collectin Abs instead of anti-185 antisera, were negative(data not shown). Most 185/333� bands from all of the sea urchinsamples analyzed (n � 13) had molecular masses ranging from 50kDa to �193 kDa, although some were as low as 20 kDa (see Fig.6). Many of the 185/333� bands on Western blots were not visibleon Sypro Ruby- and Coomassie blue-stained 1DE gels, suggestingthat the 185/333 proteins were present at extremely lowconcentrations.

FIGURE 1. 1DE SDS-PAGE and Western blot of CF proteins from anindividual sea urchin. CF proteins were loaded at 100 �g/well onto 10%SDS-PAGE gels. Lane 1, Sypro Ruby staining. Lane 2, Coomassie bluestaining. Lane 3, Western blot immunostained with an equal mixture ofanti-185 sera (anti-185-68, -66, and -71; 1/20,000). The positions of mo-lecular mass markers are shown on the left.

2205The Journal of Immunology

Two-dimensional Western blots of CF proteins

Given that cDNA analyses predicted a broad array of 185/333proteins in the CF (8, 11, 12), we extended our analysis of 185/333proteins using 2DE, which affords far greater resolution than does1DE. Sea urchin CF proteins were initially separated by isoelectricfocusing (pH 3–10) and then by 10% SDS-PAGE. Large numbersof 185/333� spots were evident after 2DE, to the extent that theyoften appeared as dense smears (Fig. 2). Most of the 185/333 pro-teins recognized by the antisera in all of the sea urchins analyzed(n � 13) had pIs between 3 and 7 with apparent molecular massesof 40 kDa to �193 kDa.

To improve the resolution of individual 185/333� proteins on2DE Western blots, additional isoelectric focusing separationswere conducted on immobilized pH gradient (IPG) strips with apH range of 3–6. Composite images obtained from film exposuresof three different time intervals were used to visualize 185/333�

proteins of varying abundance (Fig. 3). For the individual sea ur-chin shown in Fig. 3, image analysis of the different exposuresrevealed that fifty-one 185/333� spots were evident after a 1-minfilm exposure. An additional 117 spots were evident after 5 min,and a further 96 spots appeared after 10 min, making a total of 264spots that were detected on the composite image for this individ-ual. The number of discrete 185/333� spots varied substantiallyamong individuals. Three of the 13 sea urchins tested did not ex-press detectable levels of 185/333 proteins. However, the numberof discrete 185/333� spots was often �200 in other individuals.

The enhanced protein separation capabilities of 2DE alsoshowed that each discrete 185/333� band evident in 1DE con-tained numerous variants with similar molecular masses but dif-ferent pIs. For example, at least fifteen 185/333� spots with dif-

ferent pIs were evident at �75 kDa (Fig. 3). A further eight 185/333� spots appeared at �60 kDa and at least six different 185/333� spots were present at �30 kDa (Fig. 3). These pI variantswere often regularly spaced from each other, differing by �0.1–0.2 pH units. There were also numerous 185/333� spots with iden-tical pI but different molecular masses (Fig. 4). For instance, threeproteins in the pI 7.5–7.75 range each had three different molecularmass forms at �150 kDa, 193 kDa, and �193 kDa (Fig. 4).

The three different anti-185 sera, which were raised against dif-ferent regions of the most commonly predicted 185/333 polypep-tide sequences, identified subsets of 185/333 proteins (Fig. 5).Within the small region of a 2DE Western blot (pI range of 4–5and molecular masses of 30 kDa or lower), anti-185-66 resolved14 distinct variants, of which 7 were 30 kDa and had a pI range of4–5 (Fig. 5A). An additional seven anti-186-66-positive spots weresmaller than 30 kDa, with a pI range of 4.5–5. In comparison,anti-185-68 recognized five of the seven proteins at 30 kDa with apI range of 4–5 that were present on the anti-185-66 blot, but didnot recognize the set of seven lower molecular mass spots. Anti-185-71 recognized only two of the 30 kDa proteins that were rec-ognized by the other two antisera. This result is consistent with theexpression of truncated 185/333 cDNAs (11).

Diversity of 185/333 proteins between individuals

There were major differences in the expression profiles of 185/333proteins among different sea urchins. Some animals did not show185/333 expression before LPS injection (Fig. 6A, lanes 3 and 11),although expression was evident after challenge (Fig. 6B, lanes 3and 11). In other cases, 185/333 expression, which was very low

FIGURE 2. 2DE Western blot of 185/333� proteins. The gel wasloaded with 200 �g of CF proteins from a single sea urchin and the blotwas immunostained with an equal mixture of anti-185 sera (anti-185-66,-68, and -71; 1/20,000). pIs are shown as pH units on the top of the blot,and molecular masses (kDa) are indicated on the left.

FIGURE 3. Composite image of a 2DE Western blot. The gel wasloaded with 200 �g of CF proteins from sea urchin 12. The blot wasimmunostained with an equal mixture of the three anti-185 antisera (anti-185-66, -68, and -71; 1/20,000) and exposed to autoradiographic film for 1,5, or 10 min. The different exposures were merged to give a final compositeimage. pIs are shown on the top as pH units, and molecular masses (kDa)are shown on the left.

FIGURE 4. Enlarged region of a 2DE Western blot of CF proteins fromanimal 12 immunostained with an equal mixture of the three different anti-185 sera (anti-185-66, -68, and -71; 1/20,000). pIs are shown on the top aspH units, and molecular masses (kDa) are shown on the left.

FIGURE 5. Different anti-185 sera recognize subsets of 185/333 pro-teins. A, Enlarged regions of three different 2DE Western blots loaded with200 �g of CF proteins from the same sea urchin and immunostained sep-arately with anti-185-66, anti-185-68, or anti-185-71 (1/20,000). B, Sche-matic diagram of a 185/333 protein showing the peptides used to generatethe three different anti-185 sera. The rectangle represents the protein se-quence, which is composed of the leader and 25 nucleotide sequence “el-ements”. The positions and sequences of the synthetic peptides againstwhich the three anti-185 sera were raised are indicated (15).


in preinjection samples, increased significantly after LPS injection.A broad distribution of 185/333 molecular masses ranging from 20kDa to �193 kDa was apparent among the 13 animals analyzed by1DE Western blots (Fig. 6). Although many individuals expressedsome 185/333 proteins with identical molecular masses (e.g., 112kDa, Fig. 6, lanes 1–4; 100 and 120 kDa, Fig. 6, lanes 7 and 8), thesuite of 185/333� bands expressed by each individual sea urchin wasunique. Three animals did not express detectable 185/333 proteins,even after challenge with LPS (data not shown). In total, nineteen185/333� bands with distinct molecular masses were identifiedamong the 13 sea urchins analyzed by 1DE. The molecular masses ofthese proteins seemed to be evenly spaced at 8–10 kDa apart over themolecular mass range of 20 to �193 kDa (Fig. 6).

MS analysis of 185/333� proteins

Proteins on Coomassie blue-stained 2DE gels that corresponded to185/333� spots on Western blots were analyzed by MS. This anal-ysis failed to identify 185/333 proteins unambiguously using thedefault criteria on the Mascot search engine. Although MS of each

FIGURE 6. 1DE Western blots of CF from 13 different sea urchinssampled before (A) and 96 h after (B) challenge with LPS. The equivalentlane numbers in both panels refer to the same animals from which CFsamples were obtained before and after LPS challenge. The blots wereimmunostained with an equal mixture of the three different anti-185 sera(anti-185-66, -68, and -71; 1/20,000).

Table I. Mass spectrometric (LC-MS/MS) data for peptides isolated from 1DE gels of S. purpuratus CF that match known185/333 sequencesa

Animal Gel Slice Loge m � h z Sequence GenBank Accession No.

6 6 �1.30 2594.5 3 MAVLTLATMAATTSIIIATTQKVTKb gb ABK88425.117 2 �3.20 1735.8 3 GQGGFGGRPGGMQMGGPR gb ABR22418.1

�3.20 1735.8 3 GQGGFGGRPGGMQTGSPR gb ABZ10666.1�3.30 1493.6 2 FDGPESGAPQMEGR gb ABZ10664.1�9.80 2886.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGPGR gb ABZ10668.1�2.40 1735.8 3 GQGGFGGRPGGMQMGGLR gb ABK88417.1�3.30 2131.9 3 RGDGEEETDAAQQIGDGLGGR gb ABZ10666.1�1.70 1506.7 2 FDGPGFGAPQMGGPR gb ABR22467.1�4.60 1157.6 2 KPFGDHPFGR gb ABR22410.1�2.00 2819.2 3 GDGEEETDAAQQIGDGLGGSGQFDGPRR gb ABR22477.1

3 �2.40 1735.8 3 GQGGFGGRPGGMQMGGPR gb ABR22418.1�2.40 1735.8 3 GQGGFGGRPGGMQTGSPR gb ABZ10666.1�4.20 1649.7 3 RFDGPESGAPQMEGR gb ABZ10664.1�5.20 1493.6 2 FDGPESGAPQMEGR gb ABZ10664.1�3.00 2131.9 3 RGDGEEETDAAQQIGDGLGGR gb ABZ10666.1�1.50 2320.2 2 PQTDQRNNRLVSATKAAMRMb gb ABK88425.1�5.80 2887.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGPGR gb ABZ10668.1�1.40 1735.8 3 GQGGFGGRPGGMQMGGLR gb ABK88417.1�2.60 1979.9 3 MGGRNSTNPEFGGSRPDGAGb gb ABR22330.1�1.70 2744.3 3 RNSTNPEFGGSRPDGAGGRPLFGQGGRb gb ABR22330.1�4.60 2927.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGHGR gb ABZ10669.1

4 �2.40 1479.6 2 FDGPESGAPQMDGR gb ABZ10666.1�4.00 1980.0 3 FGGSRPDGAGGRPFFGQGGR gb ABZ10669.1�2.40 2886.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGPGR gb ABZ10668.1�2.00 1157.6 2 KPFGDHPFGR gb ABR22410.1�5.10 1633.7 2 RFDGPESGAPQMEGR gb ABZ10664.1�0.17 2564.1 3 FDGPESGAPQMEGRRQNGVPMGGR gb ABK88329.1�3.10 2132.0 3 RGDGKEETDAAQQIGDGLGGR gb ABR22436.1�1.40 1926.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGHGR gb ABZ10669.1

5 �1.7 3398.5 3 DFNERREKENDTERGQGGFGGRPGGMQMGGP gb ABK88476.1�3.5 1980.0 3 FGGSRPDGAGGRPFFGQGGR gb ABZ10669.1�1.5 1831.9 3 RFDGPEPGAPQMEGRR gb ABK88803.1

6 �2.7 2131.0 3 RGDGEEETDAAQQIGDGLGGR gb ABZ10666.1�1.8 2927.3 3 RGDGEEETDAAQQIGDGLGGPGQFDGHGR gb ABZ10669.1

7 �1.6 1515.7 2 ADVVEIAVNEEDVNb gb ABA19607.1�3.3 1649.7 3 RFDGPESGAPQMEGR gb ABZ10664.1�4 1493.6 2 FDGPESGAPQMEGR gb ABZ10664.1�3.5 1980.0 3 FGGSRPDGAGGRPFFGQGGR gb ABZ10664.1

22 2 �2.50 1033.5 2 FGAPQMGGPR gb ABR22467.1�1.40 2814.3 3 RGRGQGRFGGRPGGMQMGGPRQDGGPMG gb ABK88373.1�3.70 1649.7 3 RFDGPESGAPQMEGR gb ABZ10664.1

a The data are from 1DE gel slices of CF from three different sea urchins. The GenBank accession numbers of the 185/333 sequences that matched peptidesfrom 1DE gels are also shown. The amino acid sequences shown are the matching 185/333 peptides from the NCBI database. Parameter definitions are: loge,values indicate the probability that a putative peptide sequence corresponding to a mass spectrum arises stochastically. The lower the loge value, the moresignificant the assignment of the peptide sequence to the mass spectrum. The loge values listed in this table are all lower than the significance cutoff valuesassigned by the GPM algorithm, and so they are deemed to be statistically significant (43); m � h, peptide mass in Da � 1; z, peptide charge.

b Peptides that matched sequences for previous analysis of nucleotide sequences that might represent frameshift mutations (12, 13).


protein successfully identified ion fragments with mass/chargeproperties that were similar to known 185/333 sequences, thesematches did not yield sufficiently high statistical probabilities toconfirm the identity of any of the proteins (data not shown).

In contrast, MS analysis of CF proteins separated by 1DE un-equivocally identified 185/333 proteins. A total of 41 peptides iso-lated from 1DE gel slices matched with known 185/333 sequencesfrom the NCBI database (Table I). The same 185/333 peptide wasoften identified in more than one gel slice. For example, the pep-tide FDGPESGAPQMEGR appeared in gel slices 2–4 from ani-mal 17. It is unlikely that the occurrence of the same peptide inmultiple fractions was due to contamination of the different frac-tions with exactly the same 185/333 protein. If the same proteinwas present in more than one fraction, we would expect to findexactly the same combination of peptides from that protein in morethan one fraction. However, this was not the case. Only singlepeptides were found in each fraction. Given this repetitive identi-fication of the same peptide in different gel slices, a total of 23unique 185/333 peptides were identified among all of the gelslices. In some cases, the GPM algorithm allocated slightly differ-ent predicted amino acid sequences to the same MS spectrum. Forinstance, GPM identified two peptides (GQGGFGGRPGGMQMGGPR and GQGGFGGRPGGMQTGSPR; underlining identi-fies amino acids that differ) from the same MS spectrum. Thisoccurred because the amino acid differences between these twopeptides were predicted to yield peptides with indistinguishablemass/charge ratios. Matches to both of these peptides were presentin the NCBI database. In some cases, the subtle differences be-tween peptides involved the inclusion of an arginine resulting inclosely related peptides of different lengths, such as RGDGEEETDAAQQIGDGLGGR and RGDGEEETDAAQQIGDGLGGPGQFDGHGR. It is noteworthy that of the 41 peptides identified, 36were located in the glycine-rich (N-terminal) and central regions ofthe predicted proteins. This is consistent with frameshifts and earlystop codons that have been identified in about half of the cDNAs(11). In many cases, the introduction of these frameshifts or earlystop codons would have resulted in the loss of epitopes for theantiserum anti-185-68, which was raised against the C-terminalregion of full-length 185/333 proteins, and to a lesser extent, theepitope for anti-185-71, which is in the central region of predictedfull-length 185/333 proteins. As a result, these antisera would nothave identified many of the 185/333 proteins encoded by truncatedmRNAs in the Western blot shown in Fig. 5A. Some of the pep-tides shown in Table I matched sequences that previous analyseshave suggested are derived from frameshift mutations (12, 13). Allof the peptides that matched 185/333 sequences came from gelslices 2–7, which were also shown by Western blot to contain thevast majority of 185/333 proteins (Figs. 6 and 7). Of the three seaurchins analyzed by 1DE and LC-MS/MS, animal 17 had the high-est overall expression levels for 185/333 proteins (Fig. 7) andyielded 37 of the 41 peptides that matched 185/333 sequences.

185/333 protein expression increases after LPS challenge

1DE Western blots of CF from five of the six animals injected withLPS showed increases in 185/333� protein expression between 24and 192 h (representative results are shown in Fig. 8A). Repeatedinjections with LPS performed 14 days after the primary challengeincreased 185/333 protein expression to levels that were higherthan those evident after the first injection (Fig. 8A). Sea urchinsinjected with aCF either did not respond to the injection or showedweak responses in terms of 185/333 protein expression (data notshown).

These Western blot data were confirmed by ELISA (Fig. 8B).Injecting LPS into sea urchins significantly ( p � 0.05) increased

the titer of 185/333 proteins detected in CF by ELISA. By 48 hafter LPS injection, anti-185 reactivity had increased 2.4-fold com-pared with the 0 h time point ( p � 0.05). It returned to levels thatwere indistinguishable ( p � 0.05) from those before LPS chal-lenge by 96 h. In response to aCF (control) injections, the titer of185/333 proteins in CF also increased up to 48 h postinjection, butonly to a level that was 1.4-fold greater than the preinjection timepoint. This increase was significantly less than the response to LPS

FIGURE 7. 1DE Western blots of CF proteins from three different seaurchins (animals 6, 17, and 22) immunostained with an equal mixture ofanti-185 sera (anti-185-66, -68, and -71; 1/20,000). The numbers on the leftshow the approximate positions of slices cut from 1DE gels for analysis bymass spectrometry (see Table I). In total, gels were cut into 16 slices. Thepositions of only eight of those slices containing the highest molecularmass proteins are shown here.

FIGURE 8. The titer of 185/333 proteins in CF increases after immunechallenge. A, 1DE Western blots of CF from a single sea urchin collectedat various times after the animal was injected twice with LPS. The secondLPS injection was administered 360 h after the first. B, 185/333 proteinexpression levels determined by ELISA (ODU415) at various times afternaive sea urchins (n � 5) were injected with LPS or aCF (controls). Barsindicate SEM. Asterisks denote time points that differed significantly (p �0.05) between LPS-injected sea urchins and controls.


( p � 0.05). Negative controls confirmed the specificity of theELISA. Wells in which the primary (anti-185 sera) or secondary(anti-rabbit IgG) Abs were omitted, or when the primary Ab wasreplaced by an irrelevant control (anti-tunicate collectin), did notyield absorbance readings that were significantly greater thanbackground levels ( p � 0.05, data not shown).

Diversity of 185/333 protein expression after immunologicalchallenge

1DE analysis did not detect changes in the types of 185/333 pro-teins expressed by individual sea urchins in response to LPS or PGinjections, even though densitometry (data not shown) indicatedthere was an increase in the relative quantity of 185/333 proteinsafter repeated challenge (Fig. 9). The molecular masses of thepredominant 185/333 proteins in CF from animals 6 and 25 did notchange in response to an initial injection of LPS, or after a secondLPS injection 2 wk later. A similar result was apparent for animals

22 and 31, which were challenged with LPS followed by PG 2 wklater. The 185/333� bands of these two animals did not show anymajor changes in molecular mass when the challenge was switchedfrom LPS to PG.

In contrast, 2DE revealed clearly discernable changes in the pIsof 185/333 proteins when sea urchins were challenged with PGafter initially responding to LPS, even though the molecularmasses of the predominant forms remained the same (Fig. 10).This is best exemplified in the small region of the 2DE Westernblots of CF from animal 31 (pIs of 4–6, molecular masses of40–65 kDa) shown in Fig. 10B. CF from this sea urchin wascollected after an initial challenge with LPS (Fig. 10B, upperpanel), and then again after a subsequent injection of PG (Fig. 10B,lower panel). Of the thirty 185/333� spots in this region, sevenwere found only after LPS challenge (e.g., 63 kDa, pI 4.8), and sixwere found only after challenge with PG (e.g., 42 kDa, pI 5.5). Theremaining 17 proteins were present after both the LPS and PGchallenges. The relative expression levels of some of these sharedproteins also differed between challenges. For instance, one of theproteins (63 kDa, pI 5.2) had a densitometry value of 5.1 after LPSinjection and 1224 after PG challenge, representing a 240-foldincrease in expression intensity in response to PG.

DiscussionThis study has identified substantial diversity among 185/333 pro-teins, as reflected by their broad range of molecular masses andpIs. Most importantly, there are obvious differences in the suites of185/333 proteins expressed by different sea urchins, and thesesuites of proteins undergo subtle but extensive changes in responseto different types of immune challenge. The diversity of 185/333proteins and the changes in their expression detected by the currentproteomic analysis correspond with previous studies of mRNAsfrom sea urchins responding to different PAMPs (11). All of thesedata suggest that sea urchins may be capable of altering the ex-pression of 185/333 proteins to tailor specific responses againstdifferent PAMPs.

Despite the overall agreement between our current observationsof 185/333 proteins and predictions based on the prior analyses of185/333 nucleotide sequences (11, 12), there are some discrepan-cies. The predicted sizes of 185/333 proteins based on cDNA se-quences range from 4 to 55.3 kDa (11, 12), with predicted pIsranging from 5.42 to 11.54. However, the present study shows thatnative 185/333 proteins have a far wider range of molecularmasses (20 to �193 kDa using 1DE analysis), with predominantbands often being at the high end of this range. They also have farmore acidic pIs than expected, mainly between 3 and 7. It is un-likely that the discrepancy between predicted and observed mo-lecular masses is due to disulfide-bonded oligomerization becausenone of the predicted 185/333 amino acid sequences identified todate contains cysteines (8, 11). Even if cysteines were present inmissense sequences of frame-shifted proteins, the strong reducingconditions used to prepare samples would have disrupted any oli-gomers held together by disulfide bonds. Another explanation forthe discrepancy in molecular masses is that 185/333 proteins areglycosylated and form large complexes covalently linked to car-bohydrates. There are numerous conserved sites for N-linked gly-cosylation within the histidine-rich region of the 185/333 proteins(elements 11–25), and there are conserved sites for O-linked gly-cosylation in the carboxyl-terminal region (8, 11). However, deg-lycosylation of N-linked oligosaccharides failed to decrease themolecular masses of 185/333 proteins to the size of predictedmonomers (L. C. Smith, unpublished data). Given these results, itseems likely that the disparity between predicted and observedmolecular masses reflects oligomerization based on mechanisms

FIGURE 9. 1DE Western blots of CF collected after sea urchins hadbeen injected with LPS or PG. Animals 6 and 25 were injected with LPStwice with a 360-h interval, while animals 22 and 31 were injected firstwith LPS and then with PG 360 h later. CF was collected 96 h after eachinjection, and the Western blots were immunostained with an equal mixtureof the three different anti-185 sera (anti-185-66, -68, and -71; 1/20,000).

FIGURE 10. 2DE Western blots of CF collected from a single sea ur-chin (animal 31) that had been injected first with LPS and then 360 h laterwith PG. CF was withdrawn at 96 h after each injection. Western blotswere immunostained with an equal mixture of the three different anti-185sera (anti-185-66, -68, and -71; 1/20,000). A, Full proteome maps (pI of3–6; 40–150 kDa; 30-s film exposure) of the CF samples after LPS and PGchallenges. B, Enlargements (pI of 4.0–6.3; 40–85 kDa; 5-min film ex-posure) of the boxed areas in A. The numbers shown in each panel are therelative expression intensities of five 185/333� spots that differed substan-tially in expression after LPS compared with PG injections.


other than disulfide bond formation that are resistant to the reduc-ing treatments used in 1DE and 2DE. This conclusion is supportedby studies of recombinant 185/333 proteins, in which the expres-sion of a single form of 185/333 protein yields a range of ex-pressed proteins with molecular masses corresponding to mono-mers, homodimers, and higher order oligomers (18).

Other data also suggest that the diversity of molecular massesevident among 185/333 proteins is increased by the expression oftruncated molecules. Many of the SNPs and indels found previ-ously in 185/333 transcripts are predicted to result in frameshifts,and the encoded proteins may be either truncated and/or have mis-sense sequences (11, 13). No such frameshifts have been identifiedin the 185/333 genes. However, comparisons of gene and mRNAsequences from individual animals suggest that posttranscriptionalmodifications may be responsible for these missense proteins (15).If these messages are translated, it would explain why the threedifferent anti-185 antisera used in the present study detected dif-ferent subsets of 185/333 proteins. The antisera were raised againstamino acid sequences in N terminus (element 1), the middle (el-ement 7), and the C terminus (element 25a) of the predicted pro-teins. The antiserum targeted to the N terminus (anti-185-66) iden-tified the largest number of 185/333 proteins on 2DE Westernblots, presumably because its epitope is most likely to be presentin every protein, including the shortest truncated forms. Of the 689translated cDNA sequences identified to date, 676 contain theepitope within element 1 that is recognized by the anti-66 anti-serum (11). Antisera directed toward more C-terminal regions rec-ognize decreasing numbers of 185/333� spots, probably becausethese epitopes are not present on truncated proteins or those withmissense sequence at the C terminus. Current cDNA sequence datafrom Terwilliger et al. (11) show that 660 of 689 cDNA sequencescontain the anti-68 epitope in element 7, while only 375 cDNAsequences contain the anti-71 epitope in element 25a.

The variability evident in the molecular masses of 185/333 pro-teins was matched by substantial diversity in their pIs. One sourcefor this diversity is found in mRNAs where sequence variabilityresults in changes in charged amino acids at particular positionsleading to predicted proteins with very similar molecular massesbut different pIs (8, 11, 12). Terwilliger et al. (see supplementalfigures S1 and S2 in Refs. 11, 12) identified numerous sequencepositions in 185/333 mRNAs that encode two to four differentamino acids, many with different charges. In the present study,similar subtle differences were detected by MS, which identified anumber of 185/333 peptides that differ by just a single amino acid.2DE Western blots also showed a variety of pIs for 185/333 pro-teins that had very similar molecular masses. In many cases, thecharge variants making up a single molecular mass class of 185/333 proteins had pIs spanning the full pH range from 3 to 10,although most variants had a pI within the range of 3–6. Thedifferent pI forms were often equally spaced, suggesting that ad-jacent protein spots represent variants that differ by just a singlecharged residue.

Although the variability in pI detected in the present studyagrees with the diversity seen among cDNA sequences, our pro-teomic data also provide evidence for additional posttranslationalmodifications. Some 185/333 proteins have identical isoelectricpoints but significantly different molecular masses. This suggeststhat individual 185/333 proteins may be conjugated with someother molecule(s) that alters their molecular mass but not theirisoelectric point. Such conjugation would provide another expla-nation for why many 185/333� bands are much larger in molecularmass than predicted. However, the cDNA data suggest that it isalso feasible for 185/333 proteins with significantly different se-

quences and different pIs to have the same molecular mass, result-ing in a vertical ladder of spots on 2DE Western blots.

The diversity of 185/333 proteins explains our inability to un-equivocally identify 185/333 proteins by MS of individual proteinsisolated by 2DE. MS analysis of the 185/333� spots from 2DEgels did not identify any statistically significant matches to pro-teins in a custom database of 185/333 sequences. However, all ofthe 185/333 proteins isolated by 2DE yielded ion fragments thatpossessed mass/charge characteristics similar to those of known185/333 protein sequences. The available evidence suggests thatthe existing database of 185/333 sequences may represent only asmall fraction of the 185/333 variants present in sea urchin pop-ulations. Consequently, the chance of finding a statistically sig-nificant match in this restricted data set to a single 185/333protein isolated from the CF of an individual sea urchin may beextremely low, even though the purity of the proteins isolatedby 2DE would have been high. In other words, we may not haveidentified matches because the variability of 185/333 sequencemeans that many 185/333 proteins will not yet be in our data-base of 185/333 sequences and so cannot be identified by MS.Our difficulties in matching isolated proteins to the existing185/333 database highlight the problems of employing MStechniques, which search for precise similarities between pep-tides to characterize hypervariable proteins. It is interesting thateven though members of the Down syndrome cell adhesionmolecule (DSCAM) family are important proteins in the immu-nological responses of insects, proteomic analyses of hemo-lymph extracts from immunologically challenged Drosophilahave not yet been able to identify DSCAMs by MS.

To circumvent the problem of matching individual 185/333�

spots to our restricted sequence data set, we employed “shotgun”MS (LC-MS/MS), in which all of the CF proteins within a partic-ular molecular mass range from 1DE gels were analyzed simulta-neously by MS. Unlike 2DE, in which the individual proteins spotsanalyzed by MS presumably contain just a single 185/333 isotype,the 1DE gel slices subjected to shotgun MS were likely to containnumerous different 185/333 isotypes. This greatly increased thechance that at least one of these isotypes would match a previouslyidentified sequence in our 185/333 database. 1DE also has theadvantage that it is not limited by the complex separation charac-teristics of 2DE, which, for instance, is relatively inefficient inseparating hydrophobic proteins. Additionally, we used an alter-native search engine (GPM as opposed to Mascot) with slightlyaltered search stringencies (three missed tryptic cleavages) to in-crease our chances of identifying matching 185/333 peptides in theavailable databases. This process successfully identified numerouspeptides that matched precisely to sequences in the NCBI databasewithout affecting the robustness of the sequence matches. The gelslices in which matching peptides were identified corresponded toregions on 1DE and 2DE Western blots that contained high con-centrations of 185/333 proteins, which confirms the specificities ofthe anti-185 sera used in this study. However, shotgun MS prob-ably still identified just a small fraction of the 185/333 peptidespresent within the peptide mixture, because of the limited numbersof potentially matching peptides in the currently available dataset.The limited sensitivity of MS in identifying highly variable pro-teins in the absence of comprehensive sequence databases alsoexplains why our MS analysis only identified multiple forms of185/333 proteins in one of the sea urchins analyzed, even thoughboth 1DE and 2DE Western blotting revealed substantial intrain-dividual diversity in all sea urchins.

The difficulties associated with analyzing the diversity of 185/333 proteins were compounded by the substantial variation evidentin the suites of 185/333 proteins expressed by different sea urchins.


None of the 13 sea urchins analyzed by 1DE Western blottingexpressed the same pattern of 185/333� bands, even though therewere a number of molecular mass forms that were shared by someindividuals. On average, two to three major bands were evident ineach sea urchin, along with numerous minor bands. The differ-ences between individuals implies that there may be a variety ofmechanisms acting in concert to produce novel arrays of 185/333proteins in individual sea urchins. These factors could include thepresence or absence of different 185/333 genes in different seaurchins, the presence of different alleles at a given 185/333 genelocus, differential gene expression of 185/333 family members,posttranscriptional processing or editing of the transcripts that in-sert frameshifts and SNPs (15), and posttranslational processing ofthe proteins (13).

Our data suggest that these potential mechanisms for moleculardiversification may allow sea urchins to vary the suites of 185/333proteins that they express in response to different types of immu-nological challenge. ELISA provided direct evidence that 185/333expression increases after the injection of PAMPs, while 2DEanalysis of 185/333 proteins from individual sea urchins identifiedmany subtle changes in the patterns of 185/333 proteins respond-ing to different PAMPs. There were clear differences in the pIs andmolecular masses of the proteins expressed in response to LPScompared with those synthesized by the same sea urchin in re-sponse PG. These results agree with changes that are apparent inthe cDNAs from animals responding to different PAMPs, includ-ing LPS, �-1,3-glucan, or dsRNA (11). Terwilliger et al. (11)showed that a diverse size range of 185/333 messages was presentin the CF of immunoquiescent sea urchins, but this broad expres-sion profile changed to one single, major mRNA size after immunechallenge. Sequence analysis of mRNAs from animals respondingto immune challenge also indicated that the predominant 185/333variants in CF are different before and after challenge. Such alter-ations in 185/333 transcription explain the changes detected duringthe present study in the suites of 185/333 proteins found in the CFof the same sea urchin responding to different PAMPs.

The identification of PAMP-specific responses suggests that185/333 protein expression can be tailored to meet different formsof immunological challenge. The obvious implication is that 185/333 proteins are involved in some form of pathogen-specific im-munological response. There is growing evidence for this level ofimmunological specificity among a variety of invertebrates (1, 2, 5,27–29). For instance, bacterial immunization in bumble bees caninduce acquired immune responses that are highly specific to theoriginal, inoculating species of bacteria (29), although the mech-anisms underlying this response are not known. In addition to 185/333 proteins, other large gene families may be capable of fine-scale discrimination between pathogens. These gene familiesencode variable lymphocyte receptors from agnathans (30–34), Vregion-containing chitin-binding proteins (VCBPs) from cephalo-chordates and tunicates (3, 35, 36), DSCAMs in insects (37, 38),and fibrinogen-related proteins (FREPs) from molluscs (39–42).A repertoire of �19,000 different DSCAMs is expressed in hemo-cytes of Drosophila and mosquitoes (37, 38), and thousands ofFREPs are present in freshwater snails (39–42). None of thesegene families is closely related to one another, suggesting thatmechanisms for immune diversification have evolved many timesand that additional unique systems are likely to be identifiedamong other organisms in the future. In this context, our continu-ing investigations of 185/333 proteins will provide insights intohow animals other than mammals adapt in the “arms race” againstthe microbes.

AcknowledgmentsWe thank Dr. Virginia Brockton for assistance in processing and shippingprotein samples, and Dr. Katherine Buckley for suggesting improvementsto the manuscript. The research has been facilitated by access to the Aus-tralian Proteome Analysis Facility established under the Australian Gov-ernment’s Major National Research Facilities Program.

DisclosuresThe authors have no financial conflicts of interest.

References1. Flajnik, M. F. 2004. Immunology: another manifestation of GOD. Nature 430:

157–158.2. Flajnik, M. F., and L. Du Pasquier. 2004. Evolution of innate and adaptive im-

munity: can we draw a line? Trends Immunol. 25: 640–644.3. Litman, G. W., J. P. Cannon, and L. J. Dishaw. 2005. Reconstructing immune

phylogeny: new perspectives. Nat. Rev. Immunol. 5: 866–879.4. Hibino, T., M. Loza-Coll, C. Messier, A. J. Majeske, A. H. Cohen,

D. P. Terwilliger, K. M. Buckley, V. Brockton, S. V. Nair, K. Berney, et al. 2006.The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol.300: 349–365.

5. Rast, J. P., L. C. Smith, M. Loza-Coll, T. Hibino, and G. W. Litman. 2006.Genomic insights into the immune system of the sea urchin. Science 314:952–956.

6. Sodergren, E., G. M. Weinstock, E. H. Davidson, R. A. Cameron, R. A. Gibbs,R. C. Angerer, L. M. Angerer, M. I. Arnone, D. R. Burgess, R. D. Burke, et al.2006. The genome of the sea urchin Strongylocentrotus purpuratus. Science 314:941–952.

7. Fugmann, S. D., C. Messier, L. A. Novack, R. A. Cameron, and J. P. Rast. 2006.An ancient evolutionary origin of the RAG1/2 gene locus. Proc. Natl. Acad. Sci.USA 103: 3728–3733.

8. Nair, S. V., H. Del Valle, P. S. Gross, D. P. Terwilliger, and L. C. Smith. 2005.Macroarray analysis of coelomocyte gene expression in response to LPS in thesea urchin: identification of unexpected immune diversity in an invertebrate.Physiol. Genomics 22: 33–47.

9. Rast, J. P., Z. Pancer, and E. H. Davidson. 2000. New approaches towards anunderstanding of deuterostome immunity. Curr. Top. Microbiol. Immunol. 248:3–16.

10. Smith, L. C., L. Chang, R. J. Britten, and E. H. Davidson. 1996. Sea urchin genesexpressed in activated coelomocytes are identified by expressed sequence tags:complement homologues and other putative immune response genes suggest im-mune system homology within the deuterostomes. J. Immunol. 156: 593–602.

11. Terwilliger, D. P., K. M. Buckley, V. Brockton, N. J. Ritter, and L. C. Smith.2007. Distinctive expression patterns of 185/333 genes in the purple sea urchin,Strongylocentrotus purpuratus: an unexpectedly diverse family of transcripts inresponse to LPS, �-1,3-glucan, and dsRNA. BMC Mol. Biol. 8: 16.

12. Terwilliger, D. P., K. M. Buckley, D. Mehta, P. G. Moorjani, and L. C. Smith.2006. Unexpected diversity displayed in cDNAs expressed by the immune cellsof the purple sea urchin, Strongylocentrotus purpuratus. Physiol. Genomics 26:134–144.

13. Buckley, K. M., and L. C. Smith. 2007. Extraordinary diversity among membersof the large gene family, 185/333, from the purple sea urchin, Strongylocentrotuspurpuratus. BMC Mol. Biol. 8: 68.

14. Buckley, K. M., S. Munshaw, T. B. Kepler, and L. C. Smith. 2008. The 185/333gene family is a rapidly diversifying host-defense gene cluster in the purple seaurchin, Strongylocentrotus purpuratus. J.Mol. Biol. 379: 912–928.

15. Buckley, K. M., D. P. Terwilliger, and L. C. Smith. Sequence variations in 185/333 messages from the purple sea urchin suggest posttranscriptional modifica-tions to increase diversity. J. Immunol. 181: 8585–8594.

16. Tsai, H., and L. A. Bobek. 1998. Human salivary histatins: promising anti-fungaltherapeutic agents. Crit. Rev. Oral Biol. Med. 9: 480–497.

17. Xu, T., E. Telser, R. F. Troxler, and F. G. Oppenheim. 1990. Primary structureand anticandidal activity of the major histatin from parotid secretion of the sub-human primate, Macaca fascicularis. J. Dent. Res. 69: 1717–1723.

18. Brockton, V., J. H. Henson, D. A. Raftos, A. J. Majeske, Y. O. Kim, andL. C. Smith. 2008. Localization and diversity of 185/333 proteins from the purplesea urchin – unexpected protein-size range and protein expression in a new coe-lomocyte type. J. Cell Sci. 121: 339–348.

19. Clow, L. A., P. S. Gross, C. S. Shih, and L. C. Smith. 2000. Expression of SpC3,the sea urchin complement component, in response to lipopolysaccharide. Im-munogenetics 51: 1021–1033.

20. Gross, P. S., W. Z. Al-Sharif, L. A. Clow, and L. C. Smith. 1999. Echinodermimmunity and the evolution of the complement system. Dev. Comp. Immunol. 23:429–442.

21. Clow, L. A., D. A. Raftos, P. S. Gross, and L. C. Smith. 2004. The sea urchincomplement homologue, SpC3, functions as an opsonin. J. Exp. Biol. 207:2147–2155.

22. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680–685.

23. Andon, N. L., S. Hollingworth, A. Koller, A. J. Greenland, J. R. Yates, andP. A. Haynes. 2002. Proteomic characterization of wheat amyloplasts using iden-tification of proteins by tandem mass spectrometry. Proteomics 2: 1156–1168.


24. Green, P., A. Luty, S. Nair, J. Radford, and D. Raftos. 2006. A second form ofcollagenous lectin from the tunicate, Styela plicata. Comp. Biochem. Physiol. BBiochem. Mol. Biol. 144: 343–350.

25. Green, P. L., S. V. Nair, and D. A. Raftos. 2003. Secretion of a collectin-likeprotein in tunicates is enhanced during inflammatory responses. Dev. Comp. Im-munol. 27: 3–9.

26. Miller, I., J. Crawford, and E. Gianazza. 2006. Protein stains for proteomic ap-plications: which, when, why? Proteomics 6: 5385–5408.

27. Kurtz, J., and K. Franz. 2003. Innate defence: evidence for memory in inverte-brate immunity. Nature 425: 37–38.

28. Kurtz, J., and S. A. Armitage. 2006. Alternative adaptive immunity in inverte-brates. Trends Immunol. 27: 493–496.

29. Sadd, B. M., and P. Schmid-Hempel. 2006. Insect immunity shows specificity inprotection upon secondary pathogen exposure. Curr. Biol. 16: 1206–1210.

30. Litman, G. W., J. P. Cannon, and J. P. Rast. 2005. New insights into alternativemechanisms of immune receptor diversification. Adv. Immunol. 87: 209–236.

31. Litman, G. W., N. A. Hawke, and J. A. Yoder. 2001. Novel immune-type re-ceptor genes. Immunol. Rev. 181: 250–259.

32. Pancer, Z., C. T. Amemiya, G. R. Ehrhardt, J. Ceitlin, G. L. Gartland, andM. D. Cooper. 2004. Somatic diversification of variable lymphocyte receptors inthe agnathan sea lamprey. Nature 430: 174–180.

33. Pancer, Z., W. E. Mayer, J. Klein, and M. D. Cooper. 2004. Prototypic T cellreceptor and CD4-like coreceptor are expressed by lymphocytes in the agnathansea lamprey. Proc. Natl. Acad. Sci. USA 101: 13273–13278.

34. Pancer, Z., N. R. Saha, J. Kasamatsu, T. Suzuki, C. T. Amemiya, M. Kasahara,and M. D. Cooper. 2005. Variable lymphocyte receptors in hagfish. Proc. Natl.Acad. Sci. USA 102: 9224–9229.

35. Cannon, J. P., R. N. Haire, and G. W. Litman. 2002. Identification of diversifiedgenes that contain immunoglobulin-like variable regions in a protochordate. Nat.Immunol. 3: 1200–1207.

36. Cannon, J. P., R. N. Haire, J. P. Rast, and G. W. Litman. 2004. The phylogeneticorigins of the antigen-binding receptors and somatic diversification mechanisms.Immunol. Rev. 200: 12–22.

37. Dong, Y., H. E. Taylor, and G. Dimopoulos. 2006. AgDscam, a hypervariableimmunoglobulin domain-containing receptor of the Anopheles gambiae innateimmune system. PLoS Biol. 4: e229.

38. Watson, F. L., R. Puttmann-Holgado, F. Thomas, D. L. Lamar, M. Hughes,M. Kondo, V. I. Rebel, and D. Schmucker. 2005. Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309: 1874–1878.

39. Adema, C. M., L. A. Hertel, R. D. Miller, and E. S. Loker. 1997. A family offibrinogen-related proteins that precipitates parasite-derived molecules is pro-duced by an invertebrate after infection. Proc. Natl. Acad. Sci. USA 94:8691–8696.

40. Leonard, P. M., C. M. Adema, S. M. Zhang, and E. S. Loker. 2001. Structure oftwo FREP genes that combine IgSF and fibrinogen domains, with comments ondiversity of the FREP gene family in the snail Biomphalaria glabrata. Gene 269:155–165.

41. Zhang, S. M., and E. S. Loker. 2004. Representation of an immune responsivegene family encoding fibrinogen-related proteins in the freshwater mollusc Bi-omphalaria glabrata, an intermediate host for Schistosoma mansoni. Gene 341:255–266.

42. Zhang, S. M., and E. S. Loker. 2003. The FREP gene family in the snail Bi-omphalaria glabrata: additional members, and evidence consistent with alterna-tive splicing and FREP retrosequences: fibrinogen-related proteins. Dev. Comp.Immunol. 27: 175–187.

43. Fenyo, D., and R. C. Beavis. 2003. A method for assessing the statistical signif-icance of mass spectrometry-based protein identifications using general scoringschemes. Anal. Chem. 75: 768–774.


Documents

Proteomic analysis of sea urchin immune responses and characterisation of highly variable immune response proteins. Macquarie University, Sydney, Australia