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ISOLATION OF LECTINS FROM DIFFERENT PLANT SOURCES AND THEIR MITOGENIC ACTION
ON HUMAN LYMPHOCYTES
DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OP
MdLittx of pt)tlo0opl)p IN
ZOOLOGY
BY
FAUZIA KHAN
SECTION OF GENETICS DEPARTMENT OF ZOOLOGY
ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)
2001
•^r
%' ^DS^3Z55
S"-^tlJi?_'Jnr7S^
DS3255
Lecturer
Md, Asim Azfer, PhD (mpm DEPAERTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY
Section of Genetics '^^^W ALIGARH-202002 INDIA
Date:..Pi./MM?rr
CERTIFICATE
This is pleasure to certify that the work presented in this dissertation entitled "Isolation of
Lectins fron different plant sources and their mitogenic action on human
lymphoot "es" is done by Miss Fauzia Khan under my supervision, in the Department of
Zoology of Aligarh Muslim University, Aligarh. The subject matter, presented by Miss Fauzia
for the award of Master of Philosophy (M.Phil) is the actual record of work and the sanjjp
does not form the part of any other course or degree presented to or taken by her.
(Md.AsiniAzfer,PhD)
Tel. 0091-571-708325 (Res.) Email: [email protected]
Dedicated to
My Abba
CONTENTS
Acknowledgment i
List of Tables ii
List of Figures iv
1. Introduction 1-22
A General Account 1
Occurrence 4
Structure 5
Biological Roles 9
Applications 13
Mitogenic Activity 18
Objectives 21
2. Material and Methods 22-29
Isolation of lectins 24
Determination of protein concentration 24
Haemagglutination Activity 25
Purification of Lectins 25
Polyacrylamide gel electrophoresis 26
Lympiiocyte Transformation Test 27
3. Results
Protein Concentration 30
Haemagglutination Activity 30
Purification 31
Mitogenic Activity 38
4. Discussion 52-60
5. References 61-73
AKNOWLEDGEMENT
AlhamduliUah, for he gave me opportunity to express appreciation to those
who have really helped during completion of my work.
First of all I want to express my sincere gratitude to my respected supervisor
Dr. Md. Asim Azfer, Lecturer, Department of Zoolog}% AMU, Aligarh for
enthusiastic supervision, excellent guidance and proper direction he provided during
tenure of mv work.
My sincere regards and honor to Prof. A. K. Jafri, chairman, Department of
Zoology for providing necessary lab facilities for completion of my work.
I am deeply indebted to Dr. Rizwan Hasan IQian, Interdisciplinary center
for Biotechnology, AMU, Aligarh for his valuable advice and support. He has been
kind enough in providing necessary help. My Sincere regards are for all the teachers
111 Department of Zoology, who have always motivated students for good. I extend
my thanks to all the research scholars in Department of Zoology.
I owe a special thanks to my lab colleagues Dr. GGHA Shadab, Asma
Farhat Sherwani, Sameena Mohmood and Hifzur Rehman Siddique and
appreciate their cooperation, helpfulness and advice during tenure of my work. I also
want to extend sincere thanks to Aabgeena Nairn, research scholar, Interdisciplinary
center for Biotechnology, AMU, AUgarh and Dr. Adil Rehman, Department of
Biochemisti-)-, JN Medical College AMU Aligarh for the timely assistance they
provided.
My words will be falUng short when I thank my family and friends for the
constant blessings and emotional support, they have endowed.
|^X«A/V
LIST OF TABLES
Title Page
1. Lectins with Specificity for Monosaccharide 2
2. Lectins with Specificities for Oligosaccharides 3
3. Isolectins with different carbohydrate specificities 7
4. Biological Roles of lectins in Nature 10
5. Application of Lectins in biology 14
6. Lectins showing human A, B & O blood group specificity 16
7. Lectins with Mitogenic Activity to Blood Cells20
8. Quantity of Protein isolated from seeds of various 32
species
9. Agglutination activity of lectins from four species. 32
10. Mitotic index and % blastogenesis in lymphocyte cultures 39
stimulated by partially purified lectins from Phaseolus vulgaris.
11. Mitotic index and % blastogenesis in lymphocyte cultures 39
stimulated by purified lectins from Phaseolus vulgaris.
12. Mitotic index and % blastogenesis in lymphocytes 40
stimulated by partially purified lectins from Lens culinaris.
13. Comparison of mitotic indices of the lymphocytes stimulated 40
by partially purified and purified lectins {Phaseolus vulgaris) with
commercial PHA.
14. Comparison of% Blastogenesis In lymphocytes stimulated ' 41
by partially purified and purified lectins from
Phaseolus vulgaris, Lens culinaris and commercial PHA.
LIST OF FIGURES
Titles Page
1. Standard curve for the determination of Protein 24
Concentration.
2. Elution profile of lectin from Phaseolus vulgaris 33
3. Elution profile of Lectin from Vigna Radiata 34
4. Absorption Spectrum of Lectin from Phaseolus vulgaris 3 5
5. Absorption Spectrum of Lectin from Vigna Radiata 36
6. Poly acrylamide Gel Electrophoresis of lectins 37
7. Mitotic index and % blastogenesis in lymphocytes 42
cultures stimulated by partially purified lectins from
Phaseolus vulgaris
8. Mitotic index and % blastogenesis in lymphocyte cultures 43
stimulated by partially purified lectins from Phaseolus
vulgaris
9. Mitotic index and % blastogenesis in lymphocyte cultures 44
stimulated by purified lectins from Phaseolus vulgaris
10. Mitotic index and % blastogenesis in lymphocyte cultures 45
stimulated by purified lectins from Phaseolus vulgaris
11. Mitotic index and % blastogenesis in lymphocytes stimulated 46
by partially purified lecfins from Lens culinaris
12. Comparison of mitotic indices of the lymphocytes stimulated 47
by partially purified and purified lectins {Phaseolus vulgaris)
With commercial PHA
iii
13. Comparison of % Blastogenesis in lymphocytes stimulated 48
by partially purified and purified lectins fi-om Phaseohis
vulgaris, Lens culinaris and commercial PHA.
14. Human metaphase spread (46 XX) fi-om the stimulated 49
lymphocytes by purified Lectin Phaseolus vulgaris
15. Human metaphase spread (46 XX) fi-om the stimulated 50
lymphocytes by purified Lectin Phaseohis vulgaris
16. Blasts cells induced by purified lectin from Phaseohis 51
vulgaris on human lymphocytes
IV
INTRODUCTION
INTRODUCTION
A General Account
Lectins are multivalent carbohydrate binding proteins that are grouped
together because they agglutinate cells or other objects, which display more than
one saccharide of sufficient complimentarity. These were first discovered by H.
Stillmark (1888) in extracts of Ricinus communis (Castor bean) as
haemagglutinins that agglutinate erythrocytes by a factor of non-immune origin.
Later the term 'Lectin' was proposed to describe plant agglutinins with blood
alloantigen specificity (Boyd and Shapleigh, 1954). The term lectin is derived
from Greek word "legere" meaning to "choose" thus refers to the specificity of
the reaction. Afterwards in 1940s agglutinins were discovered which could select
type of cells, based on their blood group activities.
At present lectins are defined as "Multivalent carbohydrate binding
proteins of non-immune origin that agglutinate cells or precipitate
glycoconjugates." Although most lectins can agglutinate some cell types, cellular
agglutination is not a prerequisite. The obligatory property of saccharide binding
is universally accepted but opinions differ as to whether lectins must be able to
agglutinate (Goldstein et al., 1980) or not (Kocureck and Horejsi, 1983). Sugar
specific enzymes, transport proteins and toxins may classify as lectins if they
have multiple binding sites.
Lectins bind to carbohydrate moieties of complex glycoconjugates
reversibly without altering the covalent structure of any of the recognized
glycosyl bond. Binding of a lectin to a sugar residue is very specific. Typical
ligand binds preferentially to a given sugar residue being it a monosaccharide
such as a-gal, |3-gal, a-GalNAc etc., a disaccharide or an oligosaccharide
(Goldstein & Hayes, 1978). In Tables 1 and 2 lectins with different mono and
oligosaccharide specificities are shown. The specificity of a lectin is defined by
Table 1: Lectins with Specificity for Monosaccharide
Source of Lectin
Abriis precatorius
Caragana arborescens
Codium fragile
DoUchos biflovus
Glycine max
Helix aspersa
Helix pomatia
Lathyrus odoratus
Lens cuUnaris
Limulus polyphemus
Lycopersicon esculentum
Phaseolns limensis
Pisiim sativum
Pseiidomonas aeruginosa
Ptilota plumose
Sophora Japan ica
Tetragonolobus
purpureas
Viciafaba
Vigna radiata
Wisteria floribunda
Monosaccharide
D-Galactose
D-GalNAc
D-GalNAc
a-D-GalNAc
D-GalNAc
D-GalNAc
D-GalNAc
a-D-mannose
a-D-mannose
NeuNAc
D-(GlcNAc)3
D-GalNAc
a-D-mannose
D-Galactose
a-D-Galactose
p-D-galNAc
a-L-Fucose
D-Glucose
a-D-Galactose
D-GalNAc
Reference
Olsnesetal., (1974)
Blochetal., (1976)
Rogers etal., 1986
Etzler &Kabat, 1970
Lotan et al., 1974
Hammarstrom et al., 1974
Hammarstrom et al., 1974
Tichaetal., 1980
Howard et al., 1971
Fernandez-Menon et al.,
1968
Nachbar et al., 1980
Galbraith & Goldstein,
1972
Trowbridge et al., 1974
Gilboa-Garber, 1982
Rogers & Blanden, 1980
Poretz etal., 1974
Periera & Kabat, 1974
Matsumotoet al., 1983
Hankins & Shannon,
1978
Kurokawaet al., 1976
Table 2 : Lectins with Specificities for Oligosaccharides
Source of Lectin Datura stramoniuma
Dolichos biflorus
Erythrina cristaglli
Arachis hypogaea
Phaseolus vulgaris (E-PHA)
Phaseolus vulgaris (L-PHA)
Vicia graminea
Vicia villosa
Triticum vulgare
Oligosaccharide GlcNAcp4GlcNAc(34GlcNAc
GlcNAca3GlcNAc
Gaip4GlcNAc
Gaip3GlcNAc
Gaip4GlcNAcp2Mana6 GlcNAcp4 ^ M a n p 4 - R
GlcNAcp2Mana3
Galp4GlcNAcp2^ Man
Galp4GlcNAcp2^
NH2-Leu
(Gaip3GalNAca)-Ser
(Gaip3GalNAca)-Thr
Galp3Ga]NAca)-Thr
Glu-COOH
NH2
GalNAc-Ser
(Pro)2
Gly
(Ala)2
(GalNAca)-Thr-COOH
GlcNAcp4 GlcNAcp4 GlcNAc
Reference Crowley et al., 1984
Baker etal., 1983
Kaladas et al., 1982
Goldstein & Hayes, 1978
Yamashita et al., 1983
Hammarstrom et al., 1982
Prigent et al., 1984
Tollefsen & Komfeld,
1983
Goldstein & Hayes,
1978
3
inhibition of liemaggiutination or polysaccharide precipitation by the lectin.
Radioactive and fluorescent detection techniques show high sensitivity. Sugar-
binding activity of lectins can be ascribed to a single protein module within the
lectin polypeptide, designated as carbohydrate recognition domain (CRD).
OCCUIIANCE
Lectins are widely distributed in nature being found in animals, insects,
plants and microorganisms (Leiner et al 1986; Sharon and Lis, 1989). Their
resources are described as follows:
Plant lectins:
Lectins were first discovered in plants during 19"' century when extracts
from castor beans were found to agglutinate erythrocytes of different animal
species. Since then lectin containing plants have been found in many botanical
groups including mono and dicotyledons, molds and lichens. Most frequently
they have been observed in leguminoseae and Euphorbiaceae. Lectins have also
been found in other families such as Verbenaceae, Malvaceae, Polygonaceae,
Solanaceae, Acanthaceae, Ranunculaceae, Caprifoliaceae, Cucurbitaceae and
Cactaceae etc. (Sharon and Lis, 1990). The largest and the best-characterized
family is that of Leguminoseae lectins. In mature seeds of leguminous plants
lectin may constitute as much as 10% of the total protein. Bulk of protein is
located in cotyledons in organelles known as protein bodies.
Microbial lectins:
Some strains of Escheritia coli as well as other species have fimbrieae
protruding from the cell surface, which contains Lectins. Type I fimbrieated
strains of Escheritia coli are known to bind mannose and are essential for
infectivity. P fimbriated Escheritia coli bind to disaccharide gal a(l-4)gal, a
constituent of glycolipid (Vaisanen-Rhen, 1984; Karch et al., 1985), susceptible
constituent of glycolipid (Vaisanen-Rhen, 1984; Karch et al., 1985), susceptible
in urinary tract tissues. In addition there are many examples of microbial
lectins, which bind to other bacterial cells. Carbohydrates can inhibit many
interactions among bacterial species, which colonize mouth. These interactions
are thought to result from lectin binding.
Animal lectins:
In 1979 Ashwell and Morel discovered a galactose binding protein in
liver membrane while attempting to label serum glycoprotien. Shortly thereafter
a large number of lectins were discovered illustrating their ubiquitous nature.
Animal lectins that require Ca" for binding, constitute the family of C type
lectins. The first identified animal lectin is prototype of C type. All C type
lectins are large, multidomain composite protein with a carbohydrate
recognition domain (CRD) and other fiinctional domain such as transmembrane
coUagen domain, epidermal Growth Factor (EGF) domain. CRDs provide sugar
recognition activity and then other modules initiate biological processes such as
adhesion, endocytosis and complement fixation. Among C type lectin family
selectins form a distinguished subfamily by their specific fimction in leukocyte
adhesion to endothelial cells through sialyl-Lewis X recognition. CoUectins are
another subfamily specific for mannose and possess a unique collagen like
domain. They are supposed to be involved in innate immunity.
Another grov dng family of animal lectins is galectins. All of them share
galactose specificity. Annexins is a group of proteins having afifmity to lipids
but recently proved to be lectins, sharing a certain binding activity to
glycosaminoglycans.
STRUCTURE:
One major property of lectins is their specific saccharide binding sites.
Lectins from any source are detected and quantitated by their ability to
agglutinate erythrocytes and are readily purified by afifmity chromatography on
immobilized carbohydrates (Lis & Sharon, 1984; Lis & Sharon, 1981). They are
classified in small number of specificity groups, according to the
monosaccharide that is effective inhibitor of the agglutination of erythrocytes or
precipitation of carbohydrate-containing polymers by the lectin e.g. Mannose,
galactose, N-acetylglucosamine, N-acetylgalactosamine, L-fucose and N-acetyl
neuranimic acid. Usually a particular source contains lectin belonging to single
specificity group, but in considerable number of cases two lectins that differ in
their specificity are found in the same plant. Individual lectins frequently occur
as a group of closely related proteins, designated as isolectins (Etzler, 1985). A
few examples of isolectins are found in lectins of Griffonia simplicifolia,
Phaseoliis vulgaris and Dolichos biflorus (table 3).
Structurally two classes of legume lectins have been recognized. First class
is one-chain lectins, those comprised of either identical or nearly identical
subunits of MW 25000-30000 Dalton. Second class lectins consist of two chains,
made up of different subunits. Light one is called a chain and heavy (3 chain.
These include lectins from red kidney bean Phaseolus vulgaris. It is composed of
two different units combined in 5 different forms of non-covalently bound
tetramers and each combination is considered to have different function. Another
example is tetrameric lectin from Dolichos biflorus made up of two closely
related subunits only one of which appears to bind sugars.
Nevertheless all these lectins posses extensive homology when properly
aligned. Concanavalin A and lectin from Dolichos grandiflora (Richardson et.
al., 1984) both belong to Diocleae tribe and in this case homology with the other
one chain lectins is observed by aligning amino ends of legume lectin with
residues 1. 2, 3 of Diocleae lectins (Hemperley & Cumiingham, 1983). Several
positions are invariant or highly conserved in legume lectins e.g. Phe-6 and Phe
11 and in a number of other positions only conservative substitutions occur
(Richardson et. al., 1984).
Table 3 : Isolectins with different carbohydrate specificities
Source
Griffonia slmplicifolia
Bandeiraea simpUcifolia
Laburnum alpimiin
Phaseolus vulgaris
Ulex europaeus
VIcia c race a
Vic la villas a
Viscum album
Lectin
I-A4 I-B4
II IV
BS-I BS-II
BSI-B4 BSI-A4
I II
E-PHA L-PHA
I II
I II
A4
B4
I II
Carbohydrate specificity
GalNAc Gal
GicNAc L-Fuc->Gal
o2
a-D-Gal, a-D-GalNAc a-GlcNAc a-D-Gal
a-D-GalNAc
(GlcNAc)2.3 Gal
Oligosaccharide Oligosaccharide
L-Fuc GlcNAc
GalNAc Man, Glc
GalNAc Oligosaccharide
Gal GalNAc
Reference
Goldstein & Hayes, 1978 Goldstein «fe Hayes, 1978 Goldstein & Hayes, 1978 Shibata et al., 1982
Murphy & Goldstein, 1977 Ebisu & Goldstein, 1978 Murphy & Goldstein, 1977 Murphy & Goldstein, 1977
Konami et a!., 1983
Goldstein & Hayes, 1978
Goldstein & Hayes, 1978
Bauniann et al., 1979
ToUefsen & Kornfeld,
1983
Franz eta!., 1981
Most lectins contain covalently bound carbohydrate units and on this bases two
types of lectin glycoproteins have been discerned. Among first type are those
containing primarily mannose and N-acetylglucosamine. The structure of
carbohydrate unit of soyabean agglutinin has been established and it is similar to
that of oligosaccharides found in several animal glycoproteins (Dorland et al.,
1981). Several lectins of this type also contain L-Fucose and Xylose (Lis &
Sharon, 1984). The lectin from Tora bean contains the pentasaccharide core with
Xylose attached by p-2 linkage to the mannose (Ohtani & Misaki, 1980).
Glycoprotein lectins of second type contain L-Arabinose and Galactose and have
been found only in plants of solanaceae family. Both the potato and Datura
stramonium contain tri and tetra arabinofuranosides (3-linked to hydroxy proline.
These linking groups have not been found in animal glycoproteins. Carbohydrate
moieties are not required for biological activities of lectin and it was proved
when chemically deglycosylated potato (Desai et. al., 1983) and tomato
(Kilpatrick et al., 1984) lectins retained their haemagglutinating activity.
Metal ions are the essential part of the native structure of most leguminous
plant lectins and many biological activities can be attributed to them. Metal
binding sites of the most studied and fully sequenced lectin Concanavalin A are
situated in the amino terminal part of the polypeptide chain. The binding site for
Mn ^ and Ca " ^ in Con A are situated 5A apart (Sharon & Lis, 1990). Lectins of
soybean, peas, faba bean and lentils have conserved amino acids that are
involved in metal binding (Reeke & Becker, 1988).
In addition to the carbohydrate binding sites lectins frequently posses
hydrophobic binding sites. Hydrophobic interactions are responsible for the
stability of native structure of protein. One hydrophobic site is found adjacent to
the carbohydrate-binding site as is evidenced by the finding that hydrophobic
derivatives of the monosaccharides bind more strongly than the non-hydrophobic
monosaccharides (Sharon and Lis 1989; Goldstein and Poretz 1986). An example
is N-Densylgalactosamine, which binds to Erythrina cristagalli lectin 60 times
more strongly than N-acetylgalactosamine (De Boeck et al., 1984). It has been
suggested that lectins may function not only by virtue of their ability to bind
carbohydrates but also by serving as binding proteins for biologically active
hydrophobic ligands (Roberts & Goldstein, 1983).
Ligands that bind at the hydrophobic site remote from the carbohydrate-
binding site include indole acetic acid and 1-8, anilinonaphthelene sulphonic acid
and there is one such binding site per subunit. Several legume lectins contain a
specific high affinity-binding site for adenine. This site is unusual in that there is
only one such binding site per lectin molecule not per subunit (Maliarick et al.,
1989). Most lectins are generalized as glycoprotein but still a few of them are
non-glycoproteins as Con A, Lentil lectin and wheat germ agglutinin. All
glycoprotein lectins contain a peptide sequence Asparagine-X-threonine / serine
which is characteristic of glycosylation site.
BIOLOGICAL ROLES:
Lectins are found in almost all living animals but despite their ubiquity,
tlieir function in nature is not very clear. Although the lectins share the common
property of binding to defined sugar structures, their roles in various organisms
are not likely to be the same. Existence of homologous lectins in different plant
families demonstrate that these proteins have been conserved throughout the
evolution and so have an important flinction in nature (Table 4).
Plant Lectins
Two main functions of plant lectins proposed, are as mediator of symbiosis
between plants and microorganisms and in protection of plants against
phytopathogens. Lectins act as mediators of symbiosis between nitrogen fixing
microorganisms and leguminous plants (Hamblin and Kent 1973). Lectin from a
particular legume binds in specific manner to the corresponding
Table 4 : Biological Roles of lectins in Nature
Biological Roles
Recognition of Rhizobia by lectins in nitrogen fixing symbiosis
Binding to specific strains of Rhizobia (Lectin from trifoliin of wiiite clover)
Inhibit fungal growth in plants
Removal of glycoproteins from circulatory system
Pinocytosis of glycoproteins with terminal non-reducing sugar residues
Targeting of hydrolytic enzymes to lysosomes
Influence the pathogenesis of cancer metastasis
Soluble vertebrate lectins bind to glycoconjugates on and around the cell and release them
Invertebrate hemolymph lectins might function as opsonins
Bacterial surface lectins may be involved in mediating adherence
Bacterial surface lectins mediate non immune phagocytosis
Lectins from slime mold function to promote substratum attachment
Reference
Bohlool & Schmidt, 1974
Dazzo & Truchet, 1983
Mirelman et. al., 1975
Ashwell & Harford, 1982
Stall! etal., 1984
Sahagian, 1984
Lotanetal, 1985
Barondes, 1984
Renwrantz & Stahmer, 1983
Beachey, 1980
Sharon, 1984
Springer et al., 1984
rhizobial species and not to bacteria that are symbionts of other plants (Dazzo et
al., 1986). Recognition of Rhizobia, by lectin of the host plant, accounts for the
specificity in the initiation of nitrogen fixing symbiosis (Bohlool & Schmidt,
1974). Lectin isolated from root of white clover, trifoliin, can bind to a specific
nodulating strain of rhizobium (Dazzo & Truchet, 1983). This lectin has been
suggested to reversibly cross bridge receptors on the root hair cell wall with
bacterial capsular polysaccharides and/or lipopolysaccharides (Dazzo &
Sherwood, 1983). Cell surface carbohydrate of rhizobia which include extra
cellular polysaccharide, capsular polysaccharides, lipopolysaccharides and
periplasmic glycans have been found to play important role in plant infection
process leading to nitrogen fixation (Guerinot and Chelm, 1987; Long, 1989;
Geremia et al., 1987).
Role of lectins in the defense of plants against bacterial, viral and fungal
pathogens have been proposed (Barondes, 1981; Leach et al 1982; Mishkind et
a!., 1982).
Animal Lectins
Lectins serve as cell recognition molecules (Sharon and Lis, 1990), may
involve in recognition activity between cells or cells and various carbohydrate
containing molecules. Membrane lectins have been suggested to mediate the
binding of soluble extracellular and intracellular glycoproteins. Binding of
asialoglycoprotein by a galactose specific lectin on mammalian liver cells and of
asialo-agalactoglycoprotein by mannose/N-acetyl glucosamine specific lectin on
avian hepatocyte are good example. Both of these interactions are probably key
steps in the removal of these glycoproteins from the circulatory system (Ashwell
& Harford. 1982). The mannose-6-phosphate specific lectin mediates targeting of
hydrolytic enzymes to lysosomes (Sahagian, 1984). Galactose specific lectin
present on various human and murine tumors (Raz & Lotan, 1981; Lotan et al.,
1985) were suggested to influence the pathogenesis of cancer metastasis by
promoting the formation of tumor cell aggregates in the circulation and their
adhesion to the endothelial layer of capillaries.
Humoral lectins are ubiquitous within invertebrates, since these taxa lack
immunoglobulin, the possibility has been raised that humoral lectins might be
their functional analogs (Renwrantz & Stahmer, 1983).
Lectins of Microorganisms
Bacterial surface lectins appear to be involved in the initiation of infection
by mediating bacterial adherence to epithelial cells, for example in urinary and
gastrointestinal tracts (Beachey, 1980). This has been best documented for
Escheritia coli carrying type I or type P fimbrieae. Type I fimbriated strains are
considerably more infective than their isogenic non-fimbriated counterparts
(Fader & Davis, 1980; Iwahi et al., 1983). The pattern of distribution on oral
epithelial surface of actinomyces carrying the galactose specific lectin supports
tlie assumption that these are the principal mediators of adherence, colonization
and establishment of specific microbial communities in oral cavities'(Springer et
al., 1984).
Non-immune phagocytosis mediated by bacterial surface lectins may be of
clinical relevance in non-immune hosts and in tissues, such as renal medulla
where opsonic activity is poor (Sharon, 1984; Perry et al., 1983; Silverblatt &
Cohen, 1979). In case of slime molds evidences indicate that discoiden I, an
endogenous lectin from Dictyostelium discoideum functions to promote cell
substratum attachment and orders cell migration during morphogenesis (Springer
et al., 1984) rather than cell-cell adhesion.
12
APPLICATIONS
Availability of a large no of lectins with different carbohydrate specificities
has led to their extensive utilization as reagents for the study of simple and
complex carbohydrates in solution and on cell surface (Lis & Sharon, 1984),
identification and separation of cells (Sharon, 1983) and for the selection of
lectin resistant mutants of animal cells with altered glycosylation patterns
(Stanley, 1983). Table 5 gives a brief account of applications of lectins in
biology. Except a few cases in which lectins from non-plant origin are employed
(Hellstrom et al., 1984; Mureson et al., 1982), such studies are done with plant
lectins.
Lectins bind to sugar moieties in cell walls or membranes and thereby
change physiology of the membrane to cause agglutination, mitosis or other
biochemical changes in the cell. Only those cells bearing specific receptor groups
for the respective lectin would be affected. As different terminal sugar residues
specify A, B and O blood groups, lectins are extensively utilized for blood group
typing or sub grouping. A few lectins that show blood group specificity are given
in Table 6.
Agglutination of cells and precipitation of polysaccharides depend on the
multivalency of lectins, most of which are oligomeric with 2-5 or so binding
sites. Thus they can cross link cells or polysaccharides, which also have many
receptors. Low molecular weight ligands compete for the binding sites and
disrupt the precipitate or aggregate. Malignant cells are preferentially
agglutinated by Con A, wheat germ agglutination (WGA) or soybean
agglutination (SBA). Since cell surface carbohydrates have obvious role in
control of cell growth and differentiation, there were attempts to use lectins for
diagnosis and therapy in cancer.
A host of lectins have been isolated from plants and animals. Their
carbohydrate specificity has been characterized and is used as a powerful tool in
glycobiology. Lectins are used in affinity chromatography, separation of cell
types, cell biology and also in lectin isolation by affinity chromatography.
Table 5: Application of Lectins in biology
Application
Study of simple and complex carbohydrates in solution
and on cell surface
Identification and separation of cells
Selection of lectin resistant mutants of animal cells
In purification of glycoproteins
Receptor for insulin
Epidermal growth factor
Glycocalicin of human platelet membrane
Second component of human complement
Sulfated glycoproteins of calf thyroid plasma
membrane
Glycoproteins of Ehrlich ascites cells
Separation of closely related compounds
Dibranched complex oligosaccharides by
Con A
- Tribranched complex oligosaccharide by L-
PHA
Compounds containing L-Fucose links by
lentil lectin
Terminal non-reducing a-linked galactose by
lectin from Griffonia simplicifolia
Mammalian ^-adrenergic receptors on con A
References
Lis & Sharon, 1984
Sharon, 1983
Stanley, 1983
Hedoetal., 1981
Cohen etal., 1982
Tsujietal., 1983
Schultz & Arnold, 1984
Edge & Spire, 1984
Eckhardt & Goldstein, 1983
Baenziger & Fiete, 1979
Cummings & Kornfeld, 1982
Kornfeld et al., 1981
Eckhardt & Goldstein, 1983
Stiles etal., 1984
14
Giycoconjiigates in solution
Radioactively labeled lectins and various lectin conjugates serve as specific
and sensitive reagents for the detection of glycoproteins separated on
polyacrylamide gels (Gershoni, 1985; Rohringer & Holden, 1985). The
usefulness of this method can be increased by in-situ chemical or enzymatic
modifications of the separated glycoproteins (Gershoni, 1985). A few examples
of lectin purified glycoproteins are; the receptor for insulin (Hedo et al., 1981)
and epidermal growth factor (Cohen et al., 1982); glycocalicin, the predominant
glycoprotein of human platelet membrane (Tsuji et al., 1983); C2, the second
component of human complement (Schultz & Arnold, 1984); the sulfated
glycoproteins of calf thyroid plasma membrane (Edge & Spiro, 1984); and the
major plasma membrane glycoprotein of Ehrlich ascites cells (Eckhardt &
Goldstein, 1983).
The high resolving power of lectins permits separation of closely related
compounds, such as variants of glycoproteins that differ in their glycosylation
pattern, or of glycopeptides and oligosaccharides that differ in their structure to a
small extent only. Chromatography on concanavalin A is employed to separate
compounds containing dibranched complex oligosaccharides from those with
more highly branched structures (Baenziger & Fiete, 1979). While
chromatography on lentil or pea lectin serves to demonstrate the presence of L-
fucose linked to chitobiose unit of the core (Kornfeld et al., 1981). Terminal
nonreducing a-1 inked galactose can be detected by Griffonia simplicifolia
(Eckhardt & Goldstein, 1983). Lectin chromatography of glycopeptides and of
oligosaccharides has been employed to analyze changes in glycan branching
and sialytion of Thy-1 antigen (Carlsson, 1985); to demonstrate structural
changes in the carbohydrate chains of human thyrogloblin upon malignant
transformation of the human thyroid gland (Yamamoto, 1984); and to study the
15
Table 6 : Lectins showing human A, B & O blood group specificity
Blood Group
A
A,
A2
B
H
P
Le'
Sources of lectin
Phaseolus limensis
Otola lactea
Crotolaria striata
Codium fragile
Helix pomatia
Dolichus biflorus
Vicia c race a
Falcatajaponica
Griffonia simplicifolia
(BSI)
Ptilota plumose
Salmo salar
Abranis brama
Sambucus nigra
Ulex europeus
Anguilla anguilla
Systisiis ratisbonensis
Tetragonolobiis purpureus
Escherichia coJi (fimbriae)
BSI-(IV)
Reference
Boyd and Regura, 1949
Boyd and Brown, 1965
Khangetal, 1990
Roggers et al., 1977
Hammerstrom and Kabat,
1969
Bird, 1951
Rudiger, 1977
Nakejimaet al., 1986
Murphy and Goldstein, 1977
Roggers et al., 1977
Uhlenbruk and Prokop, 1967
Krajhanzal et al., 1978b
Predanov and Atanasova,
1980
Matsumo Osawa, 1969
Horesji and Kocourek, 1978
Renkonen, 1948
Cazal and Lalanrie, 1952
Korhonen et al., 1974
Shibata et al., 1982
carbohydrate moieties of various glycoprotein, such as epidermal growth factor
(Childs et al., 1984) and low density lipoprotein receptors (Cummings et al., 1983)
etc. Sugar nucleotides have also been fractionated on lectins, as illustrated by the
separation of UDP-Gal from UDP-Glc and of UDP-GalNAc from UDP-GlcNAc
by HPLC on immobilized Ricinns communis agglutinin (Tokuda et al, 1985).
Glycoconjugates on Cells and organelles
Many workers have reported applications of lectins in histochemical and
cytochemical studies (Schrevel et al., 1981). Changes in lectin binding pattern
have been observed during embryonic differentiation (Watanabe, 1981), cell
maturation (Zeiske & Bernstein, 1982), aging (Bischof & Aumuller, 1982),
metaplastic alterations (Orgad et al., 1983; Wells et al., 1984), malignant
transformations (Cooper, 1984; Kluskens et al., 1984; Walker, 1984) and many
other pathological conditions such as lysosomal storage diseases (Alroy et al.,
1984), inflammatory bowel diseases (Jacobs & Huber, 1985), psoriasis
(Kariniemi et al, 1983) and pneumococcal meningitis (Vierbuchen & Klein,
1984). Staining with lectins is of use in the identification of immature
thymocytes (Sharon, 1983). Ulex europaeus lectin I binds specifically to vascular
endothelium (Miettinen et al., 1983) and can thus facilitate the detection of
vascular invasion by tumor cells. They may also serve as an aid in the
investigation and classification of lymphocyte proliferative diseases (Strauchen,
1984).
Mapping Neuronal Pathways
Lectins conjugated to Horseradish peroxidase have proved to be useful
markers in mapping central neuronal pathways, since the conjugates are taken up
by neurons and transported within the axons (Mesulam, 1982)). Wheat germ
agglutinin transport in both anterograde and retrograde directions, while L-PHA
17
(Gerfen & Sawchenko, 1984, Luiten et al., 1985) and ricin (Harper et al, 1980)
conjugates only transport in the anterograde and retrograde directions
respectively.
Typing of Bacteria
Lectins have been shown to distinguish between microbial species (Doyle
& Keller, 1984). For example Neisseriae gonnorrhoeae can be differentiated
from other Neisseriae and related bacteria by its agglutination with wheat germ
agglutinin (Doyle et al, 1984). Bacillus anthracis can be identified with the aid of
soybean agglutinin (DeLucca, 1984). Lectins discriminate between pathogenic
and non-pathogenic strains of Trypanosoma cruzi and between different
morphological stages of Leishmania donovani (De Miranda & Pereira, 1984).
Cell Separation
Peanut agglutinin (PNA) selectively interacts with immature thymocytes
and this is the basis of widely used method for the separation of these cells from
mature thymocytes by selective agglutination (Reisner & Sharon, 1984).
Soybean agglutinin is used for the separation of mouse T and B' splenocytes
(Reisner et al, 1976).
MITOGENIC ACTVITY
One major property of a few plant lectins is ability to induce mitosis in cells
that are normally not dividing. This property has been exploited extensively in an
attempt to understand the process of lymphocyte blastogenesis and the
biochemical and structural alterations associated with mitogenesis. The
discovery of lectin mediated mitogenesis by Nowell in 1960 stimulated interest
in the properties of lectins. It is not clear why some lectins are mitogenic since
the structures to which mitogenic lectins bind are not necessarily the same and
18
not all lectins with similar binding specificities are mitogenic. It is likely that
binding to the cell surface alone is not sufficient to cause mitosis but that other
interactions on the cell surface are equally important. Mitogenic lectins
presumably bind to the T cell receptor complex and also promote a positive co-
stimulatory signal leading to the synthesis of interleukin-2 and interleukin-2
receptors.
Nowell observed that some of the mononuclear leukocytes, obtained from
phytohaemagglutinin (PHA) treated normal blood, entered mitosis after 48-72
hrs. The changes induced by the action of PHA on the morphology of leukocytes
in culture were later described (Cooper et ai., 1963; Marshall & Roberts, 1963).
Primary action of PHA is to cause some of the small lymphocytes to transform
into primitive blast like cells, which then undergo mitosis. This process requires
an increased protein synthetic capacity that depends directly on elevated
ribosomal content.
Transcription of ribosomal gene by RNA polymerase is assisted by at least
two trans-acting factors, Upstream binding factor (UBF) and SLl (Bell et al.,
1990; Learned et al., 1986). UBF is essential for basal transcription in human
systems and is also involved in growth dependent regulation of rRNA gene
transcription (Jacob, 1985; Moss & Stefanovsky, 1995). UBF is a
phosphoprotein whose activity is inhibited when dephosphorylated in vitro (Voit
et al., 1992). In vivo, UBF occurs in two forms, UBFl (97Kda) and UBF2 (94
Kda), both encoded by two different niRNAs which result from alternative
splicing of primary transcript (Hisatake et al., 1991; O'Mahony & Rothblum,
1991). Vertebrate UBF2 has been found to be completely inactive (Paule, 1993)
and marginally active in human systems (Jantazen et al., 1992). Study carried
out by Cab art and kalousek (1998) on PHA stimulated human lymphocytes
demonstrated maximal increase in UBF mRNA levels at 6 hrs and it was found
that mRNA of UBFl was expressed twice as much as that of UBF2 in time
interval of 40 hrs, after addition of PHA, UBF phosphorylation remarkably
precedes its neosynthesis. The concept that UBF must be phosphorylated at
multiple sites to be transcriptionally active has been well established (Grummt,
1999; Tuan et al 1999; Zhai & Comal, 1999). Since 1960, after Nowell's
investigation, lectins from a no. of species (both from plant and animals) have
been tested for mitogenic activity (Table 7). A few were found to induce mitosis
in cells, in vitro.
Table 7: Lectins with Mitogenic Activity to Blood Cells
Source of lectin
Axinellu plypoides
Staphylococcus aureus
Chelidonium majus
L.(CML)
Slyela plicata
Phnis parviflora
Pseudomonas aeruginosa
Hiira crepitens
Phytolacca octondra (po III
Erythrina corallodendron
Sambucus nigra
Cell type
Human peripheral blood
lymphocytes
Human peripheral blood
lymphocytes
Human lymphocytes
Mammalian peripheral blood
mononuclear cells
Mice splenocytes
Neuraminidase treated
lymphocytes & murine
splenocytes
Human T lymphocytes
Human T lymphocytes
Neuraminidase treated
lymphocytes
Neuraminidase treated
lymphocytes
Reference
Philips etal., 1976
Zhuravkov et al., 1983
Fik etal., 2001
Nairetal., 2001
Kurukata et al., 1989
Avichezer & Gilboa-
Garberetal., 1987
Barbieri et al., 1983
Bodgeretal., 1979
Gilboa-Garber & Mizrahi
1981
Broekaert et al., 1984
20
OBJECTIVES:
With the above-sited hterature, flirther research on lectins should advance our
insight into different activities and will also lead to better understanding of their
use in human cytogenetics and human clinical genetics. In view of the above
considerations we have studied different biological activities of legume lectins
from several species. Objectives of the present study are
• To isolate lectins from different plant seeds by ammonium sulphate
precipitation method. Plant species used were Phaseolus vulgaris
(Rajma), Vigna radiata (mung bean), Cajanus cajan (Arhar) and Lens
cuUnaris (Masoor).
• To purify lectins employing affinity chromatography.
• To test Haemagglutination activity of lectins with respect to different
human A, B, & O blood groups.
• To test the blastogenic activity of lectins from different species in
lymphocytes transformation using partially purified and purified extracts
• To test the Mitogenic activity of lectins from different species in
lymphocytes transformation using partially purified and purified extracts
• To compare Mitogenic activity of lectins with commercially used
phytohaemagglutinin from GIBCO, BRL, India.
21
MATERIAL AND
METHODS
MATERIALS AND METHODS
Four locally available varieties were selected for the experimental purpose.
These are edible pulses and are used routinely in our homes. Varieties are as
follows (with their biological names in parentheses)
I. Raj ma {Phaseolus vulgaris)
II. Mung {Vigna radiata)
III. Masoor {Lens culinaris)
IV. Arhar (Cajanus cajan)
Isolation of lectins:
The buffer used for isolation of lectin was Tris HCl (pH-8.0) containing metal
ions i.e. CaCl2. 20 gms seeds were soaked in 100 ml buffer overnight at 4 C. After
homogenization suspension was filtered through muslin cloth and centrifuged at
10,000 rpm for 15 min. supernatant was saved and its pH was lowered to 4.0 with
the help of IM Acetic acid. Again the precipitate was removed by centrifugation
and 30% ammonium sulphate was added to supernatant. Addition of salts resulted
in precipitation, which was removed in the same way. Salt concentration of the
supernatant was raised to 50%. Now the precipitate was saved, dissolved in Tris
HCl and dialyzed extensively against the same buffer to remove salts.
Determination of protein concentration:
Protein concentration was determined by the method of Lowry et al. (1951) usng
bovine serum albumin (BSA) as the standard, this method involves use of two
reagents
i) Folln & Ciocalteu's phenol reagent.
ii) Copper reagent.
i) Preparation ofFolin and Ciocalteu 's phenol reagent:
22
About 100 gm of sodium tungstate, 25 gms of sodium moiybdate, 50 ml of
8.5% (v/v) orthophophoric acid were added to about 700 ml of distil water in a flat
bottom ilask. The mixture was refluxed for 10 hrs. The flask was wrapped with a
black paper. After cooling, 150 gm of lithium sulfate, 50 ml of distilled water and a
few drops of liquid bromine were added. The mixture was then heated for 30
minutes without condenser to remove excess bromine. The solution was cooled and
its volume was made upto one liter with the distilled water. The bright yellow
colored reagent was filtered through whatman filter paper and stored in an amber
colored boftle. This stock solution was diluted in the ratio of 1: 4 (v/v) before use.
ii) preparation of copper reagent:
3 stock solutions were prepared:
• 4% (w/v) sodium carbonate
• 4% (w/v) sodium potassium tartarate.
• 2% (w/v) copper sulfate.
Determination of concentration:
A stock solution 0.5 mg /ml of BSA (bovine serum albumin) was taken and
serial dilutions were prepared in a range of 0.1 to 1 ml by adding required amount
of Tris HCl buffer. The 5 ml of freshly prepared copper reagent was added to all the
tubes and the contents were mixed well. Mixture was incubated for 10 minutes at
room temperature. After incubation 1.0 ml diluted folin phenol reagent was added
and contents were well mixed well. Color intensity was read at 700 nm after 30
minutes against blank prepared in the same way as that of test solution without
protein. Absorbance was plotted against the amount of protein. The linear curve as
obtained was found to fit the following equation (Fig 1).
Y = 0.28 X +0.04
23
/•o i-s"
Protein Concentration (mg/ml)
2. -i-
Fig. 1 standard curve for the determination of protein concentrationby method of Lowry et al (1951) using BSA as
standard. Straight was drawn by method of least square analysis.
24
Haemagglutination Activity:
Haemagglutination activity of tlie lectins was measured using human
erythrocytes. Blood was collected from the persons of different blood groups in the
collection tubes containing 0.1 ml of commercially used heparin. Blood was mixed
gently in normal saline (0.9% (w/v) NaCl in distil water) with the help of Pasteur's
pipette. Cells were palleted by centrifugation at 1500 rpm for 10 minutes. The
process was repeated three times for complete removal of heparin. To check
haemagglutinating activity 0.1 ml of protein solution was taken on a glass slide and
0.1 ml human erythrocytes were added to it. Agglutination was checked visually.
Purification of Lectins:
/. Packing of the column
About 50 ml of sediment commercial sepharose-6B and cross-linked alkali
treated sepharose 6B were washed with 0.20 M HCl on glass filters at room
temperature. The gel was then transferred for treatment into 250 ml conical flasks
each containing 100 ml of 0.20 M HCl. The flasks were shaken in a water bath at
50° C and aliquots were removed at intervals. Each of these gel aliquots were
immediately washed on a glass filter, first with water and then with 0.05 M sodium
acetate buffer (pH 6.0) containing 1 mole of NaCl per liter. In order to maintain a
uniform density of the gel in all the samples the washed gel was transferred to a test
tube, suspended in the buffer and allowed to sediment to a constant volume. The
supernatant was carefully removed and 1 gm of the gel was transferred to other test
tube to which 4 ml of buffer was also added. When all the aliquots and blanks
reached this stage then the gel was washed with the desired buffer (Trjs HCl buffer)
and diluted with the same. The diluted gel was packed into the column and allowed
to settle.
25
2. Purification:
The dialyzed extract was applied on the packed column, which was
equilibrated with the Tris HCl buffer prior to use. Fractions (2 ml each) were
collected at the rate 12 ml/hr. Bound protein was eluted out with 50 mM galactose
in Tris HCl.
PAGE (Polyacrylamide gel electrophoresis):
Polyacrylamide gel electrophoresis of the purified protein was performed
according to the method of Laemmli (1970) in Tris glycine buffer (0.025 M Tris,
and 0.192 M Glycine), pH 8.3 on 10% polyacrylamide gel.
Gel plates were thoroughly washed, rinsed with distil water and dried before
use. Three spacers (width) were placed along the sides and bottom of the plates. The
plates were fixed vertically and sides were sealed with 1% agarose solution. 3/4" of
the space between the plates was filled with resolving gel solution containing 7.5%
acrylamide, 0.2% N,N'-methylenebis-acrylamide, 0.08%) Ammonium per sulphate
and 0.05%) N,N,N"-N'tetramethylenediamine (TEMED). The surface was covered
with a layer of water and gel was allowed to polymerize at room temperature. After
polymerization water was removed and the remaining space was filled with
stacking gel solution containing 3.6% acrylamide, 0.096%o N, N'-methylenebis-
acrylamide, 0.075%o Ammonium per sulphate and 0.08%o N, N, N'-
N'tetramethylenediamine (TEMED). A comb was placed after pouring the stacking
gel solution between the plates. After polymerization of stacking gel spacers were
removed and plates were vertically placed in lower tank of the slab gel
electrophoresis apparatus, filled with electrophoresis buffer.
Sample was applied in the wells. Anodic current was passed till the
bromophenol blue front migrated to nearl}' bottom of the gel. Then gel was taken
out and silver staining was performed.
26
Reagents for Silver staining:
Fixative I 50% methanol with 7.5% Acetic Acid.
Fixative II 5% methanol with 7.5% Acetic Acid
Fixative III 10% Gluteraldehyde
Developer To 0.05% of Citric Acid 5 |il of 37% formaldehyde was
added for eah ml of Citric Acid.
Stain To make 62.5 ml os stain 1.25 ml of 2N NaOH was
mixed with 0.87 ml of NH4OH and 12.6 ml of distilled
water. To this solution Silver Nitrate (0.4 gm in 2ml
distilled water) was added slowly and volume was
raised to 62.5 ml.
After electrophoresis gel was put in fixative I, II, and III consecutively for 20
minutes in each. After that gel was washed with large amount of water until smell
of gluteraldehyde is gone. Then gel was placed in Silver stain for 20 minutes. When
gel started turning brown it was washed again with large amount of water. After
washing gel is put in developer until bands become visible.
Lymphocyte Transformation Test:
Mitogenic potential of any lectin i.e. ability to induce cell division was observed
using lymphocyte transformation test as per the method of Moorehead et. Al., 1960.
The activitywas compared with the commercial mitogens from Gibco BRL, India.
Preparation of Media:
The medium used for the lymphocyte culture was RPMI-1640. Stock
medium was prepared by dissolving 10 gms of powder (RPMI-1640 from GIBCO
BRl^, India) in 1000 ml of autoclaved distilled water. The working medium was
prepared by supplementing it with 10% foetal calf serum. The pH of the medium
27
was maintained at 7.2 with the help of NaHC03. The working medium was filtered
and kept at 4 C in autoclaved bottles for further use.
Lymphocyte Culture:
Cultures were planted in previously cleaned and sterile chamber of laminar
flow. All the necessary apparatus (culture vials, pipettes) were sterilized before use.
5 ml of media was taken per culture vial. Different doses of lectin from 25|ig/ml of
culture to 200^g/ml of culture were used to test the mitogenic activity.
Simultaneously, cultures with commercial phytohaemagglutinin was also planted.
To every culture vial 0.4 ml of blood was added. Then vials were closed tightly and
were put at 37°C for 68 hrs. Once in 24 hrs screw cap was loosen and air passage
was allowed.
Harvesting the culture:
Cultures were harvested at 68 hrs of plantation. 0.1 ml of colchicine (3fig/ml)
was added to each vial and kept at 37°C for 75 minutes. After that culture were
centrifuged at 1000 rpm for 10 minutes. The pallet was saved and mixed with
prewarmed 0.56% KCl (hypotonic solution) gently. Now the tubes were kept again
at 37 C for 15 minutes. It was spun at 1000 rpm for 10 minutes and pallet was
saved. To the pallet 8 ml of the fixative (methanol & acetic acid in 3:1 ratio) was
added to the tube, while the contents were kept stirred on a vertex shaker. Cells
were washed thrice with fixative and slides were prepared.
Slide preparation:
Slides were prepared by flame drying technique. Slides were washed clean
and kept at 4 C in distilled water. About 3-4 drops of cell suspension was dropped
per slide with the help of Pasteur's pipette and a brief exposure to the flame was
28
given to dry the slides. After one hour sHdes were stained in 5% giemsa stain in
phosphate buffer (pH 6.8).
Assessment of Mitogenic potential:
Stimulation potential of each lectin was assessed against two parameters
1. % Blastogenesis- percentage of blast cells per sample.
Z. Mitotic Index- percentage of metaphases per sample.
29
RESULTS
RESULTS
Lectins from four species were isolated from four plant species viz.
Phaseolus vulgaris, Vigna radiata, Cajanus cajan and Lens culinaris. These were
purified by affinity chromatography and both purified and partially purified
proteins were tested for mitogenic activity in lymphocyte transformation test and
Haemagglutination activity with respect to different A, B & O blood groups.
Results concluded from the experiments are as follows:
Protein Concentration
Around 20 gms of pulses from each species were homogenized in 100 ml
Tris HCl buffer (pH-8.0) containing CaCli to obtain a suspension which on
acidification and salt precipitation yielded a clear protein solution. Protein
concentration for each solution was estimated by the method of Lowry et al., 1951.
The amount of lectin from Phaseolus vulgaris was found to be maximum i.e.
60mg while in Cajanus cajan, Lens culinaris and Vigna radiata the amount were
45 mg, 33.6 mg and 27mg respectively (Table 8). '
Haemagglutination Activity
Protein isolated from each sample was tested for haemagglutination activity
with human red blood erythrocytes previously suspended in normal saline. Blood
cells (0.1 ml) were mixed with same amount of protein to check the activity.
Lectins from the given four species gave various responses, with the one did not
show any agglutination activity in all blood groups. Lectins were fairly stable and
retained haemagglutination activity for a month, when kept in refiigerator at 4° C.
However prolonged storage of lectins affected the activity, which may be because
of aggregation of lectins. Haemagglutination activities of lectins with respect to
human A, B and 0 blood groups are presented in Table 9. Lectin from Phaseohis
vulgaris and Lens culinaris agglutinated all human A, B and O blood groups
without any specificity. Mung bean did not agglutinate any type. Only lectin from
Cajanus cajan showed Blood group specificity. It agglutinated blood group B
erythrocytes specifically.
Purification
Acid treated sepharose (6B) column was used to perform affinity
chromatography. Lectins were purified from two species i. e. Phaseohis vulgaris
and Vigna radiata. The elution profile of the two lectins showed symmetrical
peaks. Optical densities were taken at 280 nm. Fig 2 and 3 represent elution profile
of the lectins from Phaseolus vulgaris and Vigna radiata respectively.
Optical properties were also measured in Tris HCl buffer at pH 8.0
containing CaCl2 in wavelength region 200nm to 360 nm. Absorption spectrum for
Phaseolus vulgaris is shown in Fig 4 and for Vigna radiata in Fig 5.
Electrophoresis gel is shown in Fig 6. In case o'l Phaseolus vulgaris 4 bands were
observed. Among 4 bands two were fast moving (~ 43 KD and 42 KD) and
remaining two were (~ 25 and ~24KD slow moving. While in case of Vigna
radiata only one band (~40KD) was found.
31
Table 8: Quantity of Protein isolated from seeds of various species.
Name of the Species
PhaseoJiis vulgaris
Vigna radiata
Lens culinaris
Cajaniis cajan
Protein Concentration
2 mg / ml
0.9 mg / ml
1.12 mg/ml
1.5 mg/ml
Total Amount of Protein
60 mg
27 mg
33.6 mg
45 mg
Table 9: Agglutination activity of lectins from four species with respect
Name of the Species
Phaseohis vulgaris
Vigna radiata
Lens culinaris
Cajan us cajan
Blood group
A
+
-
+
-
Blood group
B
+
-
+
+
Blood group
AB
+
-
+
+
Blood group
0
-+•
-
+
-
32
O-OtT
O'0S»'
O'dO
on a
— a-oW
u
o
0-o2t)
o-Pio
qo ^0 60 TO 80 fo loo
Elution Volume (ml)
uo uo lio
Fig. 2 Elution profile of Lectin from PItaseolus vulgaris purified on Acid treated Sepharose 6B Column.
33
0-bh
Q.bi
0-OS
O'OH
e a Q , - -O 'OS
a. O
O-02
0 - 0 /
Ho ro 60 7o 50 9t. 100 110 (1-0 \io
Elution Volume (ml)
Fig. 3 Elution profile of Lectin from Vigna radiata purified on Acid treated sepharose 6B Column.
34
O ' l D
OM
t/5
a
- 0.06
O
©•oH
O-02.
Zii 3li 3o^ 5?^ S-2 <^« ^ « •?5'-2 ^VZ 23^2
Wavelength (nm)
Fig. 4 Absorption Spectrum of Phaseolus vulgaris lectin in Tris HCl buffer.
35
O'Ol
0-08
o-o?
c <u
Q _O06O «
O
&&
oikXf
3.2?. 3/2. S02 m 282. Z?i 562. 2S Z ^'12. ^^^
Wavelength (nm)
Fig. 5 Absorption Spectrum of Lectin from Vigna radiata in Tris HCl buffer.
36
B
A B
43K
42K
40 Kd
< 25Kd
^ 23Kd
Fig. 6. Polyacrylamide Gel Electrophoresis of lectins from
Vigna radiata (A) and Phaseolus vulgaris (A).
37
Mitogenic Activity
Mitogenic activity of lectins from various species was tested employing
lymphocytes transformation tests. Lectins from Phaseolus vulgaris and Lens
cidinaris were found to induce blast formation in cells identified by their round up
comparatively larger size than others (Figure 16). However, the mitogenic activity
(Figure 15) was shown by only one species i.e. Phaseolus vulgaris. It showed
activity both in partially purified and purified extracts.
Two parameters were used to assess the activity mitotic index i.e.
percentage of metaphase plates per sample and % blastogenesis i.e. percentage of
blast like cells per sample. Mitotic index and % blastogenesis assessed for
partially purified extracts is given in Table 10. In the partially purified extract
maximum mitotic index and % blastogenesis were found at concentration of
75|ig/ml of culture (figure 7 & 8). Mitotic index in case of purified lectins was
highest at the same concentration but % blastogenesis was found to be the
maximum at 150|ig/ml. Data for partially purified extract has been summarized in
table 11 and Figures (9 & 10).
The percent (%) Blastogenesis assessment for lectin from Lens culinaris is
given in Table 12. The induction of the activity started at 75 |ig/ml of culture,
which did not show mitogenic activity (figure 11). The stimulation activities were
compared with the commercially available Phytohaemagglutinin from Gibco BRL,
India. Comparative account of mitotic index is given in table 13 (Fig 12). In table
14 and fig. 13, % blastogenesis of both species is compared with commercial
PHA.
38
Table 10: Mitotic index and % blastogenesis in lymphocyte cultures
stimulated by partially purified lectins from Phaseolus vulgaris.
Concentration
25 ).ig/ml
50|ag/ml
75|ig/ml
lOOfig/ml
150)ig/ml
Mitotic Index
2.31±1.732
5.421+1.915
6.156±1.78
5.36±1.25
3.21±1.85
% Blastogenesis
21.89±1.79
36.21±3.55
35.3513.69
33.35±2.05
27.92±4.12
Table 11: Mitotic index and "/o blastogenesis in lymphocyte cultures
stimulated by purified lectins from Phaseolus vulgaris.
Concentration
25 (.ig/ml
50^ig/ml
75ng/ml
100).ig/ml
150(_ig/ml
Mitotic Index
4.34+0.28
5.91 + 1.73
6.92+1.83
6.13+2.10
5.84+1.81
% Blastogenesis
28.56±4.65
34.25±2.58
36.15+3.192
37.42+3.32
37.81±2.14
39
Table 12: Mitotic index and % blastogenesis in lymphocytes stimulated
by partially purified lectins from Lens culinaris.
Concentration
25 \\glm\
50|ig/ml
75(ig/ml
100|ig/ml
150(ig/ml
Mitotic Index
-
-
-
-
-
% Blastogenesis
-
-
9.1311.32
12.78±1.96
15.16±3.21
Table 13: Comparison of mitotic indices of the lymphocytes stimulated
by partially purified and purified lectins {Phaseolus
vulgaris) with commercial PHA.
Cone.
25 [ig/ml
50|.ig/ml
75|ig/ml
lOO^g/ml
150|ag/ml
Partially purified lectin from P. vulgaris 2.31±1.732
5.421±1.915
6.156±1.78
5.36+1.25
3.21±1.85
Purified lectin from P. vulgaris
4.34+0.28
5.91+1.73
6.92+1.83
6.13+2.10
4.92+2.14
Commercially used PHA
5.98+1.95
7.426+1.83
6.35+1.72
5.84+1.81
5.134+1.14
40
Table 14: Comparison of % Blastogenesis in lymphocytes stimulated by
partially purified and purified lectins from Phaseolus
vulgaris, Lens culinaris and commercial PHA.
Cone.
25 |ig/ml
50|.ig/ml
75)ig/ml
lOO ig/ml
150|.ig/ml
Partially purified lectin
from P. vulgaris 21.89+1.79
36.21±3.55
35.35±3.69
33.35±2.05
27.92±4.12
Purified lectin from P. vulgaris
28.56+4.65
34.25+2.58
36.15+3.192
37.42±3.32
37.81+2.14
Commerci ally used
PHA
33.21+2.96
37.36±2.23
38.75±1.58
39.51±3.63
38.25+2.14
Partially purified lectin
from Lens culinaris
-
-
9.13±1.32
12.78+1.96
15.16±3.21
41
1
25 50 75 100
Concentration of protein per ml of culture (|j.g)
150
Figure 7: Mitotic index in stimulated lymphocyte cultures stimulated by partially purified extracts of
Phaseolus vulgaris at different concentrations.
42
40
35
30
t / )
u c <u M O i «
c: CO
^
25
20
15
10
25 50 75 100
concentration of protein per ml of culture {\xg)
150
Figure 8: % Blastogenesis in lymphocyte cultures stimulated by partially purified extracts of
Phaseolus vulgaris at different concentrations.
43
lU
c .u 4 o
25 50 75 100
Concentration of protein per ml of culture (ug)
Figure 9: Mitotic index in lymphocyte ultures stimulated by purified extracts of Phaseolus vulgaris at different concentrations.
44
40
35
30
c/) 25
B <U DXI
2 20 </)
CO
N? 15
10
25 50 75 100
Concentration of protein per ml of culture {\xg)
Figure 10: % Blastogenesis in lymphocyte cultures stimulated by purified extracts of Phaseolus vulgaris at different concentrations.
45
16
14
12
. - 10
C
o
25 50 75 100
Concentration of Protein per ml of culture ((.ig)
Figure 11: % Blastogenesis in lymphocyte cultures stimulated by partially purified lectins from Lens culinaris.
46
n Partially purified from P. vulgaris I Purified from P. vulgaris I Commercial PHA
•a
o 4
'T' . u ^ ^ H S •. ? /^^Hffi ' r : K ^ ^ ^
"'^^Bif
:^::'i^^Hi ' • •^^•as
-^Wli ;'- '>^H ' . ' ^ ^^H -'/'^H
. ' ^^K l^^i^^iinh
?^1 •^;-s\ d
<•%»*] n. ' s\ . *,• 1
25 50 75 100 150
Concentration (fig per ml of culture)
Figure 12: Comparison of mitotic indices and in lymphocyte cultures stimulated by partially purified and purified lectins from Phaseolus vulgaris
with that of commercial PHA.
47
45
lA
Vi
a> C 0)
o -^ 1/5
re S ^
40
35
30
25
20
15
10
D Partially purified lectin from P. vulgaris • Purified lectin fi-om P. vulgaris
@ Commercially used PHA • Partially purified lectin foin Lens culinaris
25
rȴi
m
50
'f¥^^^ '•,f^^^^i
. ' .» |^^B i « - . - ! ^ ^ =
" ^ l ! ^ ^ ^
v^^S
^ 1 : ^ ^ ^ ' 4 ! . , * ^ ^ ^ ^ ^
V';^^K^S T'^^^^^S
'.rf'.^^^^^S
'' ^^^^= *• ^ ^ ^ ^ ^ ^ s
xs
l B t '^^^^S ^i^^^^s ^T'.^^^^S
tl'^^S
:>rt^^^^^S
i^^B^ mm • -c^^^^s U l F ^ ^ ^ S
. t ' -^^^S ' ! ' ^ ^ ^ h^^^^S -lUH^SS
^ 7
^ ^*^il i f .^1
'itM c "'B
« > • j ^ H
v'«H " H T ^ 1
^H
fft?jT
Eg
Eg S-
75 100
Concentration ()ig/ml of culture) 150
Figure 13: Comparison of % Blastogenesis in lymphocyte cultures stimulate by partially purified and purified lectins from Phaseolm vulgaris, Lens culina,
and that of commercial PHA.
48
if £ ^%, % »**-l
Jf jBfi* » nil V «*ifc
) I 4^ •^
Figurel4: Human metaphase spread (46 XX) from the stimulated lymphocytes by purified Lectin Phaseolus vulgaris
w
/ "•^f.
s-t
• •• r
«
•
w § •
FigurelS: Number of metaphases (5) induced by purified lectin from Phaseolus vulgaris on human lymphocytes (Arrows indicate metaphases)
50
Figure 16: Blasts cells induced by purified lectin from Phaseolus vulgaris on human lymphocytes (Arrows indicate blast cells)
51
DISCUSSION
DISCUSSION
1.1 Agglutination
Agglutination of cells is a complex process likely to require binding of
lectin to cell surface, lateral movements and clustering of lateral receptors,
changes in cytoskeletal network, intercellular associations, biochemical
alterations, redistribution of cell surface changes which ultimately result in
multicellular aggregates. Age of cells, species from which cells have been taken,
whether they have been trypsinized, fixed or nueraminidase treated, the buffer
used and the type of assay performed all have a significant effect on the titer
obtained. Agglutination or precipitation depends on multivalency of lectins, most
of which are oligomeric having 2-5 binding sites. Cells have wide spectrum of
glycoproteins present on their surface. It is easy for lectins carrying many binding
sites to cross-link these cells.
Monosaccharide binding site of lectins also play important role in binding
of to complex glycans. Along this way a single monosaccharide binding unit of the
complex glycans bind to monosaccharide binding sites (Morgan & Watkins 1953),
while other non-covalent bonds occur at the interface of both molecules and
hydrophobic interactions stabilize the complex between lectins and ligands
(Sharon, 1993). When lectins from different species are mixed with blood of
different animals it is found that reactions differ according to species.
Different sugar heads present on erythrocyte surface membrane bound to
either protein or lipids specify blood groups. Blood group specific lectins are
extensively utilized in cases where antisera are not available. Morgan & Watkins,
(1953) suggested fucose was a component of H-specificity, galactose of B-
specificity and N-acetyl galactosamine of A-specificity (Morgan & Watkins
1953). Renkonen (1948) gave the first report of a blood group specific lectin i.e.
anti A from Vicia cracca (Renkonen 1948). Anti-0 lectins were also reported from
Cysticus Sessilofolius and Lotus tetragonolobus extracts. G.W. G Bird discovered
first cryptantigen lectin from Arachis hypogaea (Bird et ai., 1982). Apart from the
specificity and affinity for blood groups these can be considered as cost effective
in cases where serum or other reagents are not available for biological tests.
Lectins can give activity even in crude extracts and cost can be further reduced by
indigenous production.
1.2 Blastogenesis
Lectins in cell suspension predominantly bind to the receptors present on
cell surface. This often changes physiology of the membrane. This leads to cell
transformation, making of round up structures. The process is reffered to as
lymphocyte blastogenesis. Phytohaemagglutinin predominantly stimulates T
lymphocytes. Blastogenesis of lymphocytes induced in culture is evaluated by
flow cytometric cell cycle analysisafter propidium iodide staining of nucleus. PI
binds to DNA in cells at all stages of cell cycle and nucleus light intensity is
directly proportional to its DNA content. Stimulated and activated cells entering
the cell cycle pass through the S phase and synthesize DNA. Lymphocyte
blastogenic response to phytohaemagglutinin, evaluated by means of flow
cytometry , is a useful tool for testing functional ability of T cells to display an
immune response against alloantigens, reproducing in vitro and in vivo
mechanism of activation.
1.3 Mitogenic Activity
Another characterstic of lectins having biological and cytological
importance is their mitogenic activity i.e. their ability to induce mitosis in cell
culture. Nowell observed for the first time mitogenic potential of
phytohaemagglutinin and this stimulated interest of Cytogeneticists in various
lectins showing mitogenic potential. The lymphocyte transformation test is used as
classical method in assessing the relative efficacy of stimulants. Mitotic index for
53
metaphase plate count is calculated for the assessment of mitogenic potential and
% blastogenesis gives an account of transformed cells.
Lectins showing mitogenic potential do not belong to a particular
specificity group. Those, which carry similar monosaccharide or oligosaccharide
specificity, may not necessarily act alike in culture. The probable mechanism for
induction of lymphocytes includes binding of lectins to ceil surface receptors
complex, promoting signals for synthesis of interleukins. This first results in
formation of blast like cells, which later undergo division. Mitogenic potenfial of
lectins from different cultivars is found to vary. The difference in stimulation
potential may be due to the difference in conformation or chemical make up of
lectins. It is found that isolation and purification techniques greatly influence the
yield of mitotic index.
When working with mitogens dose concentration is also an important factor
to consider because generally dose response curve show tall, narrow base peaks. A
concetration lower than will not induce proliferation and higher will cause
toxicity.
Two galactose binding haemagglutinins were isolated irom sponge
Axmella plypoides. Axinella I only showed Mitogenic action on human
peripheral blood lymphocytes. Mitogenic response was strongly inhibited by D
galactose, D fucose, raffmose or 2-deoxygalactose added within 5 lirs of
mitogen (Phillips et al., 1976).
Some vegetable extracts also have haemagglutinating activity and are able
to transform lymphocytes in culture. Activities of Pisum sativum lectin and
PHA v/ere compared. The lectin from Pisum sativum was found to show similar
activity as that of PHA (Bernard-Griffiths et al., 1976).
Blastogenic response of normal human peripheral lymphocytes' to three
groups of mitogens was studied (Novogrodsky et al., 1977). Soyabean
agglutinin (SEA) was Mitogenic for human cells and the cell treatment with
neuraminidase (NA) enhanced the effect. Peanut agglutinin showed the activity
54
after cells have been treated with NA. Galactose oxidase (GO) was effective
before and activity was increased after lymphocytes were treated with NA. In
the study it was concluded that either different mitogens require different
lymphocyte to macrophage ratios for optimal stimulation or some mitogens
(SBA & PNA) form inliibitory complexes in the lymphocyte macrophage
mixture.
The Hura lectin agglutinates directly erythrocytes from several species
without specificity for human blood groups. The lectin has mitogenic activity
for purified human T lymphocytes but not for purified B lymphocytes (Falasca
et al., 1980). Lichtman et al. (1980) suggested calcium to be an internal
messenger when lymphocytes were stimulated by mitogens to enter the cell
cycle.
A mitogenic lectin has been isolated from saline extracts of Lathynis
sativus by Kolberg & Sletten (1982). The lectin agglutinated human
erytlirocytes of different A, B and 0 blood groups equally well. Pseudomonas
aeruginosa lectin PA-II agglutinates human peripheral lymphocytes and
stimulates mitogenesis (predominantly in T cells), like plant lectins PHA and
Con A. Murine splenocytes are also agglutinated and stimulated by PA-II as by
con A. Sialidase treatment of human and murine cells enhances their
agglutination (Avichezer & Gilboa-Garber, 1987).
According to Mc Currach & Kilpatrick (1988) the lectin from Datura
stamonium, Lycopersicon esculatum (tomato) and Solarium tuberosum (potato)
are structurally related and possess a similar carbohydrate specificity, yet the
Datura lectin is mitogenic for human lymphocytes while other two are not. Vuk
Parlevic et al. (1988) studied Cotton bract tannin, a major organic component of
cotton dust, caused polyclonal activation of human T lymphocytes in vitro.
They found the pattern of Tannin was similar to the pattern seen in lymphocytes
stimulated with PHA and Con A.
'>? 55
An acidic Pine cone extract, fr. V. of Pinus parviflora significantly
stimulated DNA synthesis of isolated splenocytes from both mice and rat but
only marginally affected the DNA synthesis of leukemic cell line (Kurakata et
al., 1989). Tulipa gesneriana lectin erythrocyte (TGL-E) showed a potent
mitogenic activity on mouse spleen cells and human peripheral blood
lymphocytes (Oda et al., 1991). It is composed of 2 subunits with a Mw of
about 26000.
According to Shinohara et al. (1991) Sclerogen a mitogen isolated from
the buffer extracts of Sclerotia of Sclerotinia sclerotiorum IFO 99395, is unique
in its characterstics, which differs from other lectins. It shows mitogenecity
after heat denaturation.
Ryder et al., 1992 concluded in their study that dietary lectins such as
PNA promote cell proliferation and thus cancerous growth, while galactose
containing vegetable fibers would inhibit this effect by competing for binding
by these lectins.
The. crude extract derived from seeds of Artocarpus integrifolia (jack
Bean) contains two fractions with different biological activities for
lymphocytes. One component is jacalin, which inhibits Con A induced cell
proloiferation, while other is responsible for the mitogenic activity of human
PBMC and Murine spleen cells. The ligand for mitogenic fraction is D-
mannose (De Miranda et al., 1992).
Mitogenic lectins currently available, PHA, con A and PWM have each
shown rare instances of hyper sensitization that hopeftilly might be eliminated
by exclusion of contaminants with recombinant DNA methods of production
(Wimer, 1996).
EHasco et al., 1996 studieed proliferative response of human peripheral
blood mononuclear cells by lectins from different Artocarpus species suggest
that lectins are similar in terms of their mitogenic activity, although their
structural features may not be identical.
56
Two lectins purified from tubers of Arisaema consangidneum schott
(ACA) and A. curvatum kunth (AcmA) belonging to the monocot family
Araceae were Mitogenic for human peripheral blood mononuclear cells
(PBMC) in the [3H]-thymidine uptake assay (Shanghary et al., 1996).
Based on in-vitro evidence that PHA (Wimer, 1997), operating through
LDCC pathway, might kill any tumor target if it remains present in adequate
concentration, the administration of mitogen for cancer therapy would be
rational. Being nonerythroagglutinating, although leukoagglutinating in higher
concentration, PPiA-L^ serves as a suitable model for immunostimulating
activities of the mitogens that can be applied directly or as in vitro activators of
adoptive leukocytes. While PHA is the mitogen clinically tested. Con A and
PWM are two most widely studied among alternatives.
Nomura et al. (1998) purified a haemagglutinin (CCA) from the
cotyledons of Japenese Chestnut Castanea crenata. The intact CCA composed
of six or eight identical subunits without disulfide bonds. The lectin showed
strong mitogenic activity similar to other lectins.
Stimulation of T cells by the T-cell receptor (TCR) /CD3 complex results
in interleukin-2 (IL-2) synthesis and surface expression of the IL-2 receptor
(IL-2R), which in turn drive T cell proliferation. TCR stimulation provides the
initial trigger to the resting T cells, which allows the cells to traverse the first
2/3'^ portions of Gi phase of cell cycle and become proliferation competent.
lL-2 action begins afterwards, delivering the actual proliferation signal (S),
allowing the cells to traverse the rest of Gi phase and enter the S phase of the
cell cycle (Chkrabarti et al., 1999).
Lectin from Chelidonium majus L (CML) significantly stimulates the
proliferation of human lymphocytes and has haemagglutinatination activity
towards group B human erythrocytes and potent antimicrobial properties
against multiresistant enterococci and staphylococci (Fik et al., 2001).
57
Nair et al. (2001) characterize one of those proteins from tunicate, Styela
pllcata, that can stimulate tunicate and mammalian cell proliferation, activate
phagocytosis; increase interleukin-2 secretion by mammalian peripheral blood
mononuclear cells and enhance IL-2 receptor expression by mammalian EL-4,11-
2 cells. S. plicata protein modulates the activities of mammalian
immunocompetent cells by interacting with carbohydrate moieties of
glycosylated cell surface receptors.
Lectin from Phaseolus vulgaris gave a positive haemagglutination test for
human A, B, 0 blood group erythrocytes. Activity was observed in both crude and
purified extracts. Mitogenic activity was tested for various concentrations ranging
from 25 fig/ml to 150)j.g/ml both in partially purified and purified extracts. In the
present study a concentration of 75|j.g/ml gave the highest mitotic index. A
concentration of 200)ig/ml showed toxicity. Activity was compared with
commercial PHA from Gibco, BRL, India. The present study is inline with the
work reported by Sengupta & Sengupta (1992). They observed the best activity at
25 []g/ml of culture in partially purified extracts. Mitogenesis using crude seed
extracts has also been reported (Hashem & Kabarity, 1964; Downing et al., 1968).
Rigas and Johnson reported that minimum amount of crude seed extract was
500i:ig/ml of culture (Rigas & Jolinson, 1982). Jaffe et al found that 3-10 Dg/ml of
extract was required for maximum mitogenic activity (Jaffe et al., 1974). •
It is well worked out that mitogenic potential is genetically controlled
(Hoffman & Donaldson, 1985). There are multiple genes in existence. Lectins are
found in closely related foms called isolectins. The molecular weight of native
Phaseolus vulgaris lectin varies from 85,000 to 150,000, made up of 4 subunits.
Phaseolus vulgaris family consists of five heterogenous lectins. Each made up of
two different subunits in various combinations. These differ in carbohydrate
content, physiochemical character and biological activity. So the variety chosen
for the experiment is also important. Lectins showing mitogenic potential are
58
employed for cytogenetic purpose, to study karyotypes/chromosomal aberrations
and different drug screening purpose.
Lectin from lens culinaris agglutinates human A, B, and O blood group
erythrocytes. There is no specific affinity to blood group antigens. When culture
for lymphocyte transformation was induced with extract from Lens culinaris blast
like cells were found. Though not very high a blastogenic index was found. The
lectin binds to multiple sugar residues more than single alpha-mannose residue. It
has an affinity for terminal D D-mannosyl and D D-glucosyl residue. LCH
comprises of two isomers LCH-A and LCH-B of molecular weight 60,000 and
42,000 respectively. LCH-A has been found to be mitogenic (Howard et al.,
1971).
Experiment with different concentration and varieties from the same may lead to
results showing a positive mitogenic potential. Lecfin from Cajanus cajan showed
positive haemagglutination test and was found specific for human blood group B
specific. It agglutinated human blood group B cells selectively. Lectins, which are
blood group specific, can be employed for blood grouping in cases where specific
antiserum, is not available.
Extract from mung bean does not agglutinated cells from human blood and
also did not show acfivity of mitogenesis or blastogenesis when tested in
lymphocyte culture. These agglutinate trypsin treated rabbit erythrocytes. Bean
agglutinin consists of single tetrameric glycoprotein composed of identical or
nearly identical subunits. lectin has an affinity for a-D-galactosyl residues and the
reversible agglutination is reported due to a-galactosidase activity of subunits
(Hankins& Shannon, 1978).
Conclusion:
From the experimental observations the conclusion can be made that the lectin
from the chosen variety from the seeds of Phaseolus vulgaris has good mitogenic
potential. After employing purification technique can be used for the routine
59
experimental work of a cytogenetic lab. High frequency of metaphases and
undamaged chromosomes indicate the suitability and potentiality of lectin from
this variety. Lectin from Lens culinaris showed blastogenic activity. Further
experiments employing purification and to test the lectin at different
concentrations may help in finding out a variety that is mitogenic. Any mitogen
prepared by in-home production will be cost effective than the one routinely used
in cytogenetic laboratories.
When testing extracts from different species for blood group agglutination
lectin from Cajanus cajan was found to be blood group specific. It showed
specificity for blood group B erythrocytes and can be used for the purpose when
antiserum is not available. Blood group specific lectins besides specificity this has
cost advantage over antiserum. These can give activity even in crude extracts.
Further work v/ill be concentrated on screening of different varieties of
legume seeds for the indigenous production of lectins, carrying mitogenic
potential and haemagglutination activity specific for blood groups. These will be
cheaper reagents for biological purposes especially in field of human cytogenetics/
karyotyping.
60
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