1

1. INTORDUCTION

1.1. Glycan protein interaction

Glycans can mediate a wide variety of biological roles by virtue of their mass,

shape, charge, or other physical properties. Nature appears to have taken full

advantage of the vast diversity of glycans expressed in organisms by evolving

proteins to recognize discrete glycans that mediate specific physiological or

pathological processes. Many of the specific biological roles of glycans are

mediated by Glycan Binding Proteins (GBPs). Indeed, there are no living

organisms in which GBPs have not been found. Excluding glycan-specific

antibodies, it is possible to classify GBPs broadly into two major groups, lectins

and glycosaminoglycan-binding proteins. Most lectins are members of families

with defined “carbohydrate-recognition domains” (CRDs) that has evolved from

shared ancestral genes, often retaining specific features of primary amino acid

sequence or three-dimensional structure. Thus, new family members can be

identified by searching protein sequence or structural databases. Lectins

recognize specific terminal glycan chains by fitting into shallow, but relatively

well-defined, binding domains. A variety of biological processes such as

fertilization, immune defense, viral replication, parasitic infection, cell-matrix

interaction and cell-cell adhesion are known to involve lectins [Sharon 2007;

Goldstein et al. 1980; Varki 2009].

1.2. Lectins

Lectins were first discovered more than 100 years ago in plants, but they are

now known to be present throughout nature. Lectins are also prevalent in the

microbial world, wherein they tend to be called by other names, such as

2

hemagglutinins and adhesins. These agglutinins were renamed as “lectins,” a

term derived from the Latin word “legere,” meaning “to select.” The definition

of lectins has been improving focused on the carbohydrate-binding properties for

several years. The most recent accepted definition establishes lectins as proteins

with at least one non-catalytic domain able to recognize and bind reversibly to

specific mono and oligosaccharides. Based on this carbohydrate binding

property, lectins are defined as “Carbohydrate binding proteins of non-immune

origin that agglutinate cells and glycoconjugates and exhibit a specific and

reversible non covalent binding activity to carbohydrates and sugar containing

substances whether free in solution or on cell surfaces without altering covalent

structure of any glycosyl ligand”[Beuth et al. 1995].

1.3. Lectin detection and assay

There are different methods to detect the presence of lectin activity.

Hemagglutination assay is one of the commonly used methods to detect the

presence of lectins [Liener and Hill 1953; Ozeki et al.1991]. Briefly the method

involves serial dilution of the lectin before incubation with human or other

animal erythrocytes. Additionally, to increase the sensitivity of the cells to lectin

agglutination, an enzymatic (trypsin, papain or neuraminidase) or a chemical

(glutaraldehyde or formaldehyde) treatment can be performed [Lee et al. 1990;

Ozeki et al. 1991]. Although the hemagglutinating assay is rapid, sensitive, semi

quantitative and simple, it suffers from several disadvantages like, it will not

detect monovalent lectin and may give false positive results due to nonspecific

agglutination of cells caused by lipids [Tsivion and Sharon 1981] or by

3

polyphenols such as tannins that are often present in plant tissues. Other methods

can also be used to identify lectin activity, such as precipitation of

polysaccharides or glycoproteins [Shibuya et al.1989].

1.4. General Classification of lectins

Lectins are classified in to different categories on the basis of structural and/or

evolutionary sequence similarities. In this type of classification the source of the

lectins, carbohydrate recognition domain has not been considered. Some of the

lectins for which the single letter code is not been accepted and hence need to

take the consensus from those scientists who study each respective family.

Therefore suggested single letter code has been given by Varki et al [2009] and

are presented in Table 1.

Table 1: General classification of lectins

a. Defined lectin families with single letter code

C-type lectins (e.g., calcium-dependent lectins such as selectins, collectins, etc.)

L-type lectins, plant legume seed lectins, ERGIC-53 in ER-Golgi pathway, calnexin family

R-type (e.g., ricin, other plant lectins, GalNAc-SO4 receptors)

M-type lectins—α-mannosidase-related lectins (e.g., EDEM)

N-type lectins- Lectin nucleotide phosphohydrolases (LNPs) with glycan-binding and apyrase domains

P-type (i.e., mannose-6-phosphate receptors)

Galectins (formerly S-type lectins) Galactose binding lectins

I-type lectins—immunoglobulin superfamily members, including the Siglec family

4

b. Defined lectin families with suggested single letter code

β-prism lectins -Jacalin-related (B-type?)

Eel fucolectins (E-type?)

Ficolins- fibrinogen/collagen-domain-containing lectins (F-type?)

Garlic and Snowdrop lectins and related proteins (G-type?)

Hyaluronan-binding proteins or hyaladherins (H-type?)

Amoeba lectins—Jacob and related chitin-binding proteins (J-type?)

Tachylectins from horseshoe crab Tachypleus tridentatus (T-type?)

Haevin-domain lectins (e.g., wheat germ agglutinin, haevin, etc.) (W-type?)

Xenopus egg lectins/eglectins (X-type?) The question marks (e.g., F-type? and X-type?) indicate suggested names for other families. Final acceptance of the latter terms will require a consensus among those scientists who study each respective family

Lectins are also classified based on the source, carbohydrate specificity

and the details are discussed elsewhere in the thesis.

1.5. Occurrence and biological significance of lectins

Lectins are biologically active proteins that are of universal occurrence

and have been isolated from humans, animals, plants, microorganisms and also

from fungi. They constitute a diverse group of proteins that specifically bind

different types of carbohydrates. The wide spread occurrence of lectins suggest

their role in various biological functions [Varki 1999].

1.5.1. Plant lectins

Lectins from plant sources were the first proteins of this class to be

studied and to date most of the lectins studied so far are mainly from plant

sources. Since the discovery of the first lectin from castor bean by Stillmark in

5

1888, many lectins from almost all parts of plants have been reported [Etzler

1986; Goldstein and Poretz 1986].

Although numerous plant lectins have been studied for their great

structural detail, the physiological role of these proteins is still poorly

understood. However there are many speculated roles for plant lectins like; ‘as

storage proteins’, ‘as defense molecules’, in symbiosis etc. A number of lectins

have been isolated from storage tissues in plants (seeds or vegetative storage

tissues) where they make for a very large proportion of the total protein content

in the tissue, it has been speculated that lectins might serve as plant storage

proteins and many of these lectins also exhibit behavior similar to other storage

proteins. For example, they are developmentally regulated in a manner very

similar to other storage proteins and, during germination some of these lectins

are degraded and appear to be important sources of nitrogen for the development

process [Dannenhoffer et al. 1997; Van Damme et al. 1995]. Some plant lectins

have been implicated in defense mechanism of plants [Etzler 1986], lectins may

protect plants against bacterial [Jones 1964], fungal [Mirelman et al. 1975] and

viral [Partridge et al. 1976] pathogens during imbibitions, germination and early

growth of the seedlings.

Another proposed role for plant lectins is in fixing the atmospheric

nitrogen. Several plants, in particular from Leguminosae family, are known to

establish a symbiosis with soil bacteria of the genus Rhizobium and related

genera which are able to fix atmospheric nitrogen, rendering plants independent

of supply of external nitrogen [Rudiger and Gabius 2001].

6

Toxic lectins such as lectins from Ricinus communis and Phaseolus

vulgaris are regarded as protectants against animal predators. Galanthus nivalis

agglutinin (GNA) is another plant lectin which is shown to be toxic against

several insects. Transgenic plants of potato [Down et al. 1996; Gatehouse et al.

1997], rice [[Rao et al. 1998; Tinjuangjun et al. 2000; Maqbool et al. 2001] and

wheat [Stoger et al. 1999] containing the Galanthus nivalis agglutinin (GNA)

gene were shown to be resistant against different insects. The other speculated

roles for plant lectins include cell wall extension and recognition [Barre et al.

2001]. In contrast to the ‘classical’ plant lectins, which are typically found in

storage vacuoles or in the extra cellular compartment, it is now reported that,

plant lectins are also located in the cytoplasm and the nucleus. Based on these

observations the concept was developed that, lectin-mediated protein-

carbohydrate interactions in the cytoplasm and the nucleus play an important

role in the stress physiology of the plant cell and are known to be stress

inducible lectins [Lanno and Van Damme 2009].

1.5.2. Animal lectins

Lectin research was focused mainly on plant lectins for nearly hundred years

however the field of animal lectins is expanding rapidly only in the recent years

[Lis and Sharon 1986]. Animal lectins reported earliest are the lectins from horse

shoe crab [Marchalonis and Edelman 1968], snail [Hammarstorm and Kabat

1969], and eel [Springer and Desai 1971]. Animal lectins have been grouped into

classes based on the nature of their carbohydrate recognition domain, the

biological processes in which they participate, their sub cellular localization, and

7

their dependence on divalent cations. Based on these properties, animal lectins

are classified into eight groups as, C-type lectins, galectins, Siglecs, R-type

lectins, M-type lectins, L-type lectins, P-type lectins and Calnexins [Drickmer

and Tayler 1993] and these are summarized along with the representative

examples in Table 2 (adapted from Drickamer 2006)

Table 2: Classification of animal lectins

Lectin family

Typical saccharide

ligands

Sub cellular location Examples of functions

Calnexin Glc1Man9 ER Protein sorting in the endoplasmic reticulum.

M-type lectins Man8 ER ER-associated degradation of

glycoproteins. L-Type lectins Various ER, ERGIC,

Golgi Protein sorting in the endoplasmic reticulum.

P-type lectins

Man 6-phosphate, others

Secretory pathway

Protein sorting post-Golgi, glycoprotein trafficking, ER-associated degradation of glycoproteins, enzyme targeting.

C-type lectins Various Cell membrane,

extra cellular

Cell adhesion (selectins), glycoprotein clearance, innate immunity (collectins).

Galectins -Galactosides Cytoplasm, extra cellular

Glycan cross linking in the extra cellular matrix.

I-type lectins (Siglecs)

Sialic acid Cell membrane Cell adhesion.

R-type lectins Various Golgi, Cell

membrane Enzyme targeting, glycoprotein hormone turnover.

F-box lectins GlcNAc2 Cytoplasm Degradation of misfolded

glycoproteins.

Ficolins GlcNAc, GalNAc Cell membrane, extracellular Innate immunity.

Chitinase-like lectins

Chito-oligosaccharides Extracellular Collagen metabolism (YKL-40).

F-type lectins

Fuc-terminating oligosaccharides Extracellular Innate immunity.

Intelectins Gal, galactofuranose, pentoses

Extracellular/cell membrane

Innate immunity. Fertilization and embryogenesis.

8

Unlike plant lectins, the physiological functions of animal lectins are

clearly defined and shown that these molecules are diverse in structure as well as

function. Animal lectins mediate several important physiological functions such

as regulation of differentiation and organ formation [Sharon 1983], in metastasis

of cancer cells [Raz and Lotan 1987, Yu et al. 2010], mediating the phagocytosis

of microorganisms [Speer et al. 1988; Sharon and Lis 1989], in the migration of

lymphocytes from the blood stream to the lymphoid organs and also some are

known as antitumor and immunomodulatory molecules [Kawagishi et al. 1990;

Beuth et al. 1992; Wang et al. 1995]. Galectins are major class of animal lectins

and have been studied in detail from diverse sources.

1.5.2.1. Galectins

Galectins (formerly known as S-type animal lectins) are the members of a highly

evolutionarily conserved family of animal lectins widely distributed in the

animal kingdom. Galectins bind to β-galactoside by means of carbohydrate

recognition domain (CRD) that has many conserved sequences [Barondes et al.

1994]. All galectins share a core sequence consisting of about 130 amino acids,

many of which are highly conserved [Cooper et al. 2002; Vasta 2009].

The evolutionarily conserved galectin sequences, their wide tissue

distribution, marked developmental regulation and abundance in particular

tissues support their involvement in important biological processes. By virtue of

their multivalency, galectins are able to cross-link cell-surface glycoconjugates

and initiate biological responses. The function of a given galectin can vary from

9

site to site depending on the nature of available ligands [Hirabayashi and Kasai

1993].

Biochemical and functional properties of different members of the

galectin family are summarized in Table 3.

Table 3: Biochemical and functional properties of different members of the galectin family (Adopted from Gabriel et. al. 2002 with updated information on their functions)

Galectins Localization Biochemical and functional properties

Galectin-1 Abundant in most organs: muscle, heart, prostate, liver, lymph nodes, spleen, thymus, placenta, testis, retina, macrophages, B cells, T cells and tumors

Non-covalent homodimer Induces apoptosis of activated T cells and

immature thymocytes Induces polarized Th2 immune response Modulates cell-cell and cell-matrix

interactions Inhibits acute inflammation: blocks

arachidonic acid release, mast cell degranulation and neutrophil extravasation

Suppresses chronic inflammation and autoimmunity

Galectin-2 Stomach epithelial cells

Non-covalent homodimers Expressed at minor levels in tumor cells

Galectin-3 Mainly in tumor cells, macrophages, epithelial cells, fibroblasts, activated T-cells

Non lectin domain linked to a CRD Anti-apoptotic and pro-inflammatory

functions Modulates cell adhesion and migrations Induces chemotaxis of monocytes Potentiates pro-inflammatory (IL-1)

cytokine secretions Inhibits nitric oxide-induced apoptosis and

anoikis Down regulates IL-5 gene transcription

Galectin-4 Gastrointestinal tract Composed of two distinct CRDs in a single polypeptide chain

Expressed at sites of tumor cell adhesion

10

Galectin-5 Erythrocytes Proto type galectin: monomer No function assigned

Galectin-6 Gastrointestinal tract Composed of two distinct CRDs in a single polypeptide chain

Closely linked to galectin-4

Galectin-7 Skin Prototype galectin: monomer Used as a marker of stratified epithelium Demonstrated as pro-apoptotic molecule Increases susceptibility of keratinocytes to

UVB-induced apoptosis

Galectin-8 Liver, kidney, cardiac muscle, prostate and brain

Composed of two distinct CRDs in a single polypeptide chain

Modulates integrin interactions with extra cellular matrix

Galectin-9 Thymus, T cells, Kidney, Hodgkin’s lymphoma

Composed of Two distinct CRDs in a single polypeptide chain

Induces eosinophil chemotaxis Induces apoptosis of murine thymocytes

Galectin-10 Eosinophils and basophils

Prototype galectin: monomer Mainly expressed by eosinophils, formerly

called “Charcot-Leyden crystal protein”

Galectin-11 Lens Also called “GRIFIN” May represent a new lens of crystalline Lacks affinity for Beta-galactoside sugars

Galectin-12 Adipocytes Composed of Two distinct CRDs in a single polypeptide chain

Induces apoptosis and cell cycle arrest Galectin-13 Recently identified in

human placenta Similar to “pro-type galectins” Also called PP-13

Galectin 14 Eosinophils Regulating the activity of eosinophils during allergic responses

Galectin 15 Endometrial luminal epithelium (LE) and superficial ductalglandular epithelium (sGE) of the ovine uterus

Regulate implantation and placentation

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1.5.3. Bacterial lectins

Although scattered reports on the ability of bacteria to agglutinate erythrocytes

appeared in the literature during the first half of the 20thcentury, systematic

research on the bacterial hemagglutinins started only in the 1950’s, with the

work of Duguid and Brinton in England and USA respectively [Duguid and Old

1980; Sharon 1989]. Duguid and his co-workers showed that hemagglutinating

activity is a property expressed by many bacterial species, most commonly by

those belonging to the family of Enterobacteriaceae [Sharon 1987] but little

attention was paid to these findings. Moreover, the idea that sugar-specific

adhesion to host cells might be a prerequisite for bacterial colonization and

infection was not considered at all at that time. The first indication of lectin

mediated host-parasite interaction emerged when Ofek et al. found that E. coli

adheres readily to buccal epithelial cells and that this adhesion was inhibited

specifically by mannose and methyl mannoside [Ofek et al. 1977; Salit and

Gotschlich 1997]. Now it is well-established fact that majority of the infectious

bacteria including human oral pathogens produce surface lectins which are

referred to as adhesins and blocking of the bacterial lectins may prevent the

infections. In addition to their role in initiation of infection, the mannose-specific

bacterial surface lectins may also have a contradictory function in protection

against infectious agents [Wallis 2010]. A similar protective role was also seen

with the surface lectins of phagocytic cells such as granulocytes and

macrophages. Bacteria and yeasts may bind to these cells in the absence of

opsonins, leading to uptake and killing of the organisms. This phenomenon,

12

named by as “lectinophagocytosis” [Ofek et al. 1977], and is an early example of

innate immunity, in which lectins are now known to be involved.

1.5.4. Viral lectins

The influenza virus hemagglutinin was the first glycan binding protein isolated

from a microorganism (~1950), and it is now one of the most thoroughly studied

of all viral lectins, which is known for its involvement in initiating pathogenesis.

Like animal lectins, most viral lectins bind to terminal sugar residues, but some

can bind to internal sequences found in linear or branched glycans. The

specificity of these interactions can be highly selective. For example, the human

influenza viruses bind primarily to cells containing Siaα2-6Gal linkages,

whereas other animal and bird influenza viruses preferentially bind to Siaα2-

3Gal termini. Influenza C, in contrast, binds preferentially to glycoproteins

containing terminal 9-O-acetylated sialic acids. Many other viruses (e.g.,

reovirus, rotavirus, Sendai, and polyomavirus) also appear to use sialic acids in

specific linkages for infection. Other viruses display glycosaminoglycan-binding

proteins that can bind to heparan sulfate proteoglycans, often with high

specificity for certain sulfated sequences [Varki et al 2009].

1.5.5. Fungal lectins

Although extensive literature is available with plant and animal lectins, very

little information is available on lectins from fungi [Gulliot and Konaska 1997;

Wang et al. 1998]. The occurrence of lectins in fungi was known as early as

1907, when Ford demonstrated strong hemagglutinating activity in the extracts

of Amanita solitaria [Ford 1907] and also in 40 species of Agaricaceae [Ford

13

1911]. In recent past fungal lectins have been receiving greater attention due to

their interesting sugar specificities and the biological activities possessed, giving

rise to a wide range of potential pharmacological and biotechnological

applications [Ng 2004; Konaska 2006; Khan and Khan 2011]. Fungal lectins

have been found in fruiting bodies, and purified from them, but very few have

been identified in vegetative mycelia. Mushroom lectins have been localized on

the caps; stipes and mycelia of mushrooms and variation in lectin content occur

depending on the age, time and place of harvest [Ng 2004].

The functional roles assigned for fungal lectins are speculative. Many

believe that fungal lectins do mediate host-parasite interactions [Rudiger 1998]

similar to bacterial adhesins. Several other roles are also put forth, mainly based

on the source selected for isolation and on the location of the lectin in the fungus

[Barak and Chet 1990; Elad et al. 1983; Inbar and Chet 1994; Kellens and

Peumans 1990]. Some of the roles assigned to fungal lectins are as storage

proteins [Kellens and Peumans 1990], fungal-fungal interactions

(mycoparasitism), and host parasite interactions [Fukazawa and Kagawa 1997;

Hostetter 1994; Rudiger 1998]. Another function gaining greater attention is the

involvement of fungal lectins in morphogenesis and development of the fungus

[Yatohgo et al. 1988; Cooper et al. 1997; Swamy et al. 2004; Li and Rollins

2010]. The physiological role of fungal lectins include participation in the

process of fruiting body formation, the creation of mycelium structures easing

the penetration of parasitic fungi into the host organism and identification of

appropriate partners during the early stage of mycorrhization [Konaska 2006].

14

There are recent reports on lectins which have been isolated from mycelia and

sclerotial bodies of the fungi. Lectin activity has been reported from the

sclerotial bodies of the fungus, Sclerotium rolfsii, and its functional role has been

demonstrated in the development and growth of the fungus by identifying its

putative endogenous receptor [Swamy et al. 2001, 2004]. Pleurotus cornucopiae

contains a developmental stage-specific mycelial lectin, and known to

participates in the process of fruiting body formation [Oguri et al. 1996]. Lectin

from Rhizopus stolonifer, RSL, is known to be produced during the development

of the fungus and is involved in the spore formation [Oda et al. 2003]. Cooper

and Barondes demonstrated the production of two different lectins by

Dictyostelium discoideum that were developmentally regulated [Barondes et al.

1985].

In the recent past fungal lectins are drawing greater attention due to their

biological activities such as lymphomitogenic effect, immunomodulatory

properties, suppression of cell proliferation and antitumor activity. Fungal lectins

have also found application in the isolation of glycoconjugates and elucidation

of changes occurring on the cell surface at various stages of physiological and

pathological development [Konaska 2006]. Following table represents some of

the recent reports on the fungal lectins and their physico chemical properties

(adapted from Khan and Khan 2011).

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Table 4: Fungal lectins

Source Molecular Mass (kDa)

Sub-unit type

pI Carbohydra

te content (%)

Specificity References

Agaricus blazei 70 α2 11.0 Glycoproteins Kawagishi et al. (1988)

Arthrobotrys aligospora

36 α2 6.5 Rosen et al. (1992)

Auricularia polytricha 23 α2 10.6 3.5 Yagi and Tadera (1988)

Amanita pantherina 43 α2 4.3 Mucin Zhauang et al. (1996)

Beauveria bassiana 15 α 7.1 12.6 Glycoprotein Kossowska et al. ( 1989)

Chlophyllum molybdites

32 α2 3.75 12 Kobayashi et al. (2004)

Clitocybe nebularis 33 α2 4.3 Asialo fetuin and Lactose

Pohlven et al. (2009)

Clitocybe nebularis 30 α2 4.3 10 D-Galctose Horejsi and Kocourek (1978)

Fusarium solani 26 α2 8.7 3.9 Glycoproteins Khan et al (2007)

Hygrophorus hypothejus

68 α2 5 00 Veau et al. (1989)

Ischnoderma resinosum

32 α2 5.5 4 Kawagishi and Mizuno (1988)

Laccaria amethystea 16 α 9.5 00 L-fucose, Lactose Guillot et al. (1983)

Lactarius deliciosus 37 αβ 6.7 00 Gal β1-3GalNAc Guillot et al. (1991)

Lactarius lignyotus 100 α4 4 Giollant et al. (1993)

Macrophomina phaseolina

34 α 16.4 N-acetylneuraminyl N-acetyllactosamine

Bhowal et al. (2005)

Peziza sylvestris 20 α Arabinose Wang an Ng (2005)

Pleurotus ostreatus 72 αβ Wang et al (2000)

Pleurocybella porrigens

56 α4 5.7 2.8 Asilo bovine submaxillary mucin

Suzuki et al (2009)

Rhizoctonia solani 31 α2 >9 Candy et al. (2001) Vranken et al (1987)

Rhizopus stolanifera 28 α5 Oda et al. (2005) Xerochomus spadiceus 32 α2 Liu et al. (2004) Schizophyllum commune

31.5 Lactose and N-acetyl-D-

Galactoseamine

Chumkhunthod et al. (2006)

Ganoderma lucidum 114 α5 9.3 Glycoprotens Thakur et al. (2007)

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1.6. Specific recognition of glycans by lectins

Glycans didn’t receive the greater attention compared to proteins, nucleic acids

and lipids till recently as they are highly complex and are not encoded in the

genome. However in the last few decades the study on the carbohydrates is

gaining momentum through the expanding field of glycobiology. Glycobiology

is an emerging field of science that tries to understand structure and functions of

glycans, and enzymes involved in the synthesis, degradation of glycans, and the

lectins involved in decoding the information coded in the glycan structure.

Glycans constitute a significant amount of the mass and structural variation in

biological systems. Carbohydrates are by far the most abundant organic

molecules found in nature, and nearly all organisms synthesize and metabolize

carbohydrates. Glycosylation is one of the most frequently occurring post-

translational modifications of proteins. The presence of an oligosaccharide

moiety in soluble and membrane bound proteins improves their solubility in

water, contributes to the proper orientation of the molecule, protects it from the

action of proteases and in some cases, it is also required for efficient intracellular

transport. Considering these important functions of glycans, the proteins that

interact with these glycans (glycan binding proteins) are becoming important

molecules to decipher the glycocode. Lectins, a well-known class of glycan

binding proteins is now being used for the identification of specific glycans that

are expressed either as a part of proteins/lipids or found in extra cellular matrix.

In order to understand the interaction of lectins with glycans and their function it

is necessary to understand the basics of glycans and their specific

alteration/expression in many pathophysiological conditions.

1.7. Glycosylation

Glycosylation is known to occur on proteins, lipids and as polymers in

extracellular matrix. Glycans bound to proteins are divided in to three well

17

defined categories; N-glycnas, O-glycans, and glycosaminoglycans (frequently

termed proteoglycans). N-glycans are linked to aspargine residues of proteins,

specifically a subset residing in the Asn-X-Ser/Thr motif, where X denotes any

amino acid except proline. N-acetylglucosamine is frequently observed first

sugar to be attached to proteins through amide linkage with Asn. O-glycans are

attached to a subset of serines and threonines, where N-acetylgalactosamine is

commonly observed as initiator glycan attached to Ser/Thr [Schachter 2000; Yan

and Lennarz 2005]. Although glycosaminoglycans are also linked to serine and

threonine, they are linear, produced by different biosynthetic pathways, and are

often highly sulfated. Glycosylation of lipids is also a prevalent modification

which creates glycolipids. Mainly sialic acid-bearing gangliosides are important

class of glycolipids. Glycosylphosphatidylinositol (GPI)-linked proteins share a

common membrane-bound glycolipid linkage structure that is attached to

various proteins. Less common types of protein glycosylation also occur, for

example, on lysine, tryptophan, and tyrosine residues of specific proteins, such

as glycogenin, which was the first identified glycoprotein. There are two

enzymes, acetyltransferase and sulfotransferase which are actually not involved

in glycosylation but frequently attach acetyl and sulfate groups to selected

saccharides residing on some oligosaccharide chains and can thereby modulate

glycan structure and function [Klein and Roussel 1998; Fukuda et al., 2001].

1.7.1. Symbolic representation of common monosaccharides

The monosaccharides most commonly known are given with symbolic

representation using different auto shapes like, round, square, diamond etc., with

specific color combinations. Following Figure 1 shows the representation of

common monosaccharides

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Fig. 1. Symbolic representation of common Monosaccharides [adapted from

Varki et al. 2009]

Glycosylation of the proteins and lipids in mammals starts with the

addition of a glycan through the nucleotide sugar donor involving a specific

glycosyltransferase. There are nine nucleotide sugar donors, six amino acid

acceptors motifs and two lipid acceptors creates a total of 14 different glycans in

stereo isomeric configurations (α or β) linked at the number 1 position. Once the

first glycan is added to the protein or lipid acceptors, the first glycan is further

elongated by the addition of nine different glycans catalyzed by forty nine

specific glycosyltransferases. This results in glycosidic bonds with α or β

configurations of the donor saccharide linked through position 1 or 2 to position

2, 3, 4, or 6 of an acceptor saccharide. The naturally occurring specific linkage,

sugar donor and acceptor, protein and lipid acceptors are tabulated in table 5

[Ohtsubo and Marth 2005].

Galactose (Gal)

N-Acetylgalactosamine (GalNAc)

Galactosamine (GalN)

Glucose (Glc)

N-Acetylglucosamine (GlcNAc)

Glucosamine (GlcN)

Mannose (Man)

N-Acetylmannosamine (ManNAc)

Xylose (Xyl)

N-Acetylneuraminic acid (Neu5Ac)

N-Glycolylneuraminic acid (Neu5Gc)

Fucose (Fuc)

Glucuronic acid (GlcA)

Iduronic acid (IdoA)

Galacturonic acid (GalA)

Mannuronic acid (ManA) Mannosamine (ManNAc)

19

Table 5: Mammalian glycan linkages produced by glycosylation

α1

-

-

-

-

-

-

-

α1-2

-

-

α1-3 α1-4 α1-6

-

-

-

-

-

-

β1

-

-

β1

-

-

α1-3 α1-4 β1-3

β1-3

β1-4

β1-3 β1-4

-

-

-

β1-4

α1

-

-

-

-

-

-

-

α1-3 β1-3 β1-4

α1-3 α1-6

-

β1-4

β1-4

-

-

-

β1

β1

-

-

α1

β1

-

β1-3

α1-2

-

α1-2 α1-3

-

-

α1-3

-

-

β1

*

-

-

-

-

α1

β1-3

β1-3 β1-6

β1-6

-

α1-6 β1-4

α1-4 β1-4

β1-2

-

-

-

-

-

-

-

-

-

-

β1-3 β1-4

β1-3

-

β1-3 β1-4

-

-

-

-

α1

-

-

α1

-

-

-

-

-

-

-

α1-4 β1-4

-

α1-2 α1-3 α1-6

-

-

-

-

-

-

-

-

-

-

α2-3 α2-6

α2-6

-

-

-

-

α2-8

-

β1

-

-

-

-

-

-

-

-

-

α1-3

-

-

-

-

α1-3

* N-glycosylation is initiated by transfer en bloc of a presynthesized dolichol lipid linked oligosaccharide precursor

SACCHARIDE ACCEPTORS PROTEINS AND LIPID ACCEPTORS

Ser/ Thr Asn hLys Trp Tyr Cer PI

20

1.7.2. N-glycans

N-glycans are covalently attached to protein at asparagine (Asn) residues by an

N-glycosidic bond. Five different N-glycan linkages have been reported, of

which N-acetylglucosamine to asparagine (GlcNAcβ1-Asn) is the most

common. N-linked glycosylation in eukaryotes is initiated by the covalent

addition of a oligosaccharide precursor with 14 monomers (2N-

acetylglucosamine, 9 mannose and 3 glucose- Dolichol phosphate – Fig. 2) to

the aspargine residue of the target polypeptide chain (core protein) as the newly

synthesized polypeptide chain is translocated into the ER.

Fig. 2. Structure of Dolichol phosphate

This 14 carbohydrate common precursor gives rise to three major classes

of N-linked oligosaccharides: (1) high-mannose oligosaccharides, (2) complex

oligosaccharides and (3) hybrid oligosaccharides. All these glycans share a

common core structure; pentasaccharide core with three mannose and two

GlcNAc residues. N-linked glycosylation is required for the proper folding of

some eukaryotic proteins in the ER. Three glucose residues are removed from

the precursor N-linked oligosaccharide of the correctly folded protein and the

glycoprotein is then exported from the ER to the Golgi apparatus. In the Golgi

21

apparatus, mannose residues may be removed and other monosaccharides (e.g.

N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose and sialic acid)

may be added in their place to elongate the N-linked oligosaccharides. These

carbohydrate residue modifications in the golgi apparatus provide the means by

which complex and hybrid N-linked oligosaccharides are synthesized. A protein

may potentially be glycosylated by all three major classes of N-linked

oligosaccharides or only two/one. Most of the N-glycans share a common core

sugar sequence, Manα1–6(Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn-X-

Ser/Thr and depending upon further elongation with different glycans gives rise

to three important classes: (1) oligomannose, in which only mannose residues

are attached to the core; (2) complex, in which “antennae” initiated by N-

acetylglucosamine attached to the core; and (3) hybrid, in which Manα1–6 is

extended by two mannose residues where as Manα1–3 is extended by addition of

N-acetyl glucosamine and further elongation with different monosaccharides

[Ruddock and Molinari 2006; Aebi et al. 2010]. The representative examples are

given in Fig. 3.

Fig. 3. Three classes of N-glycans

22

Protein glycosylation in particular N-linked glycosylation is prevalent in

proteins destined for extra cellular environments. These include proteins on the

extra cellular side of the plasma membranes, secreted proteins and the proteins

contained in the body fluids. These also include the proteins that are most easily

accessible for diagnostic and therapeutic purpose. Hence, glycoproteins with N-

linked glycans are best clinical markers and therapeutic targets.

1.7.3. O-Glycans

O-linked glycosylation is a modification of proteins that is most likely catalyzed

in the golgi apparatus as a post translation event. In O-linked glycans, the C-1 of

N-acetylgalactosamine is covalently bonded to the hydroxyl of serine or

threonine of the target polypeptide chain (core protein). These structures are

usually referred as mucin glycans. Once the N-acetylgalactosamine residue has

been added, the elongation of the O-linked oligosaccharides may then proceed

by the addition of other carbohydrate residues such as galactose, fucose, N-

acetylglucosamine and sialic acid [Schachter 2000; Yan and Lennarz 2005;

Mitra et al. 2006]. The addition of first sugar GalNAc to serine or threonine

results in Tn antigen, which can be further sialylated by addition of sialic acid to

form sialyl Tn and addition of GalNAc results in formation of Core 1 structure

(T antigen). The eight different core structures in O-linked glycans have been

identified and are known as mucin type core structures [Hounsell 1996]. The

structures of these glycans are given in the following Table 6.

23

Table 6: Core structures of O-glycans

O-Glycan Structure Core Tn antigen GalNAcαSer/Thr

Sialyl-Tn antigen Siaα2-6GalNAcαSer/Thr

Core 1 or T antigen Galβ1-3GalNAcαSer/Thr

Core 2 GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr

Core 3 GlcNAcβ1-3 GalNAcαSer/Thr

Core 4 GlcNAcβ1-6(GlcNAcβ1-3)

GalNAcαSer/Thr

Core 5 GalNAcα1-3 GalNAcαSer/Thr

Core 6 GlcNAcβ1-6 GalNAcαSer/Thr

Core 7 GalNAcα1-6 GalNAcαSer/Thr

Core 8 Galα1-3 GalNAcαSer/Thr

There are also several types of non mucin O-glycans, including α-linked

O-fucose, β-linked O-xylose, α-linked O-mannose, β-linked O-GlcNAc (N-

acetylglucosamine), α- or β-linked O-galactose, and α- or β-linked O-glucose

glycans are identified which have specific functional roles. O-linked

oligosaccharides are simple when compared to N-linked oligosaccharides and

are involved in various functions such as, leukocyte circulation involving

selectin, fertilization and clearance of glycoprotein etc. They are also involved in

immunological recognition of antigens and signal transduction [Chapman et al.

1996; Kodama et al. 1993; Springer 1994; Kojima et al. 1994; Drickamer 1991].

1.7.4. Glycosylation and cancer

Development of tumor in humans is a multistep process and these steps reflect

alterations that drive the progressive transformation of normal human cells into

24

highly malignant tissue. Cancer cells are known to have defects in regulatory

circuits that govern normal cell proliferation and homeostasis. There are more

than 100 distinct types of cancer, and subtypes of tumors known to be identified

within specific organs. The vast amount of research on cancer continues to

provide evidences for the specific reasons for transformation of normal cells.

According to D. Hanahan and R. A. Weinberg [2000] the vast catalog of cancer

cell genotypes is a manifestation of six essential alterations in cell physiology

that collectively dictate malignant growth. They are; self-sufficiency in growth

signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of

programmed cell death (apoptosis), limitless replicative potential, sustained

angiogenesis, and tissue invasion and metastasis. Each of these physiological

changes acquired during the development provides the successful escape of an

anticancer defense mechanism present in the normal cells and tissue.

Successful entry of a normal cell form its quiescent state to a proliferating

state require external mitogenic growth signals which are transmitted by the cell

membrane receptors or by diffusible growth factors. Tumors cell with the ability

to generate own growth signals reduces their dependence on such external

growth stimulatory factors and become self sufficient to proliferate in an

uncontrolled manner. In order to maintain the tissue homeostasis and cellular

quiescence, antigrowth signals are the critical factors for the normal cell cycle.

Soluble growth inhibitors or embedded extracellular signals inhibit the growth of

normal cells according to the requirement of the tissue microenvironment. These

growth inhibitory signals are known to involve specific pathway mediated by

25

retinoblastoma protein (pRb), p107 and p130. Disruption of these important

pathways makes the tumor cells insensitive to the antigrowth signals.

The defects in any molecular machinery of normal cell will trigger the

activation programmed cell death. Due to the mutation in the proapoptotic genes

and production of excess antiapoptotic molecules provides the resistance to

tumor cell apoptosis, resulting in uncontrolled tumor growth. Proliferative

potential of a tumor cell is also dependent on the persistence of the telomere

nucleotide sequence. Due to the increased expression of the telomerase enzyme,

which adds hexanucleotide repeats onto the ends of telomeric DNA, tumor cell

can replicate in a limitless manner. Such limitless growth of tumor cells needs

excess nutrients and oxygen to proliferate. The growth of normal vascular

system, which supplies the required nutrients to cells, is maintained by the

balanced expression of angiogenesis inducers and inhibitors. The decrease of the

angiogenesis inhibitors in tumor cells makes the sustained angiogenesis to

provide the required nutrients and oxygen. Once tumor acquires the above said

molecular changes to grow as a macroscopic primary tumor, then tumor cells

loses the cell to cell contact and invade the adjacent tissue to reach the vascular

system. The expression of high levels of mucins on the plasma membrane and

decreased expression of cell-cell adhesion molecules (CAM) on the tumors cells

makes them to release from the primary site. Through the successful crossover

of different steps like, invasion, increased adherence, survival in the vascular

system, extravasations, acquiring the secondary site cellular characters and the

limitless growth makes the primary tumor cells into a metastatic, lethal

26

malignant tumors at different places of the body. The involvement of these six

hallmarks for the successful development of malignant tumor is common in all

types of cancer [Hanahan and Weinberg 2000].

1.7.5. Glycosylation changes in cancer

The tumor cells undergo activation and rapid growth, adhere to a variety of other

cell types and cell matrices, and invade tissues similar to normal cells during

embryogenesis. Embryonic development and cellular activation in vertebrates

are typically accompanied by changes in cellular glycosylation profiles. Thus, it

is not surprising that, glycosylation changes are also universal feature of

malignant transformation and tumor progression. The earliest evidence came

from observation that, plant lectins (e.g., wheat germ agglutinin) showed

enhanced binding and agglutination of tumor cells [Raedler and Schreiber 1988]

reflecting the altered glycosyaltion. Several glycans, on both the tumour surface

and host elements, have now been identified as mediating key

pathophysiological events during the various steps of tumour progression.

Changes in the glycosylation in a tumor microenvironment allow neoplastic cells

to usurp many of the events that occur in development (for example, receptor

activation, cell adhesion and cell motility), which allows tumour cells to invade

and spread throughout the organism [Varki 2009]. These changes in the

glycosylation are known to express many tumor specific markers. Many of the

first-identified tumour-specific antibodies were directed against carbohydrate

oncofetal antigens presented on tumour glycoproteins and glycosphingolipids

[Feizi 1985]. In some cases, the under expression, truncation or altered

27

branching patterns of certain glycans correlate with cell growth. Following are

some of the examples of altered glycans commonly observed during cancer.

a. Increased β1–6 branching of N-glycans. The excessive branching of the

N-glycans is commonly observed during cancer due to the enhanced

expression of UDP-GlcNAc:N-glycan GlcNAc transferase V (GlcNAcT-

V) and UDP-GlcNAc:N-glycan GlcNAc transferase III (GlcNAcT-III).

These enzymes catalyses the addition of bisecting GlcNAc branch which

involves the addition of GlcNAc residue through β1–6 linkage to the

mannose residue. This increased branching is correlated with increased

frequency of tumor cell metastasis. The β1–6-GlcNAc branched N-

glycans are tri-or tetra-antenna oligosaccharides that increase the total cell

surface terminal sialylation in malignant cells, which prevents further

chain elongation. These observations are typically seen in the initial

stages of carcinogenesis induced by some of the oncogenic viruses and

also by oncogenes [Dennis et al. 1987; Gorelik et al. 2001; Ghazarian et

al. 2011]. Such terminal sialylations of β1–6 branched N-glycans are also

shown to be involved in the adhesion and motility of melanoma cells

[Reddy and Kalraiya 2006]. These oligosaccharides are invariably found

on invading trophoblasts, activated granulocytes and endothelial cells

[Granovsky et al. 1995; Pili et al. 1995; Tomiie et al. 2005; Yagel et al.

1990], and highly invasive glioma cells [Yamamoto et al. 2000;

Fernandes et al. 1991; Takano et al. 1990]. It is observed that the

invasiveness and metastatic ability of the cells will be lost when the

28

expression of β1–6 branched N-glycans is inhibited [Humphries et al.

1986; Krishnan et al. 2005]. Representative images for β1–6 branched N-

glycans are shown in the following figure 4.

Fig. 4. Examples of β1–6 branched N-glycans

b. Altered expression of mucin glycans: Mucins are the heavily

glycosylated high molecular weight proteins with a “rod-like”

conformation. Mucins are known to have conserved tandem repeat

sequences of serine and threonine which are the sites of O-glycosylation.

These mucins are known to express several tumor associated antigens

(TAAs), which are originally found as oligosaccharide structures. One of

the common consequence is altered glycosyaltion of O-glycans in mucins

is the expression of T (Galβ1–3GalNAc-α1-O-Ser/Thr) and Tn (GalNAc-

α1-O-Ser/Thr) antigens. These antigens are known to be expressed in

more than 90 % of human carcinomas [Yu 2007]. Antibodies directed

against TAAs on mucins are widely used clinically as diagnostic tools

(serum assays) for different types of cancers. To name few examples, the

monoclonal antibody (mAb) CA19-9, which recognizes NeuNAcα2-

β1-6 β1-6 β1-6

29

3Galβ1-3GlcNAc β1-3(Fucα1-4)Gal…, for colorectal and pancreatic

adenocarcinomas; DuPan2, which is produced against pancreatic tumor

cells HPAF, for pancreatic adenocarcinoma; mAb OC125, which

recognizes the CA125 antigen, for ovarian carcinomas; and mAb B72.3,

which recognizes human tumor associated antigen 72 (TAA-72), for

several different types of adenocarcinomas. Increasing concentrations of

mucin-type glycoproteins in serum are correlated with increasing tumour

burden and poor prognosis. It is observed that the over expression,

inappropriate expression or expression of aberrant forms of mucins

contribute to the pathogenesis of cancer [Hollingsworth and Swanson

2004].

c. Sialylated Lewis structures. Sialyl Lewisx and sialyl Lewisa were the

first identified tumor antigens. Over expression of Lewisx and Lewisa

structures on O-glycans as well as on N-glycans and glycosphingolipids

are frequently seen in many carcinomas as evidenced by immuno

histochemical studies. The expression of these antigens by epithelial

carcinomas correlates with tumor progression, metastatic spread, poor

prognosis in humans, and metastatic potential in mice. The sialylated

structures also form critical components of most natural ligands for the

endogenous selectins [Powlesland et al. 2009; Goetz et al. 2009;

Ghazarian et al. 2011]. Representative images are shown in the following

figure 5.

30

Fig. 5. Sialyl Lewis antigens

d. Core fucosylated N-glycans. Fucosylation is one of the most common

modifications involving oligosaccharides on glycoproteins or glycolipids.

Fucosylation comprises the attachment of a fucose residue to N-glycans,

O-glycans and glycolipids. Fucosylation is one of the most important

types of glycosyaltion observed in cancer. Several proteins with the core

fucosylation are known to be gastrointestinal cancer markers. Alfa

fetoprotein-L3 (APF-L3), fucosylated hepatoglobulin, and fucosylated α-

1-antitrypsin (Fc-AAT) are the important hepatocellular markers which

are currently in clinical use [Miyoshi 2012]. These markers are known to

express specifically core fucosylated glycans. Representative images are

shown in the following figure 6.

Fig. 6. Core fucosylated N-glycans

Sialyl Lewis a Sialyl Lewis x

31

Apart from the above said examples, there are several other glycan

changes that are known to contribute for cancer progression. Such glycans are

becoming important diagnostic and therapeutic targets. Following table

summarizes the importance of N- and O-glycans, their proposed functions and

their possible use as therapeutic targets at different stages of cancer [Fuster and

Esko 2005].

Table 7: Examples of glycan families involved in tumour progression

Glycans Involved

Proposed Major function (s)

Possible therapeutic targeting

Examples of neoplasms References

a. Growth and proliferation

N-glycans Suppresses apoptosis; growth-factor signalling

Alkaloid inhibitors for N-linked glycan processing

Breast, melanoma, Ewing’s sarcoma

Girnita et al 2000 Komatsu et al 2001

O-glycans

Mucin (MUC4)-mediated activation of ERBB2 receptors

Immunotherapy targeting MUC4 (similar to other mucin-targeting immunotherapy)

Breast Komatsu et al 2001

O-glycans Suppress apoptosis (possibly through galectin-3 binding to tumour O-glycans expressing terminal galactose)

Galectin-3 inhibitors (β-galactosides)

Colon, pancreatic

Takenaka 2004

b. Tumor Invasion

N-glycans

Alter E-cadherin-dependent tumour adhesion

Alkaloid inhibitors of N-glycan processing

Breast, colon Yoshimura et al 1996 Granovsky et al 2000

32

N-glycans

Tumour repulsion (for example, polysialylation)

Sialyltransferase inhibitors

Neuroblastoma, lung (small cell)

Seidenfaden et al 2003

O-glycans

Stimulate migration; potentiate migration of tumour cells through inhibition of cell–cell contacts (for example, sialyl Tn on mucins)

Vaccines (for example, conjugated sialyl Tn)

Breast, gastric, ovarian

Julien et al 2001

c. Tumour metastasis

O-glycans

Facilitate tumour adhesion during haematogenous metastasis (sLex, sLea and other selectin ligands);

Disaccharide primers of glycosylation (reduce tumour sLex); competition by intravenous heparin

Colon Borsig et al 2002 Fuster et al 2003 Varki et al 2002

N-linked and O-linked glycans

Promote tumour aggregation (galectin-3 binding)

Galectin-3 inhibitors (β-galactosides)

Melanoma Takenaka et al 2004

d. Tumour angiogenesis

N-glycans Promote migration of endothelia

Alkaloid inhibitors of N-linked glycosylation

Prostate Pili et al 1995

A growing body of evidence supports crucial roles for glycans at various

pathophysiological steps of tumour progression. Glycans regulate tumour

proliferation, invasion, haematogenous metastasis and angiogenesis. The

33

detailed understanding of roles of these glycans helps for developing

pharmaceutical agents that target these molecules. Such novel agents might be

used alone or in combination with operative and/or chemo radiation strategies

for treating cancer [Fuster and Esko 2005].

The present study is mainly focused on interaction of two of the lectins

with human ovarian cancer, in view of this, it is essential to understand the

present status of the ovarian cancer, altered glycosylation in ovarian cancer, and

its diagnostic and therapeutic targets.

1.8. Ovarian cancer

Ovarian cancer is the seventh most common cancer diagnosed among women in

the world and the fifth most common cancer diagnosed among women in more

developed regions. A total of 224,747 new cases of ovarian cancer were reported

worldwide in 2008 and 140,200 cancer deaths were observed. Most of the

ovarian cancer patients were diagnosed at advanced stages (stage III and IV) due

to its asymptotic nature at early stages. The late stage diagnosis of this cancer

makes the increased ovarian cancer deaths [Permuth-Wey and Sellers 2009].

Mainly there are three types of ovarian cancer depending upon the origin

of cell type; epithelial, gonadal-stromal and germ cell tissue. Among these tissue

origins, epithelial cancer constitutes for the 90% of ovarian cancers, and

gonadal-stromal and germ cell cancer accounts for 6 and 4 % respectively

[Holschneider and Berek 2000]. The epithelial ovarian cancer is divided into

four subtypes; serous (fallopian tube-like), endometrioid (endometrium-like),

mucinous (endocervical-like), and clear cell (mesonephros-like) [Auersperg et

34

al. 2001]. The serous subtype is the most commonly diagnosed and is

responsible for the majority of ovarian cancer deaths [Jemal et al. 2011]. Most of

the ovarian cancers are known to have mutated BRCA1 and/or BRCA2 genes

which are also implicated in hereditary breast cancer. These genes in normal cell

act as tumor suppressors and regulate cellular proliferation and DNA repair by

maintaining chromosome integrity [Antoniou et al. 2003].

A number of epithelial ovarian cancer markers have been studied

recently, and most extensively researched is CA125, a large mucin glycoprotein.

CA125 is the gold standard tumor marker in ovarian cancer and serum level of

CA125 is used to monitor response to chemotherapy, relapse, and disease

progression in ovarian cancer patients. CA125 levels of less than 35 U/mL are

now accepted as normal and expression above this level will be used for the

detection ovarian cancer [Gupta and Lis 2009]. Mucins are the promising,

potential tumor markers, studied in detail as diagnostic and therapeutic targets in

ovarian cancer. Stage specific biomarkers for ovarian carcinoma are

characterized in ascitic fluid, serum, urine and on tissues. The promising

biomarkers like; human epididymis protein-4 (HE4), decoy receptor-3 (DcR3),

SMRP, CA72-4, osteopontin, mesothelin, prostasin, p53, B7-H4, OVA1, and

lysophosphatidic (LPA) acid, and mucin biomarkers like; MUC1, MUC2,

MUC3, MUC4, MUC5AC and MUC16 have been studied in-detail for their

expression in different stages of ovarian cancer [Gubbels et al. 2010; Chauhan et

al. 2009; Rein et al. 2011]. Table 8 represents expression pattern of these

markers in early and late stage ovarian cancer [adapted from Chauhan et al. 2009

and Rein et al. 2011].

35

Table 8: Expression pattern of mucin and other markers in ovarian cancer

Ovarian Biomarker Early stage Late stage 1. Mucin Biomarkers

MUC16 MUC1 MUC2 MUC3 MUC4 MUC5AC MUC13

++ +++ +++ +++ +++ ++ +++ + +++ ++ ++ ++ +++ ++

2. Other protein markers

HE4 Osteopontin Mesothelin B7-H4 Prostasin VEGF p53 LPA DcR3 OVA1

+++ +++ ++ ++ ++ +++ - ++ ++ ++

+++ ++ +++ +++ +++ ++ +++ +++ ++ ++

The studies were carried out by different groups to evaluate these tumor

markers in ovarian cancer for early diagnosis. They suggested that, combination

of some of these tumor markers has provided increased sensitivity and

specificity in diagnosing ovarian cancer. Use of CA125 and HE4 in combination

for the diagnosis has improved the sensitivity and specificity to 96% and 98%

respectively and use of other biomarkers along with CA125 and HE4 has

provided high sensitivity and specificity for diagnosing ovarian cancer [Rein et

al. 2011; Nosov et al. 2009; Visintin et al. 2008]. In spite of large numbers of

biomarkers available, FDA has approved only three biomarkers; CA125, HE4

36

and OVA1 for the diagnosis of ovarian cancer. These are used in screening test

either in ELISA tests or by using specific antibodies [Rein et al. 2011].

Most of these ovarian cancer biomarkers are known to express altered

glycans during transformation. Mucin biomarkers are known to express altered

N- and O-glycans. The expression of sialyl Lewisa, sialyl Lewisx, sialyl Tn, Ley,

Tn, TF and their isomers on the N- and O-linked oligosaccharides are observed

in various human malignancies including ovarian cancer. Modification of

glycans on many glycoproteins of ovarian cancer is becoming potential target to

be considered as biomarkers [Hollingsworth and Swanson 2004]. Table 9

represents the glycosylation changes commonly observed in different ovarian

glycoproteins.

Table 9: Glycan modifications on the ovarian cancer protein markers

Glycoprotein Type of modification Modified group

AGP Sialylation sLe(x)

Hp β-chain Sialylation sLe(x)

α-antichymotrypsin Sialylation sLe(x)

CA15-3 (Muc1) Sialylation oligosaccharide replacement

sTn Tn

CA15-3 Asialylation oligosaccharide replacement

TF

CA15-3 Fucosylation Le(y)

CA125 Fucosylation Le(y)

THBS1 Fucosylation Core fucose

POSTIN β1,6 branching Bisecting GlcNAc

37

These glycans on the glycoproteins are becoming important antigenic

determinants and some of them are used in immunotherapy. Hence

understanding the altered glycans will be of great interest in developing new

cancer specific targets for diagnosis and therapy of ovarian cancer.

1.9. Applications of lectins

The growing body of literature on lectins and their interesting sugar recognition

properties has provided diverse applications of lectins in different fields of life

science. In the early 1970s it became apparent that erythrocyte agglutinating

property of lectins can also be extrapolated to other types of cells due to their

unique sugar recognition property [Sharon and Lis 2004]. The recognition of

specific glycans made them useful tools in isolating specific glycoproteins from

different body fluids. WGA and ConA are well known lectins used for the

isolation of specific class of proteins from serum, tissue and cultured cell lysates

[Ghosh et al. 2004]. Lectins are used in the identification and separation many

different cells and some lectins are under clinical use, for example, the lectin

combination of Sophora japonica agglutinin (SJA) and Erythrina cristagalli

agglutinin (ECA) are known to differentiate between cells of the proximal

tubules, distal tubules, collecting ducts, and lymphocytes in urine [Grupp et al.

2001]. Blood group typing and identification of specific blood group antigen by

lectins is an important application and is being used by clinicians routinely.

Lectin from Dolichos biflorus and Vicia cracca are used to identify cells with

A1 blood group, Lectins from Grifonia simplicipholia used to identify B blood

group antigen, Ulex europaeus lectin is used to identify the H blood group

38

antigen, Lectins from Iberis umbletta and Vicia graminea are used to identify the

M and N blood group antigens respectively [Khan et al. 2002]. Lectins are used

in the diagnosis of many diseases by recognizing specific markers in serum,

urine and on tissues. Apart from these clinical applications the lectins due to

their immunomodulatory, antiproliferative/cytotoxic effects have been used in

different research fields like, immunology, AIDS research, developmental

biology, neurobiology, proteomics, Glycomics etc. The following table 10

summarizes the application of lectins in different aspects of biology [Sharon and

Lis 2004].

Table 10: Major applications of lectinsa (adapted from Sharon and Lis 2004)

Major functions Examples References

Cell identification and separation SJA and EJA Grupp et al. 2001

Detection, isolation, and structural studies of glycoproteins WGA and ConA Ghosh, D., et al. 2004

Investigation of carbohydrates on cells and subcellular organelles; histochemistry and cytochemistry

Helix pomatia agglutinin (HPA), Ulex europeus agglutinin-I (UEA-I)

Arab et al. 2010 Sobral et al. 2010

Mapping of neuronal pathways WGA Braz et al 2005

Mitogenic stimulation of lymphocytesb PHL Nowell 1960

Purging of bone marrow for transplantationb SBA Nagler et al 2004

Selection of lectin-resistant mutants Against L-PHA, WGA Patnaik and Stanley 2006

Studies of glycoprotein biosynthesis PHA Narasimhan et al 1977

aLectins from sources other than plants are rarely in use. bIn clinical use.

39

1.10. Lectin mediated physiological responses

Lectins due to their unique glycan binding properties can recognize various cell

surface molecules and exert number of interesting physiological responses, like

mitogenecity, antiproliferative/cytotoxic effects. These interesting glycan

recognition property and physiological responses mediated by lectins has

provided direct or indirect scope for application of lectins in different fields of

life science like; Glycobiology, ontogeny, immunology, neurobiology,

metabolism, hematology, and cancer biology. The expression and unique

involvement of specific glycans in many pathophysiological conditions are

becoming important targets for diagnosis, treatment and therapeutic use in many

diseases. Hence carbohydrate binding proteins like lectins are gaining

applications in diverse fields. The various physiological responses mediated by

lectins are as follows;

1.10.1. Mitogenicity of lectins

Lectins due to their unique specificity towards saccharides and/or cell surface

glycoproteins have become valuable tools to elucidate their role in various

physiological processes and understanding the respective signaling pathways

[Ashraf 2003]. Lectins are known for their mitogenecity, they can stimulate

transformation of cells from the resting phase to active cells, which may

subsequently undergo mitotic division. The potent mitogenicity of lectins has

opened up a new arena for scientists to study the probable role of lectins in cell

growth and development. The proliferative activity of lectins plays crucial role

in understanding the relationship between chromosomal abnormality and human

40

diseases, which further helps the diagnosis. Lectin-lymphocyte interaction has

made a substantial contribution for understanding the mechanism of lymphocyte

activation and its control and further cell growth and development.

PHA (Phytohemagglutinin), the lectin from the red kidney bean was the

first lectin shown to be mitogenic towards lymphocytes that stimulate these cells

to grow and divide [Nowell 1960]. This was the first report on mitogenic

property of lectins and these findings shattered the belief, held until then, that

lymphocytes are dead end cells that could neither divide nor differentiate further.

This was followed by discovery of several other lectins that are proven to be

mitogenic, most notably Concanavalin-A (Con-A) [Haris et al. 1963], Wheat

germ agglutinin (WGA) [Aub et al. 1965] Poke Weed Mitogen (PWM)

[Brittinger et al. 1969] and all these lectins have been extensively used to study

lymphocyte function, in vitro. Later, mitogenic lectins with varied sugar

specificities were also reported from various plant parts of different taxonomic

groups, like lectins from underground tubers of Alocasia indica, Gonatanthus

pumilus, and Sauromantum guttum [Shangary et al. 2004], Cotyledons of

Castanea crenata [Nomura et al. 1998], seed integument of Saraca indica [Gosh

et al. 1999], pulp of Musa acuminate [Peumans et al. 2002], rhizomes of Smilax

gabra [Ng and Yu 2001]. Recently potent mitogenic lectins from seeds of red

cluster pepper (Capsicum frutescens) [Ngai and Ng 2007a], dark red kidney

bean; Phaseolus vulgaris cv. [Xia and Ng 2006] have been reported.

41

1.10.2. Antiproliferative activity /cytotoxicity of lectins

Apart from stimulating the resting lymphocytes, lectins are also reported to have

other physiological responses like antiproliferative activity and cytotoxicity on

different cell types. These interesting properties of lectins are gaining

applications in treating human diseases such as cancer and autoimmune

disorders that are caused by the aberrant behavior of a single cell type and

treating them by chemotherapy is a challenge. Successful therapy should

selectively eliminate the abnormal cells while leaving all normal cells

functionally undisturbed. Some of the lectins, considering their selective sugar

specificity are being investigated for their use in cancer research and therapy.

Some plant lectins are known to be toxic which kill animal cells by

arresting the protein synthesis. Ricin, the toxic lectin from Ricinus communis

seeds, has become the toxin of choice because it is easily purified and well

characterized, and it is one of the most potent cytotoxins known [Olsnes et al

1978]. In addition to ricin, other cytotoxic plant lectins are abrin from Abrus

precatorius seeds, modeccin from Adenia digitata roots, viscumin Viscum album

leaves, and volkensin from Adenia volkensii roots [Stirpe and Barbieri 1986;

Narayana et al. 2004]. Mistletoe is a common name for many species of semi-

parasitic plants which grow on deciduous trees all over the world. European

mistletoe (Viscum album L.; EM) extract is widely used in cancer therapy

[Bussing et al. 1997] and has been shown to exhibit antitumor and

immunomodulatory activity against HL60 (promyelocytic leukemia) and Jurkat

(Leukemia) cell lines [Kuttan et al. 1992]. Korean mistletoe (Viscum album C.;

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KM), a different subspecies of Viscum album from European mistletoe, was

shown to be more cytotoxic against L1210 murine leukemia cells in vitro than

EM. Recently two cytotoxic isolectins designated as KML-IIU and KML-IIL

were isolated and characterized from Korean mistletoe [Kang 2007].

Wheat germ agglutinin (WGA), an N-glycan specific lectin shown to

exhibit most deleterious effect on the viability of H3B (human hepatocellular

carcinoma), JAr (human choriocarcinoma), ROS (rat osteosarcoma), L929

(mouse Fibroblasts) and Jurtkat cells [Liu et al. 2004; Gastman et al. 2004].

WGA induces G2/M phase cell cycle arrest in L929 cells and induces apoptosis

involving caspase-3 and Bax (proapoptotic protein) [Liu et al. 2004], whereas in

Jurkat cells WGA induced apoptosis is known to involve both Caspase-8 and -9

[Gastman et al. 2004]. Concanavilin A (ConA) is another cytotoxic lectin

studied in detail for its apoptotic induction potential on many cells. ConA

induces cytotoxic effects in tumour cells in vivo and in vitro involving

mitochondrial mediated P73-Foxo1a-Bim signaling pathway for apoptosis and

BNIP3-mediated mitochondrial autophagy [Li et al. 2011]. (Autophagy is a

tightly regulated pathway involving the lysosomal degradation of cytoplasmic

organelles or cytosolic components. This pathway can be stimulated by multiple

forms of cellular stress, including nutrient or growth factor deprivation, hypoxia,

reactive oxygen species, DNA damage, protein aggregates, damaged organelles,

or intracellular pathogens [Guido Kroemer et al. 2010]. Many other plant lectins

are known for their antiproliferative effect on different cancer cells by induction

of cell cycle arrest in different phases of cell cycle leading to

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apoptosis/autophagy [Lam and Ng 2011]. Noticeably, Abrus agglutinin induces

antiproliferative activity in Dalton’s lymphoma and HeLa cells [Bhutia et al.

2008a&b], Sophora flavescens lectin induces cytotoxicity in HeLa cells [Liu et

al. 2008], Polygonatum odoratum and Polygonatum cyrtonema lectins are

known to induce apoptosis in L929 and human melanoma A375 cells (Liu et al.

2009a&b]. Pseudomonas aeruginosa hemagglutinin induces antiproliferative

activity in breast cancer cells MDA-MB-468, and MDA-MB-231HM cells [Liu

et al. 2009c], French bean hemagglutinin-induces strong cytotoxicity in breast

cancer MCF-7 cells [Lam and Ng 2010].

During recent past, fungal lectins are gaining importance largely due to

the discovery that some of these lectins exhibit potent antitumor activities. For

example, Volvariella volvacea lectin shows antitumour activity against sarcoma

S-180 cells [Lin and Chou 1984], Grifola frondosa lectin is cytotoxic to HeLa

cells [Kawagishi et al. 1990], Agaricus bisporus lectin possesses

antiproliferative activities against human colon cancer cell line HT29 and breast

cancer cell line MCF-7 [Yu et al. 1993]. A lectin from Sclerotium rolfsii

possesses strong antiproliferative effect on human colon cancer cell line HT29

and DLD1 involving caspase mediated apoptosis [Inamdar et al. 2012].

Tricholoma mongolicum lectin inhibits mouse mastocytoma P815 cells in vitro

and sarcoma S-180 cells in vivo (Wang et al. 1997). A lectin from Agrocybe

aegerita (AAL) shows strong growth inhibitory effect on number of human

tumor cell lines, HeLa (cervical cancer), SW480 (colon adenocarcinoma cell

line), SGC-7901 (gstric cancer), MGC80-3 (gstric adenocarcinoma), BGC-823

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(gstric cancer), HL-60 (promyelocytic leukemia cells) and also mouse sarcoma

S-180. Lectins from Boletopsis leucomelas, Agrocybe aegerita native and the

recombinant are known for inducing apoptosis [Zhao et al. 2003; Koyama et al.

2001]. A xylose specific lectin from the mushroom, Xylaria hypoxylon is

antiproliferative towards M1 (leukemia) and HepG2 (hepatome) cell lines [Liu et

al. 2006] and a lectin from Ganoderma capense exhibited similar effect on

leukemia cells (Patrick et al. 2004). Pleurotus citrinopileatus contains lectin with

potent antitumor activity in mice bearing sarcoma 180 and caused 80 %

inhibition of tumor growth indicating its potential as an antitumor agent [Li et al.

2008]. Recently a ricin B-like lectin from the mushroom Clitocybe nebularis

with antiproliferative activity on human leukemic T-cells is reported and this

effect is comparable to that of lectins from Agaricus bisporus (ABL) and

Agrocybe aegerita (AAL) [Pohleven et al. 2009]. AAL2 is another lectin from

Agrocybe aegerita known to induce apoptosis in H22 and Huh7 hepotoma cells

[Jiang et al. 2012]. In recent years many lectins are reported from different fungi

with different sugar specificities. The antiporoliferative/proliferative activity is

studied in many cases, whereas the detailed mechanism of action is been

deduced in only few reports [Khan and Khan 2011, Sing et al. 2010]. Enormous

reports are available on the mushroom lectins, but only limited reports are

available on the hyphae forming lower fungi. Considering the interesting sugar

recognition property, biological responses and their application in diverse fields

prompted us to study fungal lectins for their glycan specificity, interaction with

cancer cells and their diverse biological responses in order to explore them for

their diagnostic and therapeutic applications.

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1.11. Genesis of the thesis

Our laboratory has been investigating fungal lectins for understanding their

structure function relations and their cell biological applications. Sclerotium

rolfsii is a soil borne plant pathogen with a host range of over 500 species

including agricultural crops like sunflower, potato, tomato etc. [Punja 1985].

Earlier in this laboratory a lectin has been purified from the sclerotial bodies of

Sclerotium rolfsii by employing ion exchange and gel filtration chromatography,

and was shown to recognize TF antigen; Galβ1-3GalNAcα-O-Ser/Thr, an

oncofoetal, mucin Core-1 antigen [Swamy et al. 2001; Wu et al. 2001]. The

physiological role of the lectin has been demonstrated in the development and

morphogenesis of the fungus [Swamy et al. 2004]. The crystal structure of the

SRL has been determined in its free form and in complex with N-

acetylgalactosamine and N-acetylglucosamine at 1.1, 2.0, and 1.7 Ǻ resolution

respectively [Leonidas et al. 2007]. The ambiguities in the amino acid sequence

arised from X-ray crystal structure were resolved and further sequence was

confirmed by matrix-assisted laser desorption ionization (MALDI) and

electrospray ionization (ESI) techniques [Sathisha et al., 2008]. Recently the

detailed carbohydrate binding specificity of SRL has been determined by glycan

array analysis at Consortium for Functional Glycomics (CFG), USA, that

revealed its specific binding to cancer associated Thomsen-Friedenreich antigen

(TF) and its derivatives [Chachadi et al. 2011]. Since TF is a cancer associated

antigen its interaction has been studied with leukemic and colon cancer cells and

also with normal human PBMCs. The results revealed that, SRL is mitogenic

46

towards normal human PBMCs, whereas it induces antiproliferative effect on

leukemic cells (unpublished data). SRL also revealed the growth inhibitory

effect on colon cancer cells.

Rhizoctonia bataticola is a plant pathogen with a host range of more than

100 species including potato, sunflower etc. [Dhingra and Sinclair 1978]. A

lectin from plant pathogenic fungus Rhizoctonia bataticola (RBL) was purified

to homogeneity by ion exchange and affinity chromatography, and its physico-

chemical properties have been studied in detail earlier in this lab. RBL showed

complex sugar specificity when analysed by hapten inhibition assay. RBL is

strongly mitogenic to PBMCs and induces mitogenicity by secretion of Th1 and

Th2 cytokines. The detailed carbohydrate binding specificity of RBL was

determined recently by glycan array analysis at CFG, USA. The Glycan array

analysis revealed the exclusive specificity of RBL towards the N-glycans,

primarily recognizing high mannose, tri- and tetra- antennary complex N-

glycans, and also tandem repeats of sialyl Lewis antigen which are known to be

expressed during malignant transformation. The N-glycans recognized by RBL

are also the part of CA-125, a cancer associated antigen known to be expressed

in many cancers including ovarian cancer. This specificity of RBL inspired to

study its interaction with human ovarian cancer PA-1 cells. The initial results

revealed the cytotoxicity of RBL towards PA-1 cells [Nagre et al. 2010a, Pujari

et al. 2010, 2012].

Cephalosporium curvulum is a human pathogen, causing ophthalmic

infections including mycotic keratitis. Several cases of keratitis and occasional

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cases of endophthalmitis due to Cephalosporium spp. have been reported

[Fincher et al. 1991; Rao et al.1997; Read et al. 2000]. A lectin from human

infectious fungus Cephalosporium curvulum (CSL) was isolated and purified to

homogeneity by using affinity chromatography and its physicochemical

characters have been established. Hapten inhibition studies revealed the complex

sugar specificity of CSL. The interaction of CSL with normal human PBMCs

was studied, which showed that CSL is also mitogenic [Nagre et al. 2010b].

The studies made so-for with SRL and RBL, considering their interesting

sugar binding properties warranted for further in-detailed investigations on

interaction of these lectins with cancer cells. The expression of cancer associated

mucin type O-glycans on many cancers including ovarian cancer is known and

specific recognition of these glycans by SRL, necessitated the detailed

investigation on the interaction of SRL with human ovarian cancer cells. RBL is

shown to be cytotoxic to PA-1 cells and hence it is essential to investigate the

detailed signaling mechanism underlying RBL induced cell death in PA-1 cells

in order to explore for its possible clinical applications. A mitogenic lectin CSL

isolated from fungus Cephalosporium curvulum is shown to exhibit complex

sugar specificity based on hapten inhibition studies. In order to explore its

possible clinical application it is essential to determine its fine sugar specificity.

Hence the detailed carbohydrate specificity of CSL was investigated by glycan

array analysis.

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1.12. The present study encompasses the following objectives;

1. Interaction studies of Sclerotium rolfsii lectin with human ovarian cancer

PA-1 cells

2. To understand the detailed signaling mechanism involved in Rhizoctonia

bataticola lectin (RBL) induced cell death in PA-1 cells

3. To determine of fine sugar specificity of CSL by glycan array analysis.