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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
11
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).
15
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)
16
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
18
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
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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.