Abstract Galectins are a family of animal lectins with

specificity for b-galactosides and are involved in a host

of cellular activities, ranging from development to

cancer. The molecules are expressed by neural and

non-neural cells intracellularly as well as extracellu-

larly. Using two-dimensional gel electrophoresis cou-

pled to tandem mass spectrometry, the present work

aimed to identify and characterize galectins in primary

rat cerebellar astrocytes. The protein-chemical method

identified nine spots representing two members of the

galectin family, namely galectin-1 and galectin-3.

These findings suggest that high abundant expression

of galectin in astrocytes is limited to the two abundant

galectin family members. As these family members are

linked to human astrocytic tumors, their reliable

detection in astrocytes by proteomic techniques would

enable us to further understand their role in neural

development, injury, and regeneration in general and

astrocytoma in particular.

Keywords Galectin Æ Astrocytes Æ Two-dimensional

gel electrophoresis Æ Mass spectrometry

Introduction

Galectins are a major family of lectins, carbohydrate-

binding proteins that can recognize various carbohy-

drates attached to proteins and lipids known as

glycoconjugates on cell surfaces and extracellular

matrices. They are defined by their homing in on

distinct b-galactoside structures and by their conserved

sequence elements in the carbohydrate-binding site [1].

The carbohydrate-binding site contains about 130

amino acids and is designated as the carbohydrate

recognition domain (CRD). Galectins show a high

level of evolutionary conservation and members of this

family are present in organisms ranging from fungi

through nematodes to mammals [2].

To date, 15 mammalian galectins have been identi-

fied, numbered sequentially according to the accepted

numbers for their genes in the Genome database, and

classified into three different subfamilies based on the

number and organization of the CRDs [3, 4]. The

prototype group contains one CRD (galectin-1, -2, -5,

-7, -10, -11, -13, -14 and -15): the chimera group con-

tains unusual proline- and glycine-rich repeats onto the

CRD (galectin-3): and the tandem repeat group con-

tains two CRDs (galectin-4, -6, -8, -9, and -12).

Galectins interact with b-galactoside residues of sev-

eral cell surface and matrix glycoproteins through the

CRD and are also capable of peptide-peptide associa-

tions mediated by their N-terminus domain. These

structural properties endow galectins with multiple

functions, including modulation of cell adhesions,

control of the cell cycle, modulation of the immune and

inflammatory responses, and mRNA splicing [4–6].

Galectins can be located intracellularly or secreted

into the extracellular space. Their expression could be

J. W. Yang Æ S. U. Kang Æ E. Engidawork Æ G. Lubec (&)Department of Paediatrics, Medical University of Vienna,Waehringer Guertel 18-20, A-1090Vienna, Austriae-mail: gert.lubec@meduniwien.ac.at

E. EngidaworkDepartment of Pharmacology, School of Pharmacy, AddisAbaba University, Addis Ababa, Ethiopia

R. Rodrigo Æ V. FelipoLaboratory of Neurobiology, Fundacion Valenciana deInvestigaciones Biomedicas, Valencia, Spain

Neurochem Res (2006) 31:945–955

DOI 10.1007/s11064-006-9100-4

123

ORIGINAL PAPER

Mass Spectrometrical Analysis of Galectin Proteins in PrimaryRat Cerebellar Astrocytes

J. W. Yang Æ S. U. Kang Æ E. Engidawork ÆR. Rodrigo Æ V. Felipo Æ G. Lubec

Accepted: 8 June 2006 / Published online: 28 June 2006� Springer Science+Business Media, Inc. 2006

tissue-specific or developmentally regulated, and one

important fact is that almost every cell can express at

least one galectin [3]. As far as the nervous system is

concerned, it is the mammalian proto-type galectin-1

that has been thoroughly studied. Several lines of

evidence suggested that galectin-1 is expressed widely

in nervous tissues at embryonic stages but becomes

restricted mainly to peripheral nervous tissues with

maturation. Indeed, galectin-1 is selectively expressed

in sensory neurons and motoneurons [7] and it mediates

the contacts to laminin/merosin and to olfactory neu-

rons [8]. It has been shown as well that galectin-1

stimulates neurite outgrowth in an olfactory neuron line

[9] and regulates sorting and fasciculation of olfactory

axons [10]. Galectin-3 has been detected in the primary

olfactory system, probably involved in sorting of axons

as they converge towards their target [11]. It is also

expressed by a subset of dorsal root ganglion neurons

[12] and by microglial cells and/or macrophages infil-

trating in vivo at sites of brain injury [13].

Currently available data regarding galectins estab-

lish a clear link between its carbohydrate-binding

capacity and its physiological function in the nervous

system [14]. According to these data, it is a promising

concept that galectins act as effectors of neuronal

functions and that, due to their carbohydrate-binding

specificity, they can be used as optimal tools for the

systematic dissection of b-galactoside involvement in

neuronal development. To address this issue, the

selection of a suitable analytical tool is required. In this

manuscript, we show the robustness of two-dimensional

gel electrophoresis (2-DE) coupled to matrix-assisted

laser desorption/ionization (MALDI) mass spectrom-

etry (MS) followed by tandem mass spectrometry

(MS/MS) and Nano-HPLC-ESI-Q-TOF for detection

of galectins in the primary rat cerebellar astrocytes.

Material and methods

Primary cultures of rat cerebellar astrocytes

All animal procedures were approved and meet the

guidelines of the European Union for care and man-

agement of experimental animals. Astrocyte-enriched

primary cultures were prepared from cerebellum of 7-

day-old Wistar rats. Briefly, rats were killed by

decapitation and the cerebella were removed rapidly.

The dissected cerebella were then finely minced and

mechanically disrupted by pipetting in Dulbecco’s

modified Eagle’s medium (DMEM). The cell suspen-

sion was vortexed at maximum speed for 1 min and

filtered through sterile nylon mesh of 90-lm pore size,

to remove non-dissociated tissue. Dissociated cells

were re-suspended in DMEM containing 10% heat-

inactivated fetal bovine serum (FBS), 2 mM glutamine,

20 U/ml penicillin, 20 lg/ml streptomycin and 5 mg/ml

fungizone and were seeded on polystyrene plates (60-

diameter) at 20 ml/cerebellum, and were kept in

humidified atmosphere of 5% CO2 and 95% air at

37�C. Medium was changed once a week and cells were

used after 10–13 days in vitro. All reagents for cell

culture were purchased from GIBCO BRL (Life

Technologies S.A., Barcelona, Spain).

Two-dimensional gel electrophoresis (2-DE)

After three times washing with cold PBS, harvested

cells were suspended in 1 ml of sample buffer con-

sisting of 7 M urea, 2 M thiourea, 4% CHAPS (3-

[(3-cholamidopropyl)dimethylammonio]-1-propane-

sulfonate), 10 mMDL-dithiothreitol (DTT), 1 mM

EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF)

and a mixture of protease inhibitors (Roche Diagnos-

tics, Mannheim, Germany). After sonication for

approximately 15 s, the suspension was left at room

temperature for 1 h and centrifuged at 14,000 · g for

60 min at 12�C. Desalting was done with Ultrafree-4

centrifugal filter unit (Millipore, Bedford, MA, USA).

The protein content of the supernatant was determined

by the Coomassie blue method. 2-DE was performed

essentially as reported [15]. Samples of 1 mg protein

were applied on immobilized pI 3–10 non-linear gra-

dient strips (Amersham Bioscience, Uppsala, Sweden).

Focusing started at 200 V and the voltage was gradu-

ally increased to 8000 V at 4 V/min and kept constant

for a further 3 h (approximately 150,000 Vh in total).

The second-dimensional separation was performed on

9–16% gradient sodium SDS polyacrylamide gels.

After protein fixation for 12 h in 50% methanol and

10% acetic acid, the gels were stained with colloidal

Coomassie blue (Novex, San Diego, CA, USA) for 8 h

and excess of dye was washed out from the gels with

distilled water. Molecular masses were determined by

running standard protein markers (Bio-Rad Labora-

tories, Hercules, CA, USA) covering the range 10–

250 kDa. pI values were used as given by the supplier

of the immobilized pH gradient strips.

MALDI-TOF/TOF

Spots were excised with a spot picker (PROTEINEER

spTM, Bruker Daltonics), placed into 96-well microtiter

plates. In-gel digestion and sample preparation for

MALDI analysis were performed by an auto-

mated procedure (PROTEINEER dpTM, Bruker

946 Neurochem Res (2006) 31:945–955

123

Daltonics)[16, 17]. Briefly, spots were excised and

washed with 10 mM ammonium bicarbonate and 50%

acetonitrile in 10 mM ammonium bicarbonate. After

washing, gel plugs were shrunk by addition of aceto-

nitrile and dried by blowing out the liquid through the

pierced well bottom. The dried gel pieces were

reswollen with 40 ng/ll trypsin (Promega, Madison,

WI, USA) in enzyme buffer, consisting of 5 mM octyl

b-D-glucopyranoside (OGP) and 10 mM ammonium

bicarbonate and incubated for 4 h at 30�C. Peptide

extraction was performed with 10 ll of 1% trifluoro-

acetic acid in 5 mM OGP. Extracted peptides were

directly applied onto a target (AnchorChipTM, Bruker

Daltonics) that was loaded with a-cyano-4-hydroxy-

cinnamic acid (Bruker Daltonics) matrix thinlayer. The

mass spectrometer used in this work was an Ultra-

flexTM TOF/TOF (Bruker Daltonics) operated in the

reflector mode for MALDI-TOF peptide mass finger-

print (PMF) and LIFT mode for MALDI-TOF/TOF.

An accelerating voltage of 25 kV was used for PMF.

Calibration of the instrument was performed externally

with [M+H]+ ions of angiotensin I, angiotensin II,

substance P, bombesin, and adrenocorticotropic hor-

mones (clip 1–17 and clip 18–39). Each spectrum was

produced by accumulating data from 200 consecutive

laser shots for PMF. Those samples, which were ana-

lyzed by PMF from MALDI-TOF, were additionally

analyzed using LIFT-TOF/TOF MS/MS from the same

target. In the TOF1 stage, all ions were accelerated to

8 kV under conditions promoting metastable frag-

mentation. After selection of jointly migrating parent

and fragment ions in a timed ion gate, ions were lifted

by 19 kV to high potential energy in the LIFT cell.

After further acceleration of the fragment ions in the

second ion source, their masses could be simulta-

neously analyzed in the reflector with high sensitivity.

PMF and LIFT spectra were interpreted with the

Mascot software (version 2.1; Matrix Science Ltd,

London, UK). Database searches, through Mascot,

using combined PMF and MS/MS datasets were per-

formed via BioTools 2.2 software. A mass tolerance of

25 ppm for PMF and an MS/MS tolerance of 0.5 Da

and 1 missing cleavage site were allowed and oxidation

of methionine residues and/or phosphorylation of ser-

ine, threonine and tyrosine was considered. The

probability score calculated by the software was used

as criterion for correct identification [18].

Database searches

Database searches were performed based on in-house

Mascot server (version 2.1) with PMF and TOF/TOF

dataset. Prediction of phosphorylation sites in protein

was performed via the NetPhos 2.0 server http://

www.cbs.dtu.dk/services/NetPhos/) and information on

phosphorylated galectins was searched in PhosphoSite

(version 1.0; http://www.phosphosite.org/).

Nano-HPLC-ESI-Q-TOF

Excised gel spots were washed with 50 mM ammo-

nium bicarbonate and two times with 50% acetonitrile

in 50 mM ammonium bicarbonate for 30 min with

occasional vortexing. After washing, the gel pieces

were shrunk by 100 ll of 100% acetonitrile and dried

completely in a vacuum centrifuge (Eppendorf,

Hamburg, Germany). Reducing cysteines was per-

formed with 10 mM DTT in 0.1 M ammonium bicar-

bonate for 60 min at 56�C and alkylation of cysteine

residues was performed with 55 mM iodoacetamide in

0.1 M ammonium bicarbonate for 45 min at 25�C. The

reduction/alkylation solutions were removed and

washed with 50 mM ammonium bicarbonate for 10 min.

The dried gel pieces in acetonitrile followed by a

vacuum centrifugation were reswollen with 12.5 ng/ml

trypsin (Promega) in 25 mM ammonium bicarbonate

and incubated for 16 h at 37�C. Peptide extraction was

performed twice with 50 ll of 0.5% formic acid/20%

acetonitrile for 15 min in a sonication bath and

reduced to 10 ll by evaporation in a vacuum centri-

fuge and then mixed with 20 ll water. LC-MS/MS

analyses were carried out with an UltiMate 3000 sys-

tem (LC Packings, Amsterdam, Netherlands) inter-

faced to the QSTAR Pulsar mass spectrometer

(Applied Biosystems, Foster City, CA, USA). The

gradient was (A = 0.05% TFA in water, B = 80%

ACN/0.04% TFA in water) from 0% to 50% B in

30 min, 90% B in 5 min, 0% B in 25 min. Peptide

spectra were recorded over the mass range of m/z

400–1600, and MS/MS spectra were recorded in

information dependent data acquisition over the mass

range of m/z 50–1600. One peptide spectrum was

recorded and followed by three MS/MS spectra on the

QSTAR Pulsar instrument; the accumulation time was

1 s for peptide spectra and 2 s for MS/MS spectra. The

collision energy was set automatically according to the

mass and charge state of the peptides chosen for

fragmentation. Doubly or triply charged peptides were

chosen for MS/MS experiments due to their good

fragmentation characteristics. MS/MS spectra were

interpreted by the MASCOT software (Matrix

Science). A mass tolerance of 500 ppm for MS and an

MS/MS tolerance of 0.15 Da and 1 missing cleav-

age site were allowed and oxidation of methionine

Neurochem Res (2006) 31:945–955 947

123

residues and/or phosphorylation of serine, threonine

and tyrosine was considered.

Results

Total protein extracts derived from primary rat cere-

bellar astrocytes were separated by 2-DE and several

hundred spots were visualized by colloidal Coomassie

blue staining in partial 2-DE image with pI spanning

from 4 to 10 and molecular weight (MW) ranging from

12 to 30 kDa (Fig. 1). These proteins were identified

forming a proteome map of rat astrocytes with 406

spots representing 228 individual proteins [17].

The spots were excised from the 2-DE gel and

subjected to MALDI-MS analysis following trypsin

digestion to determine peptide mass. Bioinformatic

tools were used to mine databases to match peptide

masses with theoretical peptide masses. This search

identified three of the spots as galectin-1 (designated as

galectin-1a, -1b and -1c in Fig. 1) and the other three as

galectin-3 (designated as galectin-3b, -3e and -3f in

Fig. 1).

MS identifications, including matched peptide

numbers (matches), theoretical and observed pI and

MW values, number of identified spots and statistically

significant Mascot scores (P < 0.05) for the assigned

proteins are reported in Table 1. In general, there ap-

peared to be good agreement between the theoretically

predicted and experimentally found pI and MW values.

Two methionine residues appear to be oxidized in

galectin-1a and -1b spots, but not in galectin-1c (Fig. 2)

and oxidation of methionine residues was detected at

m/z 2186 (241–261), m/z 2304 (141–161), m/z 2432

(141–162) and m/z 2569 (238–261) in spectra from

galectin-3 from PMF.

A phosphorylation site was detected at m/z 2043.91

in MS spectrum of galectin-3a spot using MASCOT

database. When we applied galectin-3 to other phos-

phoprotein databases, phosphorylated serine at 308 was

predicted in rat galectin-3 with score 0.979, indicating a

phosphorylation site by the NetPhos 2.0 server (http://

www.cbs.dtu.dk/services/NetPhos/). Mouse galectin-3

was predicted to have different pI 7.16 and 7.96 for one

and two of phosphorylated residues, respectively, as

compared to basal pI of 8.5 by PhosphoSite (http://

www.phosphosite.org/).

To make an affirmative identity of the protein spots,

peptide sequencing was carried out using MS/MS to

obtain peptide sequences for database search. Precur-

sor ions from spot galectin-1a (m/z 1456.75 and

1486.70), galectin-1b (m/z 1486.70) and galectin-1c (m/z

2345.21) were selected, respectively, for peptide

sequencing from the MS spectra of the tryptic peptide

mixture of the spots. Likewise, four (m/z 1287.64,

1415.70, 1677.89 and 2186.13), four (m/z 1287.64,

1415.71, 1677.90 and 2186.13) and two (m/z, 1287.63

and 1677.89) peaks were selected from MS spectra

generated from galectin-3b, -3e and -3f for MS/MS

analysis. MS/MS spectra for all of the peptides ana-

lyzed from spots representing gal1 and gal3 are shown

in Figs. 3 and 4. The complete amino acid sequences of

the analyzed peptides are summarized in Table 1. By

database searching with combined MS and MS/MS

results, the three galectin-1 spots were found to be

galectin-1 protein (accession no. P11762) and the other

three galectin-3 spots were assigned to galectin-3 pro-

tein (accession no. P08699), confirming unambiguous

assignment of the proteins by significant MS/MS results

(Table 1).

We additionally applied nano-HPLC-ESI-Q-TOF

mass spectrometry to identify more galectin-3 spots as

well as to characterize phosphorylation site. This ESI-

MS/MS analysis allowed 3 more identifications of

galectin-3 (galectin-3a, -3c and -3d in Fig. 1, Table 2)

but did not lead to the detection of any phosphoryla-

tion site, especially from the tryptic digested partial

peptides QSAFPFESGKPFK which was estimated

from both MS analysis with Mascot search and phos-

phoprotein databases (Fig. 5).

Discussion

Cell surface glycans, such as glycocoproteins and

glycolipids, encode information that modulates

kDa

37

25

20

15

10

pI 4.0 6.0 8.05.4 6.8

Fig. 1 Partial 2-DE gel image of rat primary astrocytes withidentified galectins. Proteins were extracted and separated on animmobilized pH 3–10 non-linear gradient strip followed byseparation on a 9–16% gradient polyacrylamide gel. Gels werestained with Coomassie blue and spots were analysed byMALDI-MS and MS/MS. Spots representing the respectivegalectin-1 and -3 are designated as a, b and c

948 Neurochem Res (2006) 31:945–955

123

Ta

ble

1M

ass

spe

ctro

me

tric

al

ide

nti

fica

tio

no

fg

ale

ctin

sin

rat

pri

ma

rya

stro

cyte

s

Acc

ess

ion

no

.aP

rote

inn

am

e(S

yn

on

ym

)T

he

ore

tica

lbID

Ob

serv

ed

cM

atc

he

sS

core

dM

S/M

S

pI

MW

(Da

)p

IM

W(k

Da

)(M

+H

)+P

ep

tid

e(I

nd

ivid

ua

lM

S/M

Ssc

ore

)

LE

G1

_R

AT

(P1

17

62

)G

ale

ctin

-1(b

-ga

lact

osi

de

-bin

din

gle

ctin

L-1

4-I

)(L

act

ose

-bin

din

gle

ctin

1)

(S-L

ac

lect

in1

)(G

ala

pti

n)

(14

kD

ale

ctin

)(R

L1

4.5

)

5.1

41

47

25

.65

1a

4.9

61

3.2

15

22

51

45

6.7

5L

PD

GH

EF

KF

PN

R(4

4)

14

86

.70

DS

NN

LC

LH

FN

PR

(61

)1

b5

.15

13

.29

16

91

48

6.7

0D

SN

NL

CL

HF

NP

R(9

8)

1c

5.4

31

2.8

56

42

34

5.2

1S

FV

LN

LG

KD

SN

NL

CL

CL

HF

NP

R(2

8)

LE

G3

_R

AT

(P0

86

99

)G

ale

ctin

-3(G

ala

cto

se-s

pe

cifi

cle

ctin

3)

(Ma

c-2

an

tig

en

)(I

gE

-bin

din

gp

rote

in)

(35

kD

ale

ctin

)(C

arb

oh

yd

rate

-bin

din

gp

rote

in3

5)

CB

P3

5)

(La

min

in-b

ind

ing

pro

tein

)(L

ect

inL

-29

)

8.6

22

70

70

.34

3b

7.4

02

7.8

19

28

91

28

7.6

4G

ND

IAF

HF

NP

R(7

3)

14

15

.70

KG

ND

IAF

HF

NP

R(2

4)

16

77

.89

VA

VN

DV

HL

LQ

YN

HR

(84

)2

18

6.1

3E

ISQ

LG

IIG

DIT

LT

SA

SH

AM

I+

MO

xe

(12

)3

e8

.10

27

.82

03

11

12

87

.64

GN

DIA

FH

FN

PR

(57

)1

41

5.7

1K

GN

DIA

FH

FN

PR

(27

)1

67

7.9

0V

AV

ND

VH

LL

QY

NH

R(9

0)

21

86

.13

EIS

QL

GII

GD

ITL

TS

AS

HA

MI

+M

Ox

(41

)3

f8

.52

27

.81

32

00

12

87

.63

GN

DIA

FH

FN

PR

(62

)1

67

7.8

9V

AV

ND

VH

LL

QY

NH

R(7

4)

aS

WIS

S-P

RO

Te

ntr

yn

am

ea

nd

pri

ma

rya

cce

ssio

nn

um

be

rin

Ex

PA

Sy

we

bsi

te(h

ttp

://w

ww

.ex

pa

sy.o

rg)

bC

alc

ula

ted

for

the

corr

esp

on

din

gS

WIS

S-P

RO

Te

ntr

yu

sin

gth

eE

xP

AS

yC

om

pu

tep

I/M

Wto

ol

inth

eE

xP

AS

yw

eb

site

cE

stim

ate

dfr

om

the

po

siti

on

of

the

pro

tein

spo

to

nth

e2

Dg

el

dS

core

is–

10

*L

og

(P),

wh

ere

Pis

the

pro

ba

bil

ity

tha

tth

eo

bse

rve

dm

atc

his

ara

nd

om

ev

en

t(M

AS

CO

T,

htt

p:/

/ww

w.m

atr

ixsc

ien

ce.c

om

/he

lp/s

cori

ng

-he

lp.h

tml)

eM

Ox,

Ox

ida

tio

n(M

)

Neurochem Res (2006) 31:945–955 949

123

interactions between cells, or between cells and the

extracellular matrix, by specifically regulating the bind-

ing to cell surface-associated or soluble carbohydrate-

binding receptors, such as lectins. Galectins, a family of

animal lectins that bind b-galactoside sugar chains, are

thought to have a variety of intra- and extracellular

functions [4, 19]. Immunochemical methods, including

immunohistochemistry and Western blot analysis have

been used to understand the cellular role of galectins at

the protein level [20–22]. However, significant techni-

cal challenges, including antibody availability and

specificity limit the usefulness of these techniques. We,

therefore, employed proteomics to show galectin pro-

tein expression in a specific cell line of the central

nervous system. Although proteomic techniques have

been employed to detect galectin expression in mac-

rophages [23], prostate cancer line PC3 [24] and rat

calvarial osteoblasts [25]; this is the first report, to the

best of our knowledge, of galectin protein expression in

cells of the central nervous system using MALDI-

TOF-TOF and Q-TOF.

Although there are about 15 family members

reported in the literature, we detected only two

members of the family, galectin-1 and -3, in primary rat

Fig. 3 MALDI TOF/TOF spectra from the precursor ionsdetected at m/z 1456.75 and 1486.70 from galectin-1a (A), atm/z 1486.70 from galectin-1b (B) and at m/z 2345.21 from

galectin-1c (C). The identified sequences unambiguously as-signed these protein spots to galectin-1 (P11762)

Fig. 2 MALDI-TOF mass spectrum of galectin-1a. The inset isthe expanded view of the peptides, which contain single anddouble oxidized methionine (M). These oxidized forms were alsodetected at the same sites in galectin-1b

950 Neurochem Res (2006) 31:945–955

123

cerebellar astrocytes. This is consistent with the view

that some galectins are distributed in a wide variety of

cells, whereas others are more specific. Moreover, high

abundant expression of galectins is impacted by dif-

ferent physiological and pathological conditions.

Whilst expression of galectins is limited to galectin-1

and -3 in un-stimulated astrocytes, as shown in the

present study, astrocytes stimulated with IL-1ß pro-

duce galectin-9 [26].

Are the three spots in the 2-DE gel the same pro-

tein? Do they reflect protein modifications or splice

variants? It has been reported that a monoclonal

antibody raised against human brain galectin-1 specif-

ically reacted with the two most acidic isoforms of

galectin-1 amongst the three identified by 2-DE [27].

Moreover, galectin-1 isolated from bovine heart yiel-

ded four isoforms ranging from pI 4.53–4.73 [28],

probably indicating that the three spots of galectin-1

may represent different isoforms. Other studies also

showed that galecin-1 could be converted to an oxi-

dized form in the extracellular space, which appears to

promote axonal regeneration [29]. We detected 2

methionine oxidized forms of galectin-1 at pI 4.96 and

5.16 by MS analysis. Although we cannot rule out that

methionine oxidation is a technical artifact, the fact

that not all galectins are oxidized speaks against simple

oxidation by sample preparation and electrophoretic

steps. It has to be taken into account that analytical

steps were carried out under reducing conditions.

We observed one phosphorylated form of galectin-3

at pI 7.40 by MS in agreement with a previous report

by Cowles and coworkers [30].

Galectin-3 is demonstrated to exist as two isoelectric

variants in mouse 3T3 fibroblasts, phosphorylated and

non-phosphorylated with pI 8.2 and 8.7, respectively

[30]. MS/MS, however, failed to confirm this modifi-

cation based upon prediction and MS analysis and

therefore MS/MS analysis is mandatory for reliable

assay of galectin-3 modifications.

Galectins modulate a host of cellular processes, such

as cell growth, cell cycle progression, mitosis and

apoptosis by a number of mechanisms [4]. Extracellu-

larly, they can cross-link cell surface glycoconjugates,

thereby triggering a cascade of transmembrane sig-

naling events and they can also promote homo-or

heterotypic aggregation as well as bridging of cells to

Fig. 4 MALDI TOF/TOF spectra from galectin-3b (A) and galectin-3e (B) and galectin-3f (C). The identified sequencesunambiguously assigned these protein spots to galectin-3 (P08699)

Neurochem Res (2006) 31:945–955 951

123

extracellular matrix. Intracellularly, galectins shuttle

between the nucleus and cytoplasm and are engaged in

fundamental processes such as pre-mRNA splicing.

Thus, galectins can mediate beneficial or harmful ef-

fects depending upon the cellular context. Galectin-1

was demonstrated to induce astrocyte differentiation,

enabling differentiated astrocytes to produce more

brain-derived neurotrophic factor, a neuroprotective

polypeptide [31]. Moreover, oxidized galectin-1 is

proposed as a factor promoting recovery after neuronal

Table 2 Phosphorylation site in MS spectrum of galectin-3a spot using MASCOT database

ID Observed MS/MS results

pI MW(kDa)

Observeda Mr(expt)b

Mr(calc)c

Deltad Misse Scoref Expectg Peptide(s)

3a 7.05 27.8 429.93 1286.78 1286.62 0.16 0 33 0.012 K.GNDIAFHFNPR.F433.63 1297.86 1297.70 0.16 0 39 0.0041 K.IQVLVEADHFK.V490.65 1468.92 1468.73 0.18 0 42 0.002 R.QSAFPFESGKPFK.I560.03 1677.08 1676.87 0.20 0 47 0.0011 K.VAVNDVHLLQYNHR.M

3b 7.40 27.8 644.40 1286.78 1286.62 0.17 0 44 0.001 K.GNDIAFHFNPR.F433.63 1297.86 1297.70 0.16 0 40 0.0034 K.IQVLVEADHFK.V649.94 1297.87 1297.70 0.17 0 (29) 0.04 K.IQVLVEADHFK.V735.47 1468.93 1468.73 0.19 0 (34) 0.014 R.QSAFPFESGKPFK.I490.65 1468.93 1468.73 0.20 0 41 0.0028 R.QSAFPFESGKPFK.I560.04 1677.11 1676.87 0.23 0 37 0.01 K.VAVNDVHLLQYNHR.M724.15 2169.43 2169.14 0.30 0 (7) 10 R.EISQLGIIGDITLTSASHAMI.-729.48 2185.43 2185.13 0.30 0 16 1.2 R.EISQLGIIGDITLTSASHAMI.- + MOx

h

3c 7.60 27.8 644.40 1286.79 1286.62 0.18 0 34 0.01 K.GNDIAFHFNPR.F429.94 1286.80 1286.62 0.19 0 (33) 0.014 K.GNDIAFHFNPR.F433.63 1297.88 1297.70 0.17 0 38 0.0056 K.IQVLVEADHFK.V649.95 1297.88 1297.70 0.18 0 (12) 1.9 K.IQVLVEADHFK.V472.35 1414.04 1414.71 –0.67 1 50 0.00042 K.KGNDIAFHFNPR.F735.48 1468.94 1468.73 0.20 0 (32) 0.024 R.QSAFPFESGKPFK.I490.65 1468.94 1468.73 0.21 0 47 0.00069 R.QSAFPFESGKPFK.I560.04 1677.11 1676.87 0.23 0 42 0.0034 K.VAVNDVHLLQYNHR.M

3d 7.80 27.8 429.94 1286.81 1286.62 0.19 0 30 0.025 K.GNDIAFHFNPR.F649.96 1297.90 1297.70 0.20 0 35 0.011 K.IQVLVEADHFK.V433.64 1297.91 1297.70 0.20 0 (29) 0.039 K.IQVLVEADHFK.V735.49 1468.96 1468.73 0.23 0 (27) 0.079 R.QSAFPFESGKPFK.I490.66 1468.96 1468.73 0.23 0 38 0.0063 R.QSAFPFESGKPFK.I560.05 1677.12 1676.87 0.25 0 34 0.018 K.VAVNDVHLLQYNHR.M768.89 2303.64 2303.29 0.34 0 12 2.9 R.MLITIIGTVKPNANSITLNFK.K + MOx

3f 8.52 27.8 417.28 832.54 832.45 0.09 0 33 0.011 R.VIVCNTK.Q445.25 888.50 888.38 0.11 0 30 0.025 K.QDNNWGR.E495.35 988.68 988.55 0.13 1 51 0.00019 R.RVIVCNTK.Q644.39 1286.77 1286.62 0.15 0 37 0.0058 K.GNDIAFHFNPR.F429.93 1286.77 1286.62 0.16 0 (33) 0.014 K.GNDIAFHFNPR.F433.63 1297.86 1297.70 0.15 0 43 0.0016 K.IQVLVEADHFK.V472.63 1414.88 1414.71 0.17 1 40 0.003 K.KGNDIAFHFNPR.F490.65 1468.92 1468.73 0.18 0 51 0.00027 R.QSAFPFESGKPFK.I839.54 1677.07 1676.87 0.20 0 (32) 0.035 K.VAVNDVHLLQYNHR.M560.04 1677.09 1676.87 0.21 0 44 0.002 K.VAVNDVHLLQYNHR.M724.14 2169.41 2169.14 0.28 0 (24) 0.21 R.EISQLGIIGDITLTSASHAMI.-729.47 2185.40 2185.13 0.27 0 41 0.0042 R.EISQLGIIGDITLTSASHAMI.- + MOx

768.86 2303.57 2303.29 0.28 0 17 0.94 R.MLITIIGTVKPNANSITLNFK.K + MOx

aExperimental m/z valuebExperimental m/z transformed to a relative molecular masscCalculated relative molecular mass of the matched peptidedDifference (error) between the experimental and calculated masseseNumber of missed cleavage sitesfIons score—if there are duplicate matches to the same peptide, then score are shown in bracketsgExpectation value for the peptide match. (The number of times we would expect to obtain an equal or higher score, purely by chance.The lower this value, the more significant the result)hMOx, Oxidation (M)

952 Neurochem Res (2006) 31:945–955

123

injury [29]. On the other hand, an increasing body of

evidence indicates that levels of galectin-1 and -3

protein expression significantly change during the

progression of malignancy in astrocytic tumors [21, 32,

33]. Although the mechanisms by which galectins are

involved in cell transformation are not known, both

galectin-1 and -3 are known to interact with oncogenic

Ras [34, 35]. Galectin-1 enhances the migratory capa-

bility and biological aggressiveness of tumor astrocytes

by restructuring of the actin cytoskeleton and increas-

ing expression of the small GTPase RhoA [22], as well

as by promoting secretion of enzymes to remodel the

interstitial space so as to accommodate the forward

motion of the migrating cells [36]. Galectin-3 up-reg-

ulates integrins [37], proteins that have an important

role in controlling cell migration and therefore tumor

invasion. Moreover, Galectin-3 exhibits high affinity

binding for advanced glycosylation end products in

astrocytes [5], which have roles in enhancing prolifer-

ation, migration and invasion of tumor cells [38].

In conclusion, we herein unambiguously character-

ize galectin-1 and -3 by two independent proteomics

techniques, MALDI-TOF-TOF and Q-TOF, with high

sequence coverage and additional expression forms,

that have not been described before. This work is,

therefore, extending previous preliminary identifica-

tions and characterizations of galectins in astrocytes.

The association of galectin expressison forms with

astrocytic tumors makes unambiguous analysis and

characterization of these molecules mandatory. This

would provide a platform for better understanding of

the role of galectins in the nervous system under both

physiological and pathological conditions. The fair

identification of galectins may even provide the basis

for the search of specific markers of astrocytes and

probably astrocytic tumors.

Fig. 5 MALDI-TOF oftryptic peptides from galectin-3b; the inset is an expandedview of the peptides whichpresented phosphorylationsites by Mascot database withPMF (A). Nophosphorylation site wasfound in peptidesQSAFPFESGKPFK fromgalectin-3b by Nano-HPLC-ESI-Q-TOF, which wasestimated phosphorylationmodification from PMF (B)

Neurochem Res (2006) 31:945–955 953

123

Acknowledgments Dr. Ephrem Engidawork is grateful for thesupport of Austrian Embassy Development Cooperation, AddisAbaba, Ethiopia.

References

1. Barondes SH, Castronovo V, Cooper DNW, Cummings RD,Drickamer K, Feizi T, Gitt MA, Hirabayashi J, Hughes C,Kasai K (1994) Galectins: a family of animal beta-galacto-side-binding lectins. Cell 76:597–598

2. Kasai K, Hirabayashi J (1996) Galectins: a family of animallectins that decipher glycocodes. J Biochem 119:1–8

3. Wang JL, Gray RM, Haudeck KC, Patterson RJ (2004)Nucleocytoplasmic lectins. Biochimi Biophys Acta1673:75–93

4. Liu FT, Rabinovich GA (2005) Galectins as modulators oftumor progression. Nat Rev Cancer 5:29–41

5. Pricci F, Leto G, Amadio L, Iacobini C, Romeo G, CordoneS, Gradini R, Barsotti P, Liu FT, Di Mario U, Pugliese G(2000) Role of galectin-3 as a receptor for advanced glyco-sylation end products. Kidney Int (Suppl) 77:S31–S39

6. Califice S, Castronovo V, Van Den Brule F (2004) Galectin-3and cancer. Int J Oncol 25:983–992

7. Hynes MA, Gitt M, Barondes SH, Jessell TM, Buck LB(1990) Selective expression of an endogenous lactose-bind-ing lectin gene in subsets of central and peripheral neurons.J Neurosci 10:1004–1013

8. Crandall JE, Dibble C, Butler D, Pays L, Ahmad N, KostekC, Puschel AW, Schwarting GA (2000) Patterning of olfac-tory sensory connections is mediated by extracellular matrixproteins in the nerve layer of the olfactory bulb. J Neurobiol45:195–206

9. Mahanthappa NK, Cooper DN, Barondes SH, SchwartingGA (1994) Rat olfactory neurons can utilize the endogenouslectin, L-14, in a novel adhesion mechanism. Development120:1373–1384

10. Puche AC, Poirier F, Hair M, Bartlett PF, Key B (1996) Roleof galectin-1 in the developing mouse olfactory system. DevBiol 179:274–287

11. Storan MJ, Magnaldo T, Biol-N’Garagba MC, Zick Y, Key B(2004) Expression and putative role of lactoseries carbohy-drates present on NCAM in the rat primary olfactory path-way. J Comp Neurol 475:289–302

12. Pesheva P, Kuklinski S, Biersack HJ, Probstmeier R (2000)Nerve growth factor-mediated expression of galectin-3 inmouse dorsal root ganglion neurons. Neurosci Lett 293:37–40

13. Probstmeier R, Montag D, Schachner M (1995) Galectin-3, aß-galactoside-binding animal lectin, binds to neural recog-nition molecules. J Neurochem 64:2465–2472

14. Zanetta JP (1998) Structure and functions of lectins in thecentral and peripheral nervous system. Acta Anat 161:180–195

15. Yang JW, Czech T, Yamada J, Csaszar E, Baumgartner C,Slavc I, Lubec G (2004) Aberrant cytosolic acyl-CoA thio-ester hydrolase in hippocampus of patients with mesialtemporal lobe epilepsy. Amino Acids 27:269–275

16. Suckau D, Resemann A, Schuerenberg M, Hufnagel P,Franzen J, Holle A (2003) A novel MALDI LIFT-TOF/TOFmass spectrometer for proteomics. Anal Bioanal Chem376:952–965

17. Yang JW, Rodrigo R, Felipo V, Lubec G (2005) Proteomeanalysis of primary neurons and astrocytes from rat cere-bellum. J Proteome Res 4:768–788

18. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999)Probability-based protein identification by searchingsequence databases using mass spectrometry data. Electro-phoresis 20:3551–3567

19. Hsu DK, Liu FT (2004) Regulation of cellular homeostasisby galectins. Glycoconj J 19:507–515

20. Walther M, Kuklinski S, Pesheva P, Guntinas-Lichius O,Angelov DN, Neiss WF, Asou H, Probstmeier R (2000)Galectin-3 is upregulated in microglial cells in response toischemic brain lesions, but not to facial nerve axotomy.J Neurosci Res 61:430–435

21. Camby I, Belot N, Rorive S, Lefranc F, Maurage CA, LahmH, Kaltner H, Hadari Y, Ruchoux MM, Brotchi J, Zick Y,Salmon I, Gabius HJ, Kiss R (2001) Galectins are differen-tially expressed in supratentorial pilocytic astrocytomas,astrocytomas, anaplastic astrocytomas and glioblastomas,and significantly modulate tumor astrocyte migration. BrainPathol 11:12–26

22. Camby I, Belot N, Lefranc F, Sadeghi N, de Launoit Y,Kaltner H, Musette S, Darro F, Danguy A, Salmon I, GabiusHJ, Kiss R (2002) Galectin-1 modulates human glioblastomacell migration into the brain through modifications to theactin cytoskeleton and levels of expression of small GTPases.J Neuropathol Exp Neurol 61:585–596

23. van den Berg TK, Honing H, Franke N, van Remoortere A,Schiphorst WE, Liu FT, Deelder AM, Cummings RD,Hokke CH, van Die I (2004) LacdiNAc-glycans constitute aparasite pattern for galectin-3-mediated immune recognition.J Immunol 173:1902–1907

24. Andersen H, Jensen ON, Moiseeva EP, Eriksen EF (2003) Aproteome study of secreted prostatic factors affectingosteoblastic activity: galectin-1 is involved in differentiationof human bone marrow stromal cells. J Bone Miner Res18:195–203

25. Choi JY, van Wijnen AJ, Aslam F, Leszyk JD, Stein JL,Stein GS, Lian JB, Penman S (1998) Developmental asso-ciation of the beta-galactoside-binding protein galectin-1with the nuclear matrix of rat calvarial osteoblasts. J Cell Sci111:3035–3043

26. Yoshida H, Imaizumi T, Kumagai M, Kimura K, Satoh C,Hanada N, Fujimoto K, Nishi N, Tanji K, Matsumiya T, MoriF, Cui XF, Tamo W, Shibata T, Takanashi S, Okumura K,Nakamura T, Wakabayashi K, Hirashima M, Sato Y, SatohK (2001) Interleukin-1beta stimulates galectin-9 expressionin human astrocytes. Neuroreport 12:3755–3758

27. Cornillot JD, Pontet M, Dupuy C, Chadli A, Caron M,Joubert-Caron R, Bourin P, Bladier D (1998) Productionand characterization of a monoclonal antibody able to dis-criminate galectin-1 from galectin-2 and galectin-3. Glyco-biology 8:425–432

28. Ahmed H, Fink NE, Pohl J, Vasta GR (1996) Galectin-1from bovine spleen: biochemical characterization, carbohy-drate specificity and tissue-specific isoform profiles. J Bio-chem 120:1007–1019

29. Horie H, Kadoya T (2004) Galectin-1 plays essential roles inadult mammalian nervous tissues Roles of oxidized galectin-1. Glycoconj J 19:479–489

30. Cowles EA, Agrwal N, Anderson RL, Wang JL (1990)Carbohydrate-binding protein 35 Isoelectric points of thepolypeptide and a phosphorylated derivative. J Biol Chem265:17706–17712

31. Sasaki T, Hirabayashi J, Manya H, Kasai K, Endo T (2004)Galectin-1 induces astrocyte differentiation, which leads toproduction of brain-derived neurotrophic factor. Glycobiol-ogy 14:357–363

954 Neurochem Res (2006) 31:945–955

123

32. Gordower L, Decaestecker C, Kacem Y, Lemmers A, Gus-man J, Burchert M, Danguy A, Gabius H, Salmon I, Kiss R,Camby I (1999) Galectin-3 and galectin-3-binding siteexpression in human adult astrocytic tumors and relatedangiogenesis. Neuropathol Appl Neurobiol 25:319–330

33. Kuklinski S, Pesheva P, Heimann C, Urschel S, Gloor S,Graeber S, Herzog V, Pietsch T, Wiestler OD, ProbstmeierR (2000) Expression pattern of galectin-3 in neural tumorcell lines. J Neurosci Res 60:45–57

34. Paz A, Haklai R, Elad-Sfadia G, Ballan E, Kloog Y (2001)Galectin-1 binds oncogenic H-Ras to mediate Ras membraneanchrage and cell transformation. Oncogene 20:7486–7493

35. Elad-Sfadia G, Haklai R, Ballan E, Kloog Y (2004) Galectin-3 augments K-Ras activation and triggers a Ras signal that

attenuates ERK but not phosphoinositide 3-kinase activity.J Biol Chem 279:34922–34930

36. Giese A, Rief MD, Loo MA, Berens ME (1994) Determi-nants of human astrocytoma migration. Cancer Res 54:3897–3904

37. Matarrese P, Fusco O, Tinari N, Natoli C, Liu FT, SemeraroML, Malorni W, Iacobelli S (2000) Galectin-3 overexpres-sion protects from apoptosis by improving cell adhesionproperties. Int J Cancer 85:545–554

38. Abe R, Shimizu T, Sugawara H, Watanabe H, Nakamura H,Choei H, Sasaki N, Yamagishi S, Takeuchi M, Shimizu H(2004) Regulation of human melanoma growth and metas-tasis by AGE-AGE receptor interactions. J Invest Dermatol122:461–467

Neurochem Res (2006) 31:945–955 955

123