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1. INTRODUCTION

Sugars or saccharides are essential components of all living things

and the various roles they play in biology are researched in various medical,

biochemical and biotechnological fields, this has led to an emerging field of

science. Glycobiology is defined as the study of the structure, biosynthesis,

and biology of saccharides (sugar chains or glycans) that are widely

distributed in nature. The field includes the chemistry of carbohydrates, the

enzymology of glycan formation and degradation, the recognition of glycans

by specific proteins (lectins and glycosaminoglycan-binding proteins),

glycan roles in complex biological systems and their analysis or

manipulation by a variety of techniques [Varki et al. 1999, 2009].

Although it has long been appreciated that glycan expression changes

with cellular condition, progress toward delineating the molecular basis of

glycan function has been rather slow relative to comparable studies of

proteins and nucleic acids. This slow progress is partly due to the fact that

the biosynthesis of glycans, unlike other biopolymers, is not template-driven

[Dube et al. 2005]. However, in the past few years, glycobiology has been

blooming with increased rate, spurred on by developments in genomics and

new technologies [Drickamer, 2002]. Recent progress in glycobiology has

revealed that carbohydrates have an enormous potential for encoding

biological information. These glycoproteins and glycolipids (combination

molecules) dot the outer surface of all cells and serve as cellular

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identification tags to the surrounding world [Ambrosi et al, 2005]. The

prime reason for this is not only their positioning on the cell surface but are

also well studied as carriers of information due to their high degree of

combinational possibility in sequence arrangement. Considering the

importance of glycans in various complex biological systems, the proteins

that recognise these glycans (lectins and glycosaminoglycan-binding

proteins) are gaining more importance for deciphering the glycocodes.

In order to understand the complexity and functions of these glycans,

it is necessary to understand their synthesis, expression and alterations in

normal and pathological conditions.

1.1 Biosynthesis of glycoprotein and glycolipid oligosaccharides

Glycosylation, is an important post-translational modification (PTM)

that is critically functioned by the biosynthetic-secretory pathway in the

endoplasmic reticulum (ER) and Golgi apparatus. Majority of proteins and

lipids expressed in a cell undergo this modification, which entails the

covalent addition of a carbohydrate (a glycosyl donor) to a hydroxyl or other

functional group of another molecule (a glycosyl acceptor). In biology,

glycosylation refers to the enzymatic process that attaches glycans to

proteins, lipids, or other organic molecules. It is a feature that enhances the

functional diversity of proteins and lipids that influences their biological

activity [Marino K et al, 2010].

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In mammals, glycans are constructed from nine monosaccharide

building blocks that can be connected to one another through glycosidic

linkages in myriad combinations. Glycans are also formed on their protein

or lipid scaffolds by glycosyltransferases and glycosidases as they traffic

through the secretory pathway. The combined action of these enzymes in the

secretory pathway leads to a diverse array of glycan structures [D H. Dube,

2005]. The common classes of glycoconjugates found on eukaryotic cells

are primarily based on the nature of the linkage regions to the aglycone (a

protein or a lipid), such as N-, O- and C-linked glycosylation, glypiation

(GPI anchor attachment) and phosphoglycosylation.

N-linked Glycosylation: N-glycans are covalently attached to an asparagine

residue of a polypeptide chain, specifically a subset residing in the Asn-X-

Ser/Thr motif, where X denotes any amino acid except proline.

O-linked Glycosylation: O-glycans are covalently attached to serine and

threonine side chains of a protein in the Golgi apparatus.

Glypiation: A special form of glycosylation is the formation of a GPI

(glycosylphosphatidylinositol) anchor. In this kind of glycosylation, a

protein is attached to a lipid anchor via a glycan chain that localizes proteins

to cell membranes and is widely detected on surface glycoproteins in

eukaryotes and some archae [Kobayashi T. et al. 1997]. GPI anchors consist

of a phosphoethanolamine linker that binds to the C-terminus of target

proteins, glycan core structure and phospholipid tail that anchors the

structure in membrane.

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C-linked Glycosylation: C-mannosylation represents carbon-carbon bonds

rather than carbon-nitrogen or carbon-oxygen bonds. C-mannosyltransferase

(c-Mtf) links C1 of mannose to C2 of the indole ring of tryptophan. The

enzyme recognizes the specific sequence Trp-X-X-Trp and transfers a

mannose residue from dolichol-P-Man to the first Trp in the sequence

[J.Krieg. et al, 1998].

Phosphoglycosylation: This type of post-translational modification is

limited to parasites (e.g., Leishmania and Trypanosoma) and slime molds

(e.g., Dictyostelium) and is characterized by the linking of glycans to serine

or threonine via phosphodiester bonds [P. A. Haynes 1998].

N-linked and O-linked chains are major classes of glycans found in

glycoproteins, glycosaminoglycan of proteoglycans and glycophospholipids.

Structural details and the biosynthesis of glycoconjugates, glycoproteins and

glycolipids have been reviewed and an exhaustive account is available in

reviws [Varki et al., 1999].

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Fig.1. Types of glycosylation: Glycopeptide bonds can be categorized into specific groups based on the nature of the sugar-peptide bond and the oligosaccharide attached. Oligosaccharide transferase (OSTase) mediate N-Glycans binding to the amino group of asparagine in the ER. Glycosyltransferases (Gtfs) mediate O-linked monosaccharides binding to the hydroxyl group of serine or threonine in the ER, Golgi, cystosol and nucleus. GPI transamidase (GPIT) mediates Glycan core linking to a phospholipid and a protein. C-mannosyl transferase (c-Mtf) mediates mannose binding to the indole ring of tryptophan via C-C linkage. Phosphoglycosyl transferase (PTase) mediates Glycan binding to serine via phosphodiester bond.

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1.1.1 N- Linked Glycans

N-linked oligosaccharide is a sugar chain covalently attached to an

asparagine (Asn) residue of a polypeptide chain. N-glycans share a

common penta-saccharide core region and are classified into three main

classes; high mannose type, complex type and hybrid type (Fig. 2). The

core structure of N-linked glycans is found attached to the nitrogen (N) of

asparagine in the sequon. The sequon is a Asn-X-Ser/Thr sequence, where X

is any amino acid except proline and composed of N-acetylgalactosamine,

galactose, neuraminic acid, N-acetylglucosamine, fructose, mannose, fucose

and other monosaccharides [Mellquist et al., 1998, Aebi et al. 2010].

The synthesis of N-linked glycans or of N-linked oligosaccharides

begins by addition of a 14-sugar precursor to the asparagine residue in the

polypeptide chain of the target protein. The structure of this precursor is

common to most of the eukaryotes and contains 3 glucose, 9 mannose and 2

N-acetylglucosamine molecules. A complex set of reactions attaches this

branched chain to a carrier molecule called dolichol phosphate, a membrane

anchor for the formation of the oligosaccharide and then it is transferred to

the appropriate point on the polypeptide chain as it is translocated into the

ER lumen. Following transfer to the nascent peptide chain, N-linked

glycans generally undergo extensive processing reactions that occur in the

golgi apparatus. To begin with three glucose residues are removed, as well

as several mannose residues, depending on the N-linked glycan in question.

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The removal of the glucose residues is dependent on proper protein folding.

Glycans undergo modification reactions that may involve the addition of a

phosphate or acetyl group onto the sugars or the addition of new sugars,

such as neuraminic acid. Since processing and modification of N-linked

glycans within the golgi does not follow a linear pathway, many different

variations of N-linked glycan structure are possible depending on enzyme

activity in the golgi [Hubbard and Ivatt, 1981; Kornfeld, 1985; Ruddock and

Molinari 2006].

N-linked glycans are extremely important and contribute in proper

protein folding in eukaryotic cells. Chaperone proteins such as calnexin and

calreticulin present in the endoplasmic reticulum bind to the three glucose

residues present on the core N-linked glycans and these chaperones then

serve to aid in the folding of the protein to which the glycan is attached.

Following proper folding the three glucose residues are removed and the

glycan moves on to further processing reactions. Interestingly, if the protein

fails to fold properly, the three glucose residues are reattached allowing the

protein to re-associate with the chaperones. This cycle may repeat several

times until a protein reaches its proper conformation. If a protein repeatedly

fails to properly fold, it is excreted from the endoplasmic reticulum and

degraded by cytoplasmic proteases [Varki et al., 1999].

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Fig.2. (A) Types of N-glycans in mammals. (B) Biosynthesis of N- glycans: A preassembled oligosaccharide comprised of 2 GlcNAc, 9 Man and 3 Glc residues (left) is co-translationally transferred to an asparagine residue of a nascent protein chain. This structure undergoes processing by the sequential removal of 3 Glc residues, mediated by α-glucosidases (α Glcase) I and II and 4 Man residues mediated by α-mannosidases ( α Manase). Then, a first antenna GlcNAc is added to the Man5GlcNAc2 structure by the action of GlcNAcT1. Subsequently, 2 other Man residues are removed by α-mannosidase II (α Manase II) and a second GlcNAc residue is added by GlcNAcT2. The resulting structure can be further elongated by the addition of galactose residues mediated by members of the β1,4GalT family and/or by core-linked fucose (mediated by FucT8) and/or bisecting GlcNAc (mediated by GlcNAcT3). The addition of these modifications is not mutually exclusive. After the addition of galactose the sugar chains are frequently terminated by sialic acids which can be linked either via α-2,3 or α-2,6 to galactose.

A

B

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1.1.2 O-Linked Glycans

The synthesis of O-linked glycans or O-linked glycosylation is normally

initiated in the golgi apparatus, most commonly by the action of an N-acetyl

galactosaminyl transferase that transfers an N-acetylgalactosamine (GalNAc) to

the –OH group of side chain of a serine or a threonine residue. This structure

forms the Tn antigen, substitution of the first GalNAc in the O-6 position with

sialic acid creates the sialyl-Tn antigen, which does not undergo further

elongation. Several O-linked core structures are synthesized subsequently by a

stepwise enzymatic elongation involving specific transferases (Fig.3). For

example, core 1 disaccharide Galβ1-3GalNAc (also called T/TF antigen) can be

synthesized by addition of a galactose to Tn antigen and synthesis is catalysed

by the action of core 1 β,3-galactosyltransferase. This TF disachharide can be a

precursor to the branched core 2 [Galβ1-3 (GlcNAcβ1-6) GalNAcα1-Ser/Thr]

and core 3 [GlcNAcβ1-3GalNAcα1-Ser/Thr] O-glycans, formed by the

presence of GlcNAc linked β1,6- or β1,3- respectively to GalNAcα1-Ser/Thr.

These three core structures are often further elongated by the addition of Gal,

GlcNAc and GalNAc residues and terminated by the addition of fucose or sialic

acid or both. [Brockhausen, 1995; Spiro, 2002]. Apart from these three core

structures five other core structures have been identified [Hounsell et al., 1996],

which are formed by further elongation or modification by sialylation,

sulfation, acetylation and polylactosamine-extension. Apart from these eight

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core structures several different types of O-linked oligosaccharides have been

identified, for example O-fucose, O-mannose, O-glucose and

O-N-acetylglucosamine.

Among the different types of oligosaccharides, O-linked

oligosaccharides are simple when compared to N-linked oligosaccharides and

are involved in various functions such as, selectin binding in leukocyte

circulation [Springer, 1994], fertilization [Kojima et al., 1994] and glycoprotein

clearance [Drickamer, 1991]. Further they also influence immunological

recognition of antigens and signal transduction [Chapman et al., 1996 &

Kodama et al., 1993] and play a role in the processing and expression of

glycoproteins [Remaley et al., 1991].

Unlike N-linked glycans, which invariably down modulate the activity

of enzymes and signaling molecules like hormones and cytokines (Opdenakker

et al., 1986; Parekh et al., 1989), the O-linked glycans can either up (Naim and

Lentze, 1992 & Goto et al., 1995) or down modulate the activity. In addition to

alterations of O-linked glycosylation being associated with cancer and

autoimmune diseases, a number of “Inborn errors of O-linked glycosylation”

have been defined to cause specific pathologies (Fukuda et al., 1987, Nishio et

al., 1995; Jaeken et al., 1980). For example Congenital disorders of

glycosylation (previously called carbohydrate-deficient glycoprotein syndrome)

like muscular dystrophy and congenital cutis laxa.

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Fig. 3. Biosynthesis of O-glycans. O-glycosylation is initiated by the transfer of GalNAcα- to Thr or Ser of the polypeptide backbone. Eight O-glycan core structures can then be synthesized. The most common O-glycan core structures are cores 1 and 2. Core structures 1-4 in particular are elongated by Gal- and GlcNAc-transferase and/or terminated by Fuc-, sialyl-, Gal-, GlcNAc- GalNAc- and/or sulfo-transferase.

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1.2 Aberrant Glycosylation and Cancer

Changes in glycosylation are often a hallmark of disease states

incuding cancer. For example, cancer cells frequently display glycans at

different levels or with fundamentally different structures than those

observed on normal cells. This was first described in the early 1970s, but the

molecular details underlying such transformations were poorly understood.

In the past few decades advances in genomics, proteomics and mass

spectrometry have enabled to understand the association of specific glycan

structures with disease states. In some cases, the functional significance of

disease-associated changes in glycosylation has been revealed.

Development of tumor in humans is a multistep process and these

steps reflect alterations that drive the progressive transformation of normal

human cells into highly malignant tissue. Normal cells have to overcome

multiple levels of regulation in order to transform into metastatic malignant

cells that eventually invade neighbouring or distant tissues. The losses of

cellular regulation that give rise to most or all cases of cancer are due to

genetic damage leading to six functional characteristics of cancer: persistent

growth signals, evasion of apoptosis, insensitivity to anti-growth signals,

unlimited replication potential, angiogenesis, invasion and metastasis.

Mutations in two broad classes of genes have been implicated in the onset of

cancer, proto-oncogenes and tumor suppressor genes. Proto-oncogenes are

activated to become oncogenes by mutations that cause the gene to be

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excessively active in growth promotion, either increased gene expression or

production of a hyperactive product will do it. Tumor suppressor gene

products such as Rb and p53 normally restrain growth, so damage to them

allows inappropriate growth. Many of the genes in both classes encode

proteins that help regulate cell birth (i.e. entry into and progression through

the cell cycle) or cell death by apoptosis; others encode proteins that

participate in repairing damaged DNA. Certain genetic changes further

allow the reactivation of telomerase activity creating an extensive

replication potential (Couldrey and Green, 2000).

A primary cancer is a tumor mass present at the site of initial

conversion of a normal cell to a tumor cell. If all cells remained in the

primary tumor, cancer would be of little clinical importance. However,

tumor cells do not always remain at the primary site but move away by one

of two processes: (1) Invasion or the movement of cells into neighbouring

space occupied by other tissues and (2) Metastasis, the spread of cells to

distant sites, usually via the bloodstream, lymphatic system, or through body

spaces. Substantial invasion usually occurs before any metastasis takes place

(Willis, 1972; Nguyen, 2009). Metastasis is facilitated by cell-cell

interactions between tumor cells and the endothelium in distant tissues.

Tumor cells in circulation interact also with platelets and leukocytes that

further contribute to tumor cell adhesion, extravasation and the

establishment of metastatic lesions. The metastatic capacity of tumor cells

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correlates with their ability to exit from the blood circulation, to colonize

distant organs and to grow in distant organs (Chambers et al, 2002). The

organ-specific character of metastasis has been already observed by S. Paget

more than a century ago and the “seed and soil” hypothesis postulates

specific interactions of tumor cells with the “friendly” environment of

distant organs that enables the establishment of metastasis and subsequent

growth (Fidler, 2003). In addition to genetic alterations, phenotypic

alterations also provide malignant cells the ability to escape tissue

boundaries through engulfment, invasion and angiogenesis. Other

phenotypic changes provide malignant cells with mechanisms to escape

immune surveillance (Couldrey and Green, 2000; Schwartz-Albiez et al,

2008).

The phenotypic alterations of interest in cancer cells are those of the

cell surface carbohydrates. Nearly all types of malignant cells and cells of

many types of diseased tissues demonstrate alterations in their glycosylation

patterns when compared to their normal counter parts (Mody et al., 1995;

Ghazarian et al, 2011). These alterations in glycosylation patterns arise as

result of altered activities of glycosyltransferases and glycosidases that is

evident in the specific and preferential display of certain glycoconjugates on

cancer cells (Mody et al, 1995; Couldrey and Green, 2000; Gorelik et al,

2001; Schwartz-Albiez et al, 2008; Blomme et al, 2009). These aberrations in

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carbohydrate patterns are observed in glycolipids, glycosphingolipids and

glycoproteins and are based on two mechanisms:

(1) Inhibition or incomplete synthesis of normally existing carbohydrate

chains and accompanying precursor accumulation.

(2) Neosynthesis through activation of new glycosyltransferases that are

characteristic of tumor cells and are absent or present only in small

quantities in normal cells.

Embryonic development and cellular activation in vertebrates are

typically accompanied by changes in cellular glycosylation profiles. The

changes that occur in normal cell during embryogenesis are almost similar

to changes during transformation to cancer. Like normal cells during

embryogenesis, tumour cells undergo activation and rapid growth, adhere to

a variety of other cell types and cell matrices and invade tissues. Thus,

glycosylation changes are universal features of malignant transformation

and tumour progression (Varki et al. 2009). The relation between altered

glycosylation and cancer biology has been studied using one of two

approaches: either by the modification of the glycosylation pattern of cancer

cell membranes by using glycosylation inhibitors or glycosidase treatments;

or by studying the subpopulations of cancer cells selected for a given

phenotype for instance, increased or reduced metastatic ability or resistance

to a given lectin [Olio FD, 1996]. The best characterised glycosylation

changes of glycoproteins associated with malignancy include the incomplete

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synthesis of glycans, altered glycosylation of mucins, alterations in synthesis

of histo- blood group related antigens, increased synthesis of

polylactosamine chains and increased (β1,6) branching of N-linked glycans,

changes in sialylation and fucosylation.

1.2.1 Incomplete synthesis of glycans

The frequency of O-glycosylation on glycoproteins is high,

particularly on secreted or membrane bound mucins, which are rich in serine

and threonine [Reis et al. 2010]. O- Glycosylation generally begins with

attachment of a single GalNAc residue to a Ser/Thr residue of the

polypeptide backbone to yield Tn antigen; GalNAcα-Ser/Thr. This results

from increased expression/activity of one of the family of

glycosyltransferases that attach the first GalNAc to the polypeptide, the

ppGalNAc transferase or decrease in activity of the core β1,3

galactosyltransferase (Freire T, 2005) that is involved in synthesis of TF

antigen. When Tn antigen is capped by sialic acid at the terminal, another

cancer-associated antigen, sialyl Tn (sTn) is formed. The synthesis of sialyl

Tn is a result of the activity of α-2,6 sialyltransferase I (ST6GalNAc1), the

glycosyltransferase which is responsible for adding sialic acid to α-GalNAc-

Ser/Thr (Brockhausen, 2001 & Marcos, 2004) and this enzyme in fact

competes with the core 1 beta 1,3 galactosyltransferase, the first enzyme

involved in the alternative route of chain elongation of O-glycans

(Julien S, 2001). Alternatively, Tn antigen may be extended in various ways

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to yield a range of mucin core structures. There are totally eight mucin core

structures synthesized by the action of different glycosyl transferases and Tn

is the precursor for their synthesis. The addition of (β1,3) linked Gal yields

Gal(β1,3)GalNAcα-Ser/Thr, the Thomsen-Friedenreich or T/TF antigen,

also called core 1 and this may be sialylated by addition of sialic acid to

terminal Gal to give Neu5Ac(α2,6)Gal(β1,3)GalNAcα-Ser/Thr, sialyl T

antigen (sTF). Further chain extension is terminated due to sialylation.

Under normal circumstances, the Tn/TF structures are concealed by terminal

sialic acids, sulphates or by addition of other sugar chains to form branched

and complex O-glycans. This is necessary because O-glycosylation

pathways play a key role in biological activity of glycoproteins involved in

the control of cell differentiation and the regulation of cell growth through

apoptosis and proliferation pathways (Gallegos et al. 2010; Patsos et al.

2009). However, the structures of these glycans get altered or changed

resulting in altered O-glycosylation, for example expression of the Tn, sTn,

and TF have been implicated in various processes such as inflammatory

responses, angiogenesis, autoimmunity and cancer metastasis (Ju et al.

2008; Mc Cluskey et al. 2010 and LG Yu, 2007). These antigens also appear

to be significant markers in cancers arising from tissues that produce high

levels of mucins, such as those of the gastrointestinal tract, breast, ovary,

uterus and bladder where their expression is reported to be associated with

poor prognosis.

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1.2.2 Changes in glycosylation of mucins

Mucins are heavily O-glycosylated high molecular proteins that are

secretory or membrane bound. At least 19 members of the MUC gene

family have been characterised and they code for mucins that are

differentially expressed by a range of tissues [Dekker J et al, 2002; Perez-

Vilar , 2004]. All the MUC gene products in general consist of a polypeptide

backbone with a degree of shared homology example apomucin, which is

heavily glycosylated by multiple O-linked glycan chains that can make up as

much as 80% of the total molecular weight of the protein [Brockhausen I et

al,1995]. All MUC family mucins are secreted except MUC1, which is

membrane-bound. The expression and glycosylation of MUC mucins are

altered in many cancer types, for example, several MUC family mucins have

been examined in colon cancer and changes in their expression has been

reported. MUC2 is decreased in colon cancer compared to normal colon and

MUC5AC, which is absent in normal colon and is shown to be produced by

pre-malignant colonic adenomas and colon cancers [Byrd J.C, 2004].

Amongst all, MUC1 also called as polymorphic epithelial mucin (PEM) or

epithelial membrane antigen (EMA) or episialin has been intensively studied

as alterations in its expression and its glycosylation appear to be a feature of

many cancer types. Glycosylation of these mucins is also altered in

pathophysiological conditions giving rise to increased expression of Tn, TF,

Lex antigens and altered O-glycans of core 3 structures due to decreased

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sialylation and sulfation. These cancer-associated glycosylation changes

observed and reported may hold promise as diagnostic and prognostic

markers and also eventually as potential targets for developing anti-cancer

vaccines.

1.2.3 Alterations in Histo-Blood group-related antigens

ABH antigens are carried by both glycoproteins and glycolipids on

red blood cells, endothelial and epithelial cells of many tissues and

secretions. Aberrant expression of ABH antigens is known in tumours and it

is consequence of the following: (1) Deletion of an antigen normally present

in the corresponding adult tissue with accumulation of the precursor sugar

chain (2) Re-expression of fetal antigens that are normally absent in the

adult tissue and (3) "Incompatible" antigen expression or in other words an

individual expressing the B or O phenotype on erythrocytes may carry the A

antigen on colon cancer cells. ABH antigens are expressed by fetal human

colon according to the individual's blood type. The expression of A, B and H

become restricted to the proximal colon after birth. In colon cancer this

proximal-distal gradient of expression of ABH antigens is lost because of

enhanced distal expression of these antigens (M Yuan et al 1985).

The Lewis family of related, small, mono- or di-fucosylated histo-

blood group antigens is one of the most researched areas. Lewis a (Lea) and

Lewis b (Leb) are based on type 1 chains, Gal(β1,3)GlcNAc/GalNAc,

whereas Lewis x (Lex) and Lewis y (Ley) are based on type 2 chains, that is

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Gal(β1,4)GlcNAc/GalNAc; all these antigens may occur in sialylated or

unsialylated form. Lewis antigens are frequently synthesised by cancer cells

[T Nakagoe. et al, 1991 and E Dabelsteen, 1996] and their profiles may be

different from that seen in adjacent normal tissues. Increased amounts of

Lewis antigens have been reported in various cancers including those of the

breast, lung, oesophagus and colon, serve as independent prognostic

markers as these antigens are frequently shed into the blood stream. Both

sLex and sLea are recognised binding ligands for E-selectin, an endogenous

lectin [Isacke, 2000] which is known to be involved in the adhesion of

circulating leukocytes to endothelium at sites of inflammation and may also

be implicated in the metastatic cascade [Sawada. et al 1994; Tomlinson. et

al 2000].

1.2.4 Increased (β1,6) branching of N-Linked glycans

The excessive branching of the N-glycans is commonly observed

during cancer due to the enhanced expression of the enzymes, UDP-

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

N-glycan GlcNAc transferase III (GlcNAcT-III). These enzymes catalyse

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 either tri- or tetra-

antennary oligosaccharides that contribute for increasing the total cell

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surface terminal sialylation in malignant cells, which prevents further chain

elongation. In the initial stages of carcinogenesis induced by some of the

oncogenic viruses and also by oncogenes typically reveal such increased

terminal sialylation [Dennis et al. 1987; Gorelik et al. 2001; Ghazarian et al.

2011]. Apart from such increased 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 terminal sialylated β1–6

branched N-glycans are invariably found on invading trophoblasts, activated

granulocytes, 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]. Further,

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

expression of β1–6 branched N-glycans is inhibited [Humphries et al. 1986;

Krishnan et al. 2005].

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

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1.2.4 Changes in Sialylation

Sialic acids, N-acetylneuraminic acid (Neu5Ac) and

N-glycolylneuraminic acid (Neu5Gc) are naturally occurring sugars with lot

of biological significance. Sialic acids are typically found as terminal

monosaccharides attached to mammalian cell surface glycoconjugates. They

play essential roles in many physiological and pathological processes

(Goswami et al. 2007). Changes in the amount, type, distribution and

linkage of certain sialic acid types can have prognostic significance in

human cancer (Hedlund et al. 2008). Increased sialylation is one of the most

commonly reported altered glycosylation of cancer cell surface

glycoprotein’s that is often manifested by an increase in sialylated Lewis

family antigens or sialylated T and Tn antigens. This increase in sialylated

structures may result from increased availability of appropriately presented

oligosaccharide acceptors for sialylation such as those resulting from

increased (β1,6) branching. An increase in the amount of one type of sialic

acid, N-acetylneuraminic acid or Neu5Ac as a part of sLex in colorectal

cancer has been reported and is associated with increased metastatic

competence in a colorectal cancer model (Yousefi, 1991 & Olio, 1993).

Increased amounts of another sialicacid, N-glyconeuraminic acid or Neu5Gc

has been described in melanoma and colorectal cancer (Furukawa,1989 &

Higashi, 1985) on O-linked glycans of MUC1 mucin in the aggressive

breast cancer cell line MCF-7 (P.L Devine, et al. 1991) and on N-linked

glycans of the metastatic lymphoma cell line MDAY-D2 (Takano et. al,

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1994). Sialic acid is functionally involved in various biological processes

like normal cellular adhesion, cellular motility and immunogenicity, as well

as in the metastatic progression of cancer. This is mainly because sialic acid

endows glycoproteins or cellular membranes, with a negative charge and

thus influences the physiochemical properties of glycoproteins in

de-adhesion and protective functions. Conversely, a failure in normal

sialylation mechanisms may result in the exposure of cancer-associated

antigens such as T and Tn antigens that are cryptic in normal cells or of the

polypeptide. These alterations may have implications for the adhesion of

cancer cells to endothelia and thus metastatic competence (Altevogt. et al,

1983).

1.3 Features of protein–carbohydrate interactions: The third alphabet of life

Recognition of glycoconjugates by glycan binding proteins (GBPs) is

an important event in all biological processes and is frequently mediated by

carbohydrate-protein interactions. There are two fundamental reasons to

support view. First, it is a well-known fact that all the eukaryotic cells are

covered with carbohydrates and these carbohydrates as well as those present

on some circulating glycoproteins appear to be modulated depending on the

physiological status, such as during developmental processes or oncological

transformation. The second reason is, unlike proteins and nucleic acids,

carbohydrates, due to the presence of multiple functional groups,

particularly hydroxyl groups on each monomeric unit are capable of forming

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many different combinatorial structures including branched ones from

relatively small numbers of sugar units. Hence, each structure could

potentially carry a specific biological message, thus widening the spectrum

of reactivity that is possible from a limited number of monomers. A variety

of regulatory processes including cell growth, apoptosis, folding, routing of

glycoprotein’s, cell adhesion/migration and metastatic cascade have been

unraveled and found to be mediated or modulated by specific protein–

carbohydrate interactions. Proteins involved in mediating such interactions

occur widely in nature, including carbohydrate-specific enzymes,

periplasmic receptors and anti carbohydrate antibodies are known to

recognize carbohydrates non-covalently which is possible due to their

various topologies. Lectins are another major class of such proteins (Liener

et al., 1986; Drickamer & Taylor 1993; Sharon & Lis, 1995) that have come

into the forefront of biological research due to their significant application in

unraveling biological processes and in elucidation of protein and

carbohydrate interactions involved in them.

A well-known characteristic of protein–carbohydrate interactions is the

low affinity of binding, usually in the millimolar range. These interactions

are driven by a favourable enthalpy (Bundle, 1992 & Toone, 1994), offset by

the multiple contact points involving hydrogen bonds, van der Waals

interactions and hydrophobic stacking between the carbohydrate and the

protein. In fact, recent studies have shown that in the binding site,

25

polar–polar interactions are stronger than in the corresponding

protein–protein interactions. The ‘small area and strong bonding’ are the

characteristic of protein–carbohydrate interactions that provides the basis for

a fast on/off rate which is essential under physiological conditions, as it

allows for fast and highly changeable interactions to occur between cells

(J.Holgresson. et.al; 2005).

1.4 Lectins, Versatile proteins of recognition

The occurrence of erythrocyte-agglutinating proteins in nature has

been known since the turn of the 19th century. By 1960’s it became

apparent that such proteins also agglutinate other types of cells and that

many of them are sugar specific. These cell-agglutinating and sugar-specific

proteins have been named as lectins (Sharon and Halina Lis, 2004). The

word lectin is derived from Latin word ‘Legere’ meaning ‘to pick’ or select.

Lectins are ubiquitous natural proteins that bind carbohydrates with

characteristic specificities. The most recent accepted definition for lectins,

based on their carbohydrate binding property is, 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).

26

1.4.1 Lectin detection and assay

The presence of lectins is mainly detected by hemagglutination assay

(Kellens et al., 1989; Ozeki et al., 1991). Hemagglutinating activity of the

lectin is determined by two fold serial dilution technique in 96 well

microtitre assay plates using trypsinized erythrocytes. The highest dilution

of the extract causing visible agglutination is orbitararily considered as the

"titre” and the minimum concentration of the protein required for

agglutination (MCA) as “one unit of hemagglutinating activity”. The

specific hemagglutinating activity is expressed as activity unit per mg of

protein. In this assay, lectin is a serially diluted before incubation with

human, rabbit 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

is generally performed (Lee et al, 1990; Ozeki et al, 1991). Although the

hemagglutinating assay is rapid, sensitive, semi qunatitative and simple but

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 polyphenols such as

tannins (Makela, 1957) that are often present in plant tissues. Other methods

can also be used to detect the presence of lectins, such as a photometric

assay (Teichberg et al, 1988) precipitation of polysaccharides or

glycoproteins (Shibuya et al., 1989).

27

1.4.2 Hapten inhibition assay

Carbohydrate binding specificity of the purified lectin is determined

by hapten inhibition assay by incubating the lectin sample (diluted to give

titre value: 8) with serially diluted sugar (200mM) /glycoprotein (1mg/ml)

in a total volume of 50 µl, prior to the addition of erythrocytes and the

hemagglutination is visually observed. The lowest concentration of the

sugar/glycoprotein, which inhibited the agglutination is taken as the

inhibitory titre of the hapten.

1.4.3 General Classification of lectins

Generally lectins are classified into four groups based on their affinity

to different monosacharides or their derivatives as follows;

1. Glucose\Mannose

2. Galactose\N-acetylgalactoseamine

3. L-Fucose

4. Sialic acid

Lectins are also classified into different categories on the basis of

structural and/or evolutionary sequence similarities (Kumar et al, 2012).

Type I: Depending upon structural and evolutionary sequence similarities.

Type II: Depending upon proteins without established evolutionary

sequence similarities.

28

Type-I

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 (ER-associated degradation-enhancing alpha-mannosidase-like proteins)

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

P-type (mannose-6-phosphate receptors)

Galectins (formerly S-type lectins) Galactose binding lectins

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

B-type lectins: β-prism lectins -Jacalin-related lectins

F-type lectins: Ficolins- fibrinogen/collagen-domain-containing lectins

G-type lectins: Garlic and Snowdrop lectins and related proteins

H-type: Hyaluronan-binding proteins or hyaladherins

J-type: Amoeba lectins—Jacob and related chitin-binding proteins

T-type: Tachylectins from horseshoe crab Tachypleus tridentatus

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

X-type: Xenopus egg lectins/eglectins

Type- II

Annexins: Annexin lectins (Annexin-V binds tosialoglycoproteins and glycosaminoglycans)

Pentraxins with penta valent domain: C-reactive protein and serum amyloid P component

G-Domains: GTP binding protiens (Glycans on alpha dextroglycan)

CD11b/CD18 (β Integrins, CR3 ): fungal glycans and exposed GlucNAc residues on glycoproteins

29

1.4.3 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. The wide spread occurrence of lectins

suggest their role in various biological functions (Varki 1999).

1.4.3.1 Plant lectins

Majority of the lectins studied in the past are from plants. Several

hundreds of these proteins have been isolated and many of them are well

characterized also a large number of lectins have been sequenced. The three

dimensional structures of many plant lectins have been elucidated.

Although numerous plant lectins have been studied in great detail, the

physiological role of these proteins is still poorly understood. It is argued

that, lectins which accumulate in large quantities probably combine a

defense-related role against phytopathogeinc invertebrates or herbivorous

animals with a function as storage proteins [Van Damme et al., 1998;

Rudiger, 1998]. Apart from this some plant lectins are also shown to

mediate symbiotic association of leguminous plants and nitrogen fixing

bacteria [Brock & Madigan 1991] and they function as mitogenic

stimulators of plant embryonic cells [Ozato, 1977]. A large number of lectins

possessing mitogenic activity towards human and animal lymphocytes are

reported and have been presented in many reviews [Lis and Sharon, 1977,

1981; Ashraf and Khan 2004].

30

1.4.3.2 Animal lectins

The first lectin of animal origin was ‘‘mammalian hepatic lectin’’

which was discovered while studying the mechanisms that controlled the

turnover of glycoproteins in the blood circulation, however, the first animal

lectin activity was observed in snakes venom. Previously most of the animal

lectins were thought to belong to one of two primary structural families, the

C-type and S-type (presently known as galectins) lectins. Based on the

nature of their carbohydrate recognition domain, the biological processes in

which they participate, their sub cellular localization and their dependence

on divalent cations. Animal lectins are now classified into 12 families such

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.

31

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.

32

A frequent finding with animal lectins is an additional ability to bind

to structures other than carbohydrates via protein–protein, protein–lipid or

protein–nucleic acid interactions. This is often found when a carbohydrate

recognition domain is combined with an additional domain, a structural

feature that led to describe animal lectins in general as ‘‘bifunctional

molecules’’. There are times, however, when the carbohydrate binding site

or a locus close to it is responsible for binding to non-saccharide ligands.

Both phenomena are exhibited by collectins such as surfactant protein A or

mannan-binding lectin. Their collagen-like domains can bind to

C1q/collectin receptors by protein–protein interactions; the separate

carbohydrate recognition domain can bind to phospholipids as well as

saccharides, possibly using the same active site, for example, the non-

integrin elastin/laminin binding protein of 67 kDa binds to laminin and

elastin via hydrophobic and protein–protein interactions but also has a

separate galactose-binding site. These two activities, however, are not

independent: elastin binds with high affinity in the absence of galactose but

the presence of the latter or related sugars induces a conformational change

and a concomitant drop in affinity for elastin (D C. Kilpatrick, 2002). This is

an excellent example of how lectin–carbohydrate interaction can regulate

protein–protein interaction. Animal lectins have been drawing a great

attention for their multifaceted roles implicated in variety of biological

processes as indicated by large number of publications. Few examples of

endogenous lectins mediated biological processes includes, cell–cell self-

33

recognition, cell–extracellular matrix (ECM) interactions, gamete

fertilization, embryonic development, cell growth, cell differentiation, cell

signalling, cell adhesion, migration, apoptosis, inflammation, host–pathogen

interactions, glycoprotein folding and routing, mitogenic induction,

homeostasis and also some are known to function as antitumor and

immunomodulator molecules (Kawagishi et al., 1990; Beuth et al., 1992;

Wang et al., 1995; Ghazarian et al.,2011). Most of the animal lectins are

thought to belong to one of two primary structural families, the C-type and

S-type (presently known as galectins) lectins. Galectins are major class of

animal lectins and have been studied in detail from diverse sources.

1.4.3.3 Galectins

Galectins are animal lectins which are a structurally related family of

proteins widely distributed from lower invertebrates to higher vertebrates.

Galectins are generally small, non-glycosylated, soluble, Ca2+ independent

proteins found intracellularly or extracellularly and have at least one

characteristic carbohydrate recognition domain (CRD) with an affinity for β-

galactosides (Barondes et al. 1994). Galectins show a highly conserved tight

fold with two anti-parallel β-pleated sheets forming a sandwich-like

structure. Amino acid side chains on one of these sheets form the core

carbohydrate-binding site contain cysteine residues but no disulfide bond

and all SH groups are in a free state. Galectins studied so far have their N-

termini blocked due to acetylation (Cooper, 2001). To date, 15 different

34

galectins have been characterized; they are numbered according to the

chronology of discovery (galectin-1 to -15). On the basis of the spatial

arrangement of the CRDs, galectins are classified into three types, the

prototype galectins also called dimeric, contain one CRD (galectins-

1,2,5,7,10,11,13,14,15), the tandem repeat galectins also called biCRD

containing two CRDs (galectins-4,6,8,9,12) and galectin-3, the chimera

type galectin consisting of a large N-terminal region connected to a CRD.

The function of a given galectin can vary from site to site depending on the

nature of available ligands (Hirabayashi and Kasai 1993). Galectins are

mainly involved in a wide variety of cellular processes that include pre

mRNA splicing, cell growth regulation, cell adhesion, embryogenesis,

inflammation, immune function, apoptosis, angiogenesis and tumor

metastasis etc (Liu, 2012; Vasta, 2009). The biochemical and functional

properties of different members of the galectin family are summarized in

Table 3.

35

Table 3: Biochemical and functional properties of members of the galectin family

Galectins Localization Biochemical properties and functions

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

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

36

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

37

1.4.3.4 Bacterial lectins

Although 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 (Duguid

and Old 1980; Sharon 1989). Duguid and his co-workers showed that many

bacterial species, most commonly those belonging to the family of

Enterobacteriaceae showed hemagglutinating activity (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 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

mannosides (Ofek et al. 1977; Salit and Gotschlich 1997). Of course, 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.

Apart from their role in initiation of infection, 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

38

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

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

example of innate immunity, in which lectins are now known to be

involved.

1.4.3.5 Viral lectins

Influenza virus hemagglutinin is one of the most thoroughly studied

of all viral lectins. It is the first glycan binding protein was isolated from a

microorganism in 1950 that 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 like retrovirus, rotavirus, Sendai, and

polyomavirus also appear to recognize 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].

39

1.4.3.6 Fungal lectins

The discovery of fungal lectins was started by research on the

toxicology of higher fungi and the first fungal lectin Phallin was reported by

Kobert in 1981 from Amanita phalloides, which was a hemolytic agent. A

fungal hemagglutinin was discovered in the fly agaric later in 1911

(Ford, 1911). Although extensive studies are carried out and lot of literature

is available on plant and animal lectins very little information is available on

lectins from fungi. However, it is evident that the occurrence of lectins in

fungi is wider than in higher plants (Coulet et al., 1970). Some species have

been shown to contain not just one but several lectins; these lectins can be

closely related, differing for example in their isoelectric points (isolectins),

such as those isolated from Agaricus bisporus (Sueyoshi et al., 1985) or in a

small variation in molecular weight from changes in a few amino acids in a

subunit, such as is the case for lectins PCL-a, PCL-b and PCL-c from

Pleurotus cornucopiae (Yoshida et al.,1994). They also differ more widely

and display separate specificities as observed in Laccaria amethystea where

one lectin (LAF) binds to L-fucose while the other (LAL) is inhibited by

lactose (Guillot et al., 1983). Fungal lectins have been found mainly in

fruiting bodies and purified from them but very few have also been

identified in vegetative mycelia. Mushroom lectins have been localized on

different parts of the fungi like the caps, stipes and mycelia of mushrooms

and variation in lectin content occur depending on the age, time and place of

40

harvest (Ng, 2004). The presence of lectins may be related to the physiology

and ecology of the fungus (Khan, 2011).

Structural investigations of fungal lectins by X-ray crystallography

demonstrated that fungal lectins are unique class with approximately 140

amino acids with unique folding similar bacterial “porins”, a family of pore-

forming toxins (Cooper et al., 1997; Birck et al., 2004). Majority of them

are developmentally regulated and galactose specific, hence they are called

as fungal galectins (Oda et al., 2003; Swamy et al., 2004).

Various physiological roles have been postulated for fungal lectins;

none of them need be mutually exclusive and like plant lectins those in fungi

probably play differing roles in different circumstances. Some of these roles

seem to be concerned with the fungal metabolism itself, while other

activities are implicated in symbiotic or parasitic relationships with other

organisms. In addition, various roles have also been ascribed to the lectins in

lower fungi, such as in parasitic behaviour involving plants

(Hohl and Balsiger, 1986) or insects (Ishikawa, 1983), predation by many

species towards soil nematodes (Rosenzweig, 1983), involvement in growth

and morphogenesis (Kaneko et al.1993), involvement in dormancy

(Peumans et al, 1985) and involvement in molecular recognition during

early stage of mycorrhization (Giollant et al., 1991, 1993). Apart from this

fungal lectins are attracting scientists to work with them because of their

immunomodulatory effects and possible clinical applications.

41

1.4.4 Application of lectins in diagnosis and therapy of cancer

Within the past few years, lectins have become a well-established

means for understanding varied aspects of cancer and metastasis. Evidence

is now emerging that lectins are dynamic contributors to tumor cell

recognition (surface markers), cell adhesion and localization, signal

transduction across membranes, mitogenic stimulation, apoptosis,

cytotoxicity, and augmentation of host immune defence. To advance

understanding of these lectin-dependent processes, attempts are being made

to discover new lectins that have one or more of these functions and to

develop lectin (or glycoconjugate) based tools that could be used to home in

on tumor cells. The recent advances in the development of lectin-based

diagnostic and therapeutic tools for cancer are summarised as follows.

1.4.4.1 Use of lectins as diagnostic probe

Some lectins were previously used as simple tumor recognition tools

to differentiate malignant tumors from benign and the degree of

glycosylation associated with metastasis (Mody et al., 1995). In the recent

years, they have been developed for utilization as sophisticated microarray

for better recognizing malignant tumors in diagnosis and prognosis of

cancer (Gemeiner et al, 2009).

Tumors derived from epithelial tissues showing glandular

differentiation (tumors of colon, breast and female genetalia) are mostly

used for the study of lectin histochemistry. Glands are differentiated as

42

serous and mucous by analyzing their secretary product. Serous glands are

negative for general carbohydrate stain and the Periodic Acid Schiff’s

reagent (PAS) reaction, while mucous glands are PAS positive. Their lectin

binding patterns also reflect this property. Accordingly, lectins that

recognize the N-linked sugars in the cell membrane glycocalyx bind with

serous glandular cells. Such lectins include Concanavalin-A (Con A),

Phytohaemagglutinin-L (PHA-L) (Fernandes, 1991; Cummings, 1982) etc.

On the other hand lectins that identify O-linked glycoprotein bind to mucous

cells. These lectins are either Nacetylgalactosamine specific (Soyabean

agglutinin- SBA, Dolichos biflorus agglutinin-DBA and Helix pomatia

agglutinin-HPA) or Fucose specific (Ulex europaeus agglutinin-UEA-I).

Cell surface carbohydrate chains of human germ cell tumors were

investigated histochemically using peanut agglutinin (PNA), Dolichos

biflorus agglutinin (DBA), Ulex europaeus agglutinin-I (UEA-1), and anti-

(Ma) antibody. Peanut agglutinin, a lectin specific for terminal beta-

galactosyl residues, bound to the surface of tumor cells, D. biflorus

agglutinin was found to be specific for terminal alpha-N-acetyl

galactosamine residues and U. Europaeus agglutinin was tested to bind to

terminal alpha-L-fucosyl residues.

43

Table.4. Specificity of lectins towards different cancer [Sherwani, 2003]

Type of Cancer Specific Lectins Site of Action

Breast cancer HPA Roman snail (Helix pomatia)

Membrane glycoprotein

Transformed mammary cells

Cra Iso-I Cratylia mollis Carbohydrate binding patterns

Colon cancer PNA Peanut, VFA Vicia faba

Clonic mucin

Colorectal cancer SNA-I Sambucus nigra Epithelial cells,mucin

Pancreatic& Gastric cancer

PNA Peanut, BPA Bauhinia purpurea, VVA Vicia villosa

Sera glycoprotien, Secreted mucin

Hepatocellular carcinoma

AAL, Aleuria aurantia, AFP-L3Lens culinaris TJA-I Trichosanthes Japonica

Blood Serum

Genitourinary tract tumors

UEA-I, Ulex europaeus, PNA, Peanut, HPA. Helix pomatia

Bladder carcinoma

VAA Viscum album

Prostate cancer SBA, Soya bean HPA Helix pomatia

Tissues Bones

Endometrial carcinoma

UEA-I, Ulex europaeus PNA Peanut

Lung cancer VAA Viscum album HPA L, Helix pomatia, UEA-I, Ulex europaeus PVL Phasoeolus Vulgaris

Apart from their application in cancer diagnosis lectins are also

gaining significance as diagnostic agents for detection of other disease-

related alterations of glycoconjugates.

44

Table.5. Detection of disease-related alterations of glycoconjugates with lectins [Mislovicova, 2009]

Lectins Alterations of glycosylation

Diseases or physiological states Methods

LCA, Con A, SNA Transferrin in human serum; sialylation of transferrin

Traumas or chronic inflammations

CAIE, ELLA

LEA, RCA, Con A Pluripotent hESC Human pluripotent embryonic stem cells

Flow cytometry

MAA, SNA MAA

Post translational modification of monocyte derived dendritic cells.

Sialylation of surgical specimenof thyroid gland.

α1-acidoglycoprotein in seminal plasma Lysosomal sialic glycoproteins

Immature tolerogenic State

Thyroid gland Inflammatory in reproduction tract of men. Sialidosis and galactosialidosis

Flow cytometry

Histochemistry

Western .blott

CAIE, ELLA

DBA, SBA

Glycosylation of mucin goblet cells

Acute phlegmonous appendicitis

Histochemistry

LTG Ileal mucus of infants Cystic fibrosis Histochemistry WGA, SBA, SJA,BSL1, PHA, Con A, PSA, LCA

Glycosylation of jejunal mucosa

Celiatic disease Histochemistry

RCA, Con A, UEA,AAL

IgG in human serum, synovial fluid

Rheumatoid arthritis and juvenile chronic arthritis, RA, JIA,GA,ReA

Lectin blotting

RCA, GSA, WGA, SNA

Human serum glycoprotein

Rheumatoid arthritis Surface Plasma Resonance

HAA, HPA, HAA Serum IgG and IgGA

Rheumatoid arthritis, IgA nephropathy

ELLA, Western blotting,ELLA

Con A Follicle-stimulating hormone (FSH). Conjuctiva keratoconus

Pubertal anorchid boys Pterygium conjunctiva

LAC Histochemistry

TML, SNA, MAA Lungs during intrauterine development

Maturity of fetus Histochemistry

GNA, LPA, LCA, PSA, PSL

Tissues of human placenta

Glycan composition of human placenta

Histochemistry

45

1.4.4.2 Lectins as antitumor agents

Cancer is one of the major life threatening diseases worldwide.

Cancer therapy is pursued with great scientific vigour. The successful

identification of any novel effective anticancer molecules/drugs is largely

dependent on testing its efficacy by the use of appropriate preclinical

experimental models. These models should possibly mimic the complexity

of different cancer diseases and investigate mode of action of drugs and

antitumor activity, in order to select them for further clinical investigation.

The typical development plan for a new anti-cancer agent involves

sequential steps: in vitro assays, both cell based and molecular target-driven

for the identification of an active compound; in vivo studies to assess the

potential antitumour activity; pharmacological studies to define drug

absorption, distribution, metabolism, and elimination; finally, toxicological

studies to define a safe starting dose in humans [Burger A.M, 2004; Aherne

W, 2002].

For the initial screening generally cell lines derived from all the major

human neoplasms that might mimic the heterogeneity of the biological

features of human tumours are being used to evaluate a large number of

anticancer agents in a disease oriented manner and also to clarify

mechanism of action of drugs and to identify their exact molecular targets

[Goldin A, 1983]. In 1990 the US National Cancer Institute (NCI)

introduced an initial panel of 60 cell lines representing nine distinct tumour

46

types, including leukaemia, colon, lung, CSN, renal, melanoma, ovarian,

breast and prostate cancer [Holbeck SL, 2004]. Compounds are generally

tested over a 5-log concentration range against each of these cell lines for

their ability to inhibit the growth or to kill the cells in a 2-day assay

generating dose-response curves. To facilitate the analysis and interpretation

of the data, three end-points are calculated for each cell line:

(1) The GI50 value that is the negative log10 of the concentration required

to inhibit the growth of that cell line by 50% (relative to untreated

control cells).

(2) The TGI that is the negative log10 minimum concentration that causes

total growth inhibition and

(3) The LC50 that reflects the negative log10 concentration needed to kill

50% of the cells.

These data generate a characteristic fingerprint of cellular response,

the ‘Mean Graph’, profiling the sensitivity/resistance of the compound on all

the cell lines. Generally compounds with similar mechanisms of action tend

to have similar patterns of growth inhibition [Ma WW, 2009; Paull KD,

1989; Sausville EA, 1999].

Once selected compound has demonstrated robust cytotoxic activity

against a panel of human cancer cell lines, it deserves further investigation

in in vivo models. The in vivo models are used to select compounds for

further clinical development. These in vivo models mainly include

subcutaneously implanted human tumour xenografts. Xenograft tumours

have originally been established by inoculating subcutaneous tumour cells

47

growing in vitro into nude mice or SCID (Severe combined immuno-

deficiency) mice which are immuno-deficient and allow the growth of a

human tumour.

Establishment of subcutaneous (s.c) human tumours has been obtained

either by inoculum of cells derived from human tumour cells cultured in

vitro and by direct implantation of patient’s tumour biopsy derived

fragments or cells. The s.c implant allows the monitoring of tumour growth

using in situ calipers and allows testing the activity of a compound in

different settings: agents can be administered at the same time as tumour

implantation (chemoprevention strategy), treatment can start when tumours

are just palpable (‘early stage’ strategy) although in such cases one needs to

be aware that the residual immune capacity of the host (mainly natural killer

activity) may influence the tumour response and finally, the treatment can as

well start when tumour masses are bigger (‘late stage’ strategy) [Kelland

LR., 2004; Fiebig HH, 1990; Sausville EA, 2006; Kerbel RS, 2003].

Activity is generally defined as tumour growth delay, optimal % T/C

(median treated tumour mass/median control tumour mass expressed in %)

or net log cell kill. Toxicity can be evaluated by monitoring loss in

bodyweight and drug-related deaths. Several studies reported so far have

supported the value of the s.c. xenograft model in predicting the clinical

activity of cytotoxic agents. Generally, the determination of maximum

tolerated dose (MTD) is performed in a way that conserves compound and

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minimizes the number of animals sacrificed. Thus, a single mouse is given a

single injection by any of the different routes (IP, IV, SC, IM or Oral) of

400 mg/kg (or lower if the compound is anticipated to be extremely potent);

a second mouse receives a dose of 200 mg/kg and a third mouse receives a

single dose of 100 mg/kg. The mice are observed for a period of 2 weeks

and are sacrificed if they lose more than 20% of their body weight or if there

are other signs of significant toxicity. If all 3 mice must be sacrificed, the

next 3 dose levels (50, 35 and 12.5 mg/kg) are tested in a similar manner.

This process is repeated until a tolerated dose is found [Rathod C.P,2011;

Olive KP, 2006].

Lectins are known to possess antitumor activities and effect is

mediated by different mechanisms. Some of the lectins s are known to

possess antitumor activities via targeting programmed cell death (PCD),

which is a cell-intrinsic mechanism for eliminating harmful cells and

maintaining homeostasis, including apoptosis and autophagy (Li et al.,

2009). The word apoptosis derived from Greek ‘apo’, meaning from, and

‘ptosis’, meaning falling or type I programmed cell death, is a complex but

highly defined program of cell death (Cotter, 2009). Autophagy, a term

derived from Greek “auto” (self) and “phagy” (to eat), refers to an

evolutionarily conserved, multi-step lysosomal degradation process in which

a cell degrades long-lived proteins and damaged organelles (Wang et al.,

2011b). There are multiple connections between apoptosis and autophagy

pertaining to cancer that may jointly seal the ultimate fate of cancer cell

49

(Giansanti et al., 2011). As some lectins are known to posess anti-

proliferative activities, they have been investigated to elucidate their

complicated mechanisms, especially highlighting PCD pathways, via (1)

directly inactivating ribosome of cancer cell (2) endocytosis and selectively

localizing on some organelles such as mitochondrion in cancer cell or (3)

binding certain sugar-containing receptors on the surface of cancer cell.

Many lectins are reported to elicit apoptosis in different cancer cell lines,

examples include Mistletoe lectins (MLs), a well-studied type II ribosome

inactivating proteins (RIPs II), possess anti-proliferative activities toward

various types of cancer cells (e.g., human acute lymphoblastic leukemia

cells, human hepatocarcinoma cells, human A549 lung cancer cells and

human myeloleukemic U937 cells) ML-I is shown to possess more

sensitivity to apoptosis induction by Tumor Necrosis Factor (TNF-α), which

further suggests the cooperation between ML-I and TNF-family death

receptors in determining cancer cell death (Pryme et al., 2006; Hoessli and

Ahmad, 2008). In addition, ML-I was found to induce apoptosis by

activating caspase-8 via the extrinsic apoptotic pathway, but independent of

death receptor pathway (Bantel et al, 1999). Additionally, Korean mistletoe

lectin (KML), belonging to ML-I, has been further demonstrated to induce

apoptosis by a mitochondrial pathway independently of p53 in

hepatocarcinoma cells (Lyu et al., 2002) by the breakdown of mitochondrial

membrane potential (MMP) and caspase-3 activation independently of p53,

but apoptosis-associated factor-1 (Apaf-1)-dependent pathway (Hostanska et

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al., 2003). Also, ML-I has been shown to alter MMP; thereby resulting in

cytochrome c release and increase of the levels of reactive oxygen species

(ROS) in Hep3B cells (Lavastre et al., 2002).

Recent studies have demonstrated that ConA, one of the most

investigated Glc/Man specific lectin bears apoptosis-inducing activities in

human melanoma A375 cells and human hepatocellular liver carcinoma

HepG2 cells. Initiation of this apoptotic cell death shown to be mediated by

mitochondria apoptotic pathway: MMP collapse, cytochrome c release and

caspase 9/3 activation, and thus eventually culminating in apoptosis (Liu et

al., 2009d, 2010c). In addition, Src homology 2 (SH2) domain-containing

protein tyrosine phosphatase substrate 1(SHPS-1) has been reported for the

first time to be regarded as an important receptor for ConA. This lectin

directly binds to the extracellular region of SHPS-1 and this interaction

mediates ConA-dependent activation of Akt and secretion of MMP-9,

indicating that SHP-2 is recruited to SHPS-1 upon ConA-stimulation

required for ConA-dependent Akt activation. However, to date, only one

report has demonstrated that ConA can induce autophagic cell death in

hepatoma cells through a mitochondria-mediated pathway. ConA after

associating with mannose moiety on the cell membrane glycoprotein, lectin

preferentially internalized to the mitochondria via clathrin-mediated

endocytosis, and then initiates the autophagic cell death (Chang et al., 2007).

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Some lectins with potential antitumour activities are also in clinical trial. For

example, ML-I has been widely utilized in clinical trials as potential

antineoplastic drug or adjuvant therapeutic agent. Notably, this lectin has

been indicated for the reduction of treatment-associated side effects as

adjuvant agents during chemotherapy and radiotherapy in Europe for several

decades (Liu et al., 2010). Recently, European mistletoe lectins have been

tested for the safety and efficacy during post-surgical after care of primary

intermediate to high-risk malignant tumor patients (stages II–III), comparing

with untreated control group. And, the long-term treatment of these lectins

are reported to be safe and without any further tumor enhancement [De

Mejía and Prisecaru, 2005; Liu et al., 2010] that encourages to explore

lectins as anticancer drugs. Todate we have sizable number of lectins which

excecute antiproliferative or cytotoxic effect on cancer cells and hence have

antittumour effects which are briefly summarised in the following table.

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Table.6. Cytotoxic and anticancer effect of lectins on malignant cells

Lectin Tumor cells Effect exerted AAL H3B human hepatoma, human

choriocarcinoma, and ROS rat osteosarcoma

Cytotoxicity/tumor inhibition, Apoptosis

ABA

LS174T, SW1222, and HT29 human colon cancer

Cytotoxicity/tumor inhibition, cell agglutination/aggregation

JAC

HT29 human colon cancer Adenomatous polyps and colorectal neoplasms

Cytotoxicity/tumor inhibition Direct contact/adhesion/binding to cell membrane or receptors

WGA Human pancreatic carcinoma U937 human monoblastic leukemia. LS174T, SW1222, and HT29 human colon cancer, H3B human hepatoma, Jar human choriocarcinoma, Adenomatous polyps and colorectal neoplasms

Direct contact/adhesion/binding to cell membrane or receptors, internalization of lectin, apoptosis, chromatin condensation/ nuclear fragmentation/DNA release. Cytotoxicity/tumor inhibition, apoptosis, G2/M phase cell cycle arrest, cell agglutination/aggregation

VCA

SK-Hep-1 (p53+), Hep 3B (p53−) hepatic cancer, Human breast cancer

Apoptosis, downregulation of Bcl-2/upregulation of Bax, downregulation of telomerase activity. Increased TNF-α, IL-6, IFN-γ and/or IL-4 secretion, Th1- shift in the Th1/Th2 balance

ML-I

Molt-4 human lymphocyte Cytotoxicity/tumor inhibition, Ribosome binding/inhibition of protein synthesis, direct contact/adhesion/binding to cell membrane or receptors, internalization of lectin and apoptosis

ML-II

Molt-4 human lymphocyte, U937 human monoblastic leukemia, U937 human myeloleukemic, Jurkat T, RAW 264.7, HL-60, DLD-1, primary acute myelocytic leukemic, Malignant melanoma

Cytotoxicity/tumor inhibition, Apoptosis, activation of extracellular signal-regulated kinases, activation of p38 mitogen-activated protein kinase, alteration of cellular signaling pathways, Apoptosis, activation of the caspase cascade

Con A

Merkel cell skin carcinomas, SK-MEL-28, HT-144, and C32 human melanoma Hs729 (HTB-153) human rhabdomyosarcoma, SK-UT-1 and SK-LMS-1 human leiomyosarcoma

Direct contact/adhesion/binding to cell membrane or receptors, Cytotoxicity/tumor inhibition

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Considering their anti cancer effect in vitro with different cell lines

some of the lectins are also further explored and reached to pre-clinical and

clinical trials. The available updates are summarised in the following Table.

Table.7. Pre-clinical and clinical studies of lectins for treating human cancers:

Lectin Effect Tested with Cancer type Effect Stage ConA Murine B16 melanoma cells, Murine

BALB/c hepatoma ML-1 and colon CT-26 cells; human hepatocellular liver carcinoma Huh-7 cells and HepG2cells Autophagic death

Cytotoxicity/tumor inhibition

Pre-clinical

PHA-L

Murine non-Hodgkin lymphoma tumor, Human melanoma cells, human rhabdomyosarcoma cells, human leiomyosarcoma cells, SP2 myeloma cells, Lox-2 Ab-producing hybridoma cells, B-DLCL human large B-cell lymphoma cells

Cytotoxicity/tumor inhibition and apoptosis

Pre-clinical

ML-I

Murine non-Hodgkin lymphoma (NHL) tumors, human ovarian cancer in SCID mice, chemically induced urinary bladder cancer in mice, murine melanoma, urinary bladder carcinoma MB49, and B16-BL6 melanoma cells. Human molt-4 lymphocyte cells, human cervical carcinoma HeLa, and human breast carcinoma MCF-7 cells

Cytotoxicity/tumor inhibition and apoptosis Cytotoxicity/tumor inhibition and apoptosis

Pre-clinical Clinical stages II–III

VAA

Urinary bladder carcinoma MB49 in mice Cytotoxicity/tumor inhibition, reduction of malignant phenotype,(inhibition of metastasis)

Pre-clinical

WGA Colon carcinoma in F-344 rats, Human colorectal cancer Inhibition of metastasis, better prognosis/longer survival times

Cytotoxicity/tumor inhibition

Pre-clinical

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1.4.4.3 Application of lectins in lectin-mediated drug targeting/delivery

Success in the treatment of cancer with chemotherapy is often limited

by the non-specific toxicity of anticancer drugs on normal cells. Intravenous

administration of anti-cancer chemotherapeutic reagents produces severe

tissue and organ damage due to their cytotoxic effects on normal cells.

Current therapeutic approaches to the treatment of cancer are thus focused

on developing novel drug delivery systems to increase the therapeutic

efficacy of anticancer agents by targeting them to malignant cells. A variety

of strategies and carrier molecules have been used to direct therapeutic

agents to tumor sites [F. Alexis et al, 2010]. One such approach to specific

drug delivery is lectin-based targeting of Drug Delivery System (DDS),

which may be accomplished via two mechanisms: direct lectins targeting

and reverse lectin targeting (Plattner et al, 2009). In direct lectin targeting,

the DDS has carbohydrate moieties that are recognized by endogenous cell

surface lectins. In reverse lectin targeting, the DDS has exogenous lectins

that recognize endogenously synthesized carbohydrate moieties on

glycolipids and glycoproteins (Bies et al., 2004; Minko, 2004). Lectin-based

DDSs could be greatly beneficial in cancer therapy, not only due to their

specific binding abilities but also their cytotoxic and apoptosis inducing

potentials (Kim et al., 1993; Gorelik, 1994; Mody et al., 1995; Ma et al.,

1999; Minko, 2004; Thies et al., 2005). Another strategy to DDS is to

synthesize lectin–monoclonal antibody conjugates that can specifically bind

to target tumor cells and induce cytotoxic effects (Mody et al., 1995). In this

55

system the lectin is the toxic entity and the antibody is a monoclonal tumor-

specific antibody. Hence virtually any tumor can be neutralized by using

tumor-specific monoclonal antibodies. The toxic lectins typically used in

such trials are plant lectins such as ML-I or the A-chain of ricin (Tonevitsky

et al., 1991; Paprocka et al., 1992).

Table.8. Lectins and their application as drug delivery systems:

Lectin Specificity Use

Viscum album agglutinin

D-Gal; GalNAc Targeting to GI

Lycopersicum esculentum agglutinin

(GlcNAc)3 Targeting to the lungs (alveoli, type I cells)

Urtica dioica agglutinin GlcNac Targeting to GI and uptake

Triticum Vulgaris agglutinin

(D-GlcNAc)2; NeuNAc Targeting to GI, lung, eye, blood-brain barrier and uptake

Galanthus nivalis agglutinin

Manα3Man Targeting to GI

Bandeireae simplifolia isolectin B4

α-D- Gal Targeting to nasal mucosa

Maclura pomifera agglutinin

GalNAc Targeting to the lungs (alveoli, type II cells)

Ricinus communis agglutinin

D-Gal Targeting to the lungs (alveoli, type I cells)

Cholera toxin agglutinin GM1 ganglioside Targeting to GI

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1.5 Genesis of the Present Thesis

Research in our Glycobiology laboratory has been mainly focused on

investigating plant and fungal lectins for their structure, biological functions

and various physiological and cell biological applications. Two lectins

namely Sclerotium rolfsii lectin (SRL) and Rhizoctonia bataticola lectin

(RBL) which are purified and characterized in our laboratory are known to

recognize tumor associated glycans/antigens.

Sclerotium rolfsii is a soil borne plant pathogenic fungus with a host

range of over 500 species including agricultural crops like sunflower,

potato, tomato etc. [Punja, 1985]. SRL was purified from the sclerotial

bodies of Sclerotium rolfsii by conventional chromatographic techniques

and lectin has been shown to recognize cancer associated Thomsen-

Friedenreich antigen (TF); Galβ1-3GalNAcα-O-Ser/Thr, [Swamy et al.

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

physiological role of the lectin has been demonstrated in 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], showing the presence of two

carbohydrate binding sites per SRL monomer. The primary binding site of

SRL recognizes the TF disaccharide and GalNAc and the secondary binding

site recognizes GlcNAc. The detailed carbohydrate binding specificity of

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SRL has been determined by glycan array analysis at Consortium for

Functional Glycomics (CFG), USA, which revealed its specific binding not

only to cancer associated TF but also towards TF-associated O-linked

glycans. [Chachadi et al; 2011]. Since the TF antigen is expressed in all

types of human cancer cells and the specificity of SRL to this cancer-

associated carbohydrate antigen prompted us to investigate its interaction

with and physiological effect on human colon and breast cancer models as

well normal mammary epithelial cells.

Rhizoctonia bataticola is another plant pathogenic fungus with a host

range of more than 100 species including potato, sunflower etc. [Dhingra

and Sinclair 1978]. A lectin 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.

The Glycan array analysis of RBL revealed its exclusive specificity 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. Hence

interaction of RBL has been studied with human ovarian cancer PA-1 cells

revealed that RBL is cytotoxic to PA-1 cells. [Nagre et al. 2010; Eligar et.

58

al. 2013]. As RBL showed potent cytotoxicity its effect on

chemotherapeutic drug resistant ovarian cancer cells is studied further and in

vivo antitumor effect of RBL tested in mice model to exploit its potential in

combating ovarian cancer. Considering the exquisite binding specificities of

both SRL and RBL towards the cancer associated antigens, present study

was carried out with the proposal of following objectives.

To investigate the effect of SRL on the proliferation of human colon

cancer cells using in vitro and in vivo models.

To investigate the effect of SRL on proliferation of human breast

cancer in comparison with normal epithelial cells.

To evaluate antitumor efficacy of RBL using ovarian cancer

xenografts in mice models.