ISOLATION AND CARBOHYDRATE BINDING SPECIFICITY OF CAJANUS

CAJAN LECTIN

ABSTRACT

BY

SHAMA SIDDIQUI INTERDISCIPLINARY BIOTECHNOLOGY UNIT

ALIGARH MUSLIM UNIVERSITY THESIS SECTIO^ A L I G A R H

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology

of the Aligarh Muslim University ALIGARH

1994

ABSTRACT

Legume lectins represent by far the largest and best

characterized family of plant lectins. They have been

implicated in specific cell recognition processes such as

rhizobium-legume symbiosis. Since physicochemical

characterization of a lectin is an essential prerequisite

for understanding its biological role on a molecular

level, we have isolated and characterized a lectin from a

hitherto less investigated source viz. Cajanus caian

pulse. After purification of the lectin a detailed study

on its physicochemical property as well as on

carbohydrate binding specificity has been carried out.

For the isolation of lectin, pulse homogenate was

prepared in 10 mM Tris HCl buffer pH 7.0 containing 0.5 M

NaCl and 1 mM each of CaCl2, MnCl2, and MgSO^ (metallized

Tris buffer). The homogenate was acidified with 0.3 M

acetic acid to pH 4.0 for 6 hours at 7°C and the

supernatant was made 50% with respect to ammonium sulfate

concentration. The precipitate was collected by

centrifugation and dissolved in metallized Tris buffer pH

7.0. Salt fractionated crude lectin thus obtained was

further purified by affinity chromatography on IgM-

Sepharose 6B column (2.2 x 6.0 cm) equilibrated with

metallized Tris buffer pH 7.0. The bound protein which

was eluted specifically with 0.25 M glucose in metallized

Tris buffer pH 7.0 constituted 3.4% of the total

- iii -

protein in the acid homogenate.

SDS-polyacrylamide gel electrophoresis of the lectin

showed a single band and gel chromatography on

Sepharose 6B column gave a single symmetrical peak,

suggesting that the affinity purified lectin was

homogenous with respect to size. The relative mobility of

the lectin was 0.79 both in the presence and absence of

2-mercaptoethanol, which corresponded to a molecular

weight of 18,000.

Molecular weight of the lectin under native

conditions was determined by analytical gel

chromatography on a Sepharose 6B column (1.8 x 78 cm)

which was calibrated with the help of marker proteins

which included bovine serum albumin (68,000), ovalbumin

(43,000), chymotrypsinogen A (25,700), myoglobin (17,200)

and cytochrome c (11,700). A curve was drawn between

Ve/Vo vs log M by the method of least squares. The curve

was found to fit the equation:-

Ve/Vo = -0.69 log M +"5.102

The value of Ve/Vo for Caianus caian lectin was

found to be 1.93, which corresponded to a molecular

weight of 39,500. These results suggested that the

Caianus caian lectin is a dimer and that the two subunits

are linked together only by non covalent interactions.

Neutral carbohydrate content of Caianus caian

lectin was determined by phenol sulfuric acid method to

- 1 V -IV

be 3% and the specific precipitation of the lectin with

con A and dissolution of the precipitate in 0.25 M

glucose suggested that the carbohydrate moiety of the

Cajanus cajan lectin contained mannose/glucose residues

for which con A is known to be specific. The optical

properties of lectin were indicative of the presence of

tyrosine and tryptophan residues. The specific extinction

coefficient of the lectin was determined to be 10.2

(g/ml)"-^ cm"- at 278 nm and 10.1 (g/ml)"-^ cm"-*" at 280 nm;

the corresponding value of the molar extinction

coefficient of the lectin at 278 nm was 39,780 M"-'- cm"- .

Results on amino acid analysis of 24 hours

hydrolyzate showed that the lectin contains high amounts

of threonine, serine, proline, glycine, alanine, valine

and lysine. Cysteine, methionine and tryptophan could not

be determined by this method. A comparison of amino acid

composition of Cajanus cajan lectin with con A showed

that the lectin had a similar content of threonine,

proline, glycine, alanine and phenylalanine.

Cajanus cajan lectin agglutinated rabbit erythrocytes and

precipitated goat IgM specifically. A precipitin cuirve

was obtained between fixed amount of lectin and

increasing concentration of IgM. To investigate the

saccharide binding specificity of the lectin the

hemagglutination and precipitin reaction was carried out

in the presence and absence of twelve saccharides.

- V -

studies on inhibition of lectin mediated hemagglutination

of rabbit erythrocytes showed that the lectin from

Caianus cai an pulse is mannose/glucose specific. The

concentration required for 50% inhibition was more for

glucose (19.5 mM) than for mannose (9.0 mM) which

indicated that the lectin showed higher affinity for

mannose than for glucose. This showed that at C-2, axial

hydroxyl group was preferred over equatorial hydroxyl

group. Substitution of hydrogen of hydroxyl at C-2

position by methyl group considerably enhanced the

affinity of lectin for the saccharide derivative.

Interaction with glucosamine and N-acetyl glucosamine

suggests that variation at C-2 position alters the

affinity of lectin for the saccharide but does not

abolish it. The C-4 hydroxyl group was crucial for

binding so that galactose is not bound to the lectin.

Replacement of -OH group of C-1 position by para

nitrophenyl derivative in mannose enhanced the affinity

of lectin for binding. Infact, Caianus caian lectin

showed maximum affinity for P-NO2 phenyl mannopyranoside

(Cj = 0.4 mM) among the twelve sacccharides used in the

inhibition of hemagglutinating and glycoprotein

precipitating activity of the lectin. Four disaccharides

viz. sucrose, maltose, isomaltose and cellobiose were

also tested for their inhibitory potency. Cellobiose was

found to be least effective inhibitor suggesting the

- VI -

^F-

i Tiportance of alpha linkage in disaccharides for

inhibition. Isomaltose (Cl, = 3.0 iiiM) having (X 1.6 linked

glucose was most potent inhibitor. Comparison of

carbohydrate binding specificity of Cajanus can an lectin

with con A showed that both in hemagglutination as well

as in glycoprotein precipitation behaviour,Cajanus caian

lectin is similar to con A. Cajanus caian was also able

to precipitate out dextran like con A. As compared to con

A the pulse lectin showed reduced affinity for specific

sacchairides.

- vii -

ISOLATION AND CARBOHYDRATE BINDING SPECIFICITY OF CAJANUS

CAJAN LECTIN

BY

SHAMA SIDDIQUI INTERDISCIPLINARY BIOTECHNOLOGY UNIT

ALIGARH MUSLIM UNIVERSITY A L I G A R H

Date

Approved

A. SALAHUDDIN (Supervisor)

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology

of the Aligarh Muslim University ALIGARH

1994

T4592

3 ^ AuC 1394

y\ a: VSdS fn

r—« T

J

CERTIFICATE

I certify that the work presented in the following pages

has been carried out by Ms. Shama Siddiqui and that it is

suitable for the award of Ph.D. degree in Biotechnology

of the Aligarh Muslim University, Aligarh.

(A. Salahuddin)

Professor and Coordinator Interdisciplinary Biotechnology Unit,

Aligarh Muslim University, Aligarh.

11 -

ABSTRACT

Legume lectins represent by far the largest and best

characterized family of plant lectins. They have been

implicated in specific cell recognition processes such as

rhizobium-legume symbiosis. Since physicochemical

characterization of a lectin is an essential prerequisite

for understanding its biological role on a molecular

level, we have isolated and characterized a lectin from a

hitherto less investigated source viz. Caianus caian

pulse. After purification of the lectin a detailed study

on its physicochemical property as well as on

carbohydrate binding specificity has been carried out.

For the isolation of lectin, pulse homogenate was

prepared in 10 mM Tris HCl buffer pH 7.0 containing 0.5 M

NaCl and 1 mM each of CaCl2, MnCl2, and MgSO^ (metallized

Tris buffer). The homogenate was acidified with 0.3 M

acetic acid to pH 4.0 for 6 hours at 7°C and the

supernatant was made 50% with respect to ammonium sulfate

concentration. The precipitate was collected by

centrifugation and dissolved in metallized Tris buffer pH

7.0. Salt fractionated crude lectin thus obtained was

further purified by affinity chromatography on IgM-

Sepharose 6B column (2.2 x 6.0 cm) equilibrated with

metallized Tris buffer pH 7.0. The bound protein which

was eluted specifically with 0.25 M glucose in metallized

Tris buffer pH 7.0 constituted 3.4% of the total

- iii -

protein in the acid homogenate.

SDS-polyacrylamide gel electrophoresis of the lectin

showed a single band and gel chromatography on

Sepharose 6B column gave a single symmetrical peak,

suggesting that the affinity purified lectin was

homogenous with respect to size. The relative mobility of

the lectin was 0.79 both in the presence and absence of

2-mercaptoethanol, which corresponded to a molecular

weight of 18,000.

Molecular weight of the lectin under native

conditions was determined by analytical gel

chromatography on a Sepharose 6B column (1.8 x 78 cm)

which was calibrated with the help of marker proteins

which included bovine serum albumin (68,000), ovalbumin

(43,000), chymotrypsinogen A (25,700), myoglobin (17,200)

and cytochrome c (11,700). A curve was drawn between

Ve/Vo vs log M by the method of least squares. The curve

was found to fit the equation:-

Ve/Vo = -0.69 log M +'5.102

The value of Ve/Vo for Caianus cajan lectin was

found to be 1.93, which corresponded to a molecular

weight of 39,500. These results suggested that the

Caianus cajan lectin is a dimer and that the two subunits

are linked together only by non covalent interactions.

Neutral carbohydrate content of Caianus cajan

lectin was determined by phenol sulfuric acid method to

- IV

be 3% and the specific precipitation of the lectin with

con A and dissolution of the precipitate in 0.25 M

glucose suggested that the carbohydrate moiety of the

Cajanus cajan lectin contained mannose/glucose residues

for which con A is known to be specific. The optical

properties of lectin were indicative of the presence of

tyrosine and tryptophan residues. The specific extinction

coefficient of the lectin was determined to be 10.2

(g/ml)"-'- cm"- at 278 nm and 10.1 (g/ml)"-"- cm~-'- at 280 nm;

the corresponding value of the molar extinction

coefficient of the lectin at 278 nm was 39,780 M"-'- cm"" .

Results on amino acid analysis of 24 hours

hydrolyzate showed that the lectin contains high amounts

of threonine, serine, proline, glycine, alanine, valine

and lysine. Cysteine, methionine and tryptophan could not

be determined by this method. A comparison of amino acid

composition of Cajanus cajan lectin with con A showed

that the lectin had a similar content of threonine,

proline, glycine, alanine and phenylalanine.

Cajanus cajan lectin agglutinated rabbit erythrocytes and

precipitated goat IgM specifically. A precipitin curve

was obtained between fixed amount of lectin and

increasing concentration of IgM. To investigate the

saccharide binding specificity of the lectin the

hemagglutination and precipitin reaction was carried out

in the presence and absence of twelve saccharides.

- V -

studies on inhibition of lectin nediated hemagglutination

of rabbit erythrocytes showed that the lectin from

Cajanus cai an pulse is mannose/glucose specific. The

concentration required for 50% inhibition was more for

glucose (19.5 mM) than for mannose (9.0 mM) which

indicated that the lectin showed higher affinity for

mannose than for glucose. This showed that at C-2, axial

hydroxyl group was preferred over equatorial hydroxyl

group. Substitution of hydrogen of hydroxyl at C-2

position by methyl group considerably enhanced the

affinity of lectin for the saccharide derivative.

Interaction with glucosamine and N-acetyl glucosamine

suggests that variation at C-2 position alters the

affinity of lectin for the saccharide but does not

abolish it. The C-4 hydroxyl group was crucial for

binding so that galactose is not bound to the lectin.

Replacement of -OH group of C-1 position by para

nitrophenyl derivative in mannose enhanced the affinity

of lectin for binding. Infact, Cajanus cajan lectin

showed maximum affinity for P-NO2 phenyl mannopyranoside

(Cjjj = 0.4 mM) among the twelve sacccharides used in the

inhibition of hemagglutinating and glycoprotein

precipitating activity of the lectin. Four disaccharides

viz. sucrose, maltose, isomaltose and cellobiose were

also tested for their inhibitory potency. Cellobiose was

found to be least effective inhibitor suggesting the

VI -

importance of alpha linkage in disaccharides for

inhibition. Isomaltose (CI, = 3.0 laM) having oc 1.6 linked

glucose was most potent inhibitor. Comparison of

carbohydrate binding specificity of Cajanus cajan lectin

with con A showed that both in hemagglutination as well

as in glycoprotein precipitation behaviour,Caianus cajan

lectin is similar to con A. Cajanus cajan was also able

to precipitate out dextran like con A. As compared to con

A the pulse lectin showed reduced affinity for specific

saccharides.

- vii -

ACKNOWLEDGEMENTS

I owe my sincerest and most profound gratitude to

Prof. A. Salahuddin, Coordinator, Interdisciplinary

Biotechnology Unit, Aligarh Muslim University, Aligarh,

the supervisor of the present study. These words are the

barest acknowledgement of all that I owe to him towards

the completion of my research work. I have no words to

express my sense of gratitude to him for his patience,

constant encouragement, unstinted help and readiness to

provide the facilities needed for my experiment.

I am extremely grateful to Dr. A. Shoeb, Assistant

Director, Central Drug Research Institute, Lucknow for

his help and encouragement. I wish to also thank Dr.

P.V. Sane, Director, National Botanical Research

Institute, Lucknow and Dr. Dhan Prakash, Assistant

Director, NBRI, Lucknow for providing the facilities for

amino acid analysis. In this context, I would also like

to mention the help received from Dr. Sarita Singh and

Dr. Manisha Gupta.

I wish to record my gratitude to Dr. Saad Tayyab,

Dr. Parveen Salahuddin, Dr. Seema Hasan, Ms. Sadhna

Sharma and Ms. Zoya Galzie for their encouragement and

moral support.

Mr. Aftab Ahmad deserves special mention for all the

help he has rendered me during the course of my research

work. I am highly grateful to him for his help,co-

- viii -

operation and moral support.He has always been a source

of inspiration to me.

I am thankful to all those who have directly or

indirectly helped me in my work. Lest the list appears

cumbersome I would mention the names of Mr. Rizwan Hasan

Khan, Ms. Sakhra Shah, Mr. Ashok B.T., Ms. Kiran Misra,

Ms. Deeba Tahirah, Dr. Pratima Srivastava, Ms. Zeba

Mahmood, Ms. Ruwaida Hassan and Ms. Farah Deeba Jafri.

I owe my thanks also to the supporting staff of the

department specially Mr. Lai Mohammed, Mr. Amir Ali, Mr.

Aqtedar and Mr. Faisal as well as M/s Integrated

Computers, Aligarh for typing out the manuscript.

But for the SRF provided by the UGC, the work

perhaps would not have seen the light of the day. I

thank the UGC for their financial help.

Aligarh (SHAMA SIDDIQDT) Dated: SJijiu,

IX -

DEDICATION Dedicated to my parents,

brother and sister.

CONTENTS Page No.

ABSTRACT Hi

ACKNOWLEDGEMENTS viii

DEDICATION X

LIST OF ABBREVIATIONS. xiii

LIST OF TABLES. xv

LIST OF FIGURES. xvii

I. INTRODUCTION 1

II. EXPERIMENTAL 2 6

A. Materials 26

B. Methods 28

1. Measurement of pH. 2 8

2. Measurement of light absorption. 28

3. Determination of protein concentration.28

4. Determination of carbohydrate content. 29

5. Determination of amino acid composition. 29

6. Gel chromatography. 31

7. Sodium dodecyl sulphate polyacrylamide 31 gel electrophoresis

8. Equilibrium dialysis 33

9. Measurement of lectin activity. 3 3

10. Isolation of lectin. 34

III.RESULTS 38

1. Isolation of lectin. 38

2, Molecular weight under native conditions.41

XX -

3. Molecular weight under denaturing condition. 50

4. Optical properties. 50

5. Carbohydrate content. 56

6. Amino acid composition. 56

7. Carbohydrate binding specificity. 60

8. Interaction of lectin with dextran. 96

9. Equilibrium dialysis. 96

IV. DISCUSSION 100

V. REFERENCES 106

VI. BIOGRAPHY 114

VII. LIST OF PUBLICATIONS/PRESENTATIONS 115

- xii -

LIST OF ABBREVIATIONS

A^

cDNA

Con A

DDL

EcorL

E-PHA

Fuc

Gal

Gal NAc

Glc

GIN

GlcNAc

HCl

IgM

kD

Lac

LCL

L-PHA

Man

ittRNA

NaCl

NaOH

Neu SAC

PBS

Angstrom

Complementary deoxy ribonucleic acid

Concanavalin A

Dolichos biflorus lectin

Erythrina corallodendron lectin

Erythroagglutinin from Phaseolus vulgaris

Fucose

Galactose

N-acetyl galactosamine

Glucose

Glucosamine

N-acetyl glucosamine

Hydrochloric acid

Immunoglobulin M

Kilodalton

Lactose

Lens culinaris lectin

Leukoagglutinin from Phaseolus vulgaris

Mannose

Messenger ribonucleic acid

Sodium chloride

Sodium hydroxide

N-acetyl neuraminic acid

10 mM sodium phosphate buffer pH 7.0 containing 0.5 M NaCl

- XI11 -

PNA Peanut agglutinin

PSL Pisum sativum lectin

SBA Soybean agglutinin

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tris Tris (hydroxymethyl) amino methane

- XIV -

LIST OF TABLES

Page No.

Table I : Origin and sugar spec i f i c i ty of p u r i f i e d l e c t i n s from legume seeds.

Table I I : Class i f ica t ion of l ec t i n s in to spec i f i c i ty groups according to the monosaccharide t h a t i s t he bes t hapten i n h i b i t o r of the l e c t i n .

Table I I I : Lec t ins with pronounced 8 spec i f i c i ty for oligosaccharides

Table IV: Common molecular proper t ies of legume l ec t in s

12

Table V: Molecular properties of some mannose specific legume lectins

13

Table VI: Purification of Caianus caian lectin

39

Table VII: Gel filtration data 44

Table VIII: Relative mobility of marker proteins on SDS-PAGE

51

Table IX: Amino acid composition of Caianus caian lectin.

61

T a b l e X: E f f e c t of c a r b o h y d r a t e s on t h e 70 h e m a g g l u t i n a t i n g a c t i v i t y o f Ca ianus c a i a n l e c t i n .

- XV -

Table XI: Effect of carbohydrates on the 81 IgM precipitating activity of Can anus cajan lectin.

Table XII: Effect of carbohydrates on 83 the hemagglutinating activity of concanavalin A

Table XIII: Effect of carbohydrates on the 95 IgM precipitating activity of concanavalin A.

Table XIV: Comparison of amino acid composi- lO: tion of Cajanus cajan lectin with con A.

- XVI -

LIST OF FIGURES

FIGURE 1: Structure of oligosaccharide unit of legume lectins

FIGURE 2: Schematic representation of the structure of con A monomer.

FIGURE 3: A view of saccharide binding site of con A.

Page No,

16

20

21

FIGURE 4

FIGURE 5:

FIGURE 6:

FIGURE 7

FIGURE 8:

Determination of carbohydrate concentration by the method of Dubois et al, 1956,

FIGURE 9

FIGURE 10;

FIGURE 11:

FIGURE 12

FIGURE 13

FIGURE 14

Determination of amino acid concentration from the peak area.

Precipitin reaction of Caianus caian lectin with IgM

Precipitin reaction of concanvalin A with IgM

Purification of salt fractionated Caianus caian lectin by affinity chromatography on IgM Sepharose 6B column.

Gel filtration behaviour of affinity purified Caianus caj an lectin on Sephadex G-lOO column.

SDS-PAGE of affinity purified Caianus caian lectin

Gel chromatography of proteins on Sepharose 6B column.

Gel chromatography of cytochrome c (4) , myoglobin (5) and glucose (6) on Sepharose 6 B column.

Gel filtration behaviour of con A (7) and Caianus cajan lectin (8) on Sepharose 6 B column.

Elution profile of Blue Dextran on Sepharose 6 B column.

30

31

35

36

40

42

43

45

46

47

48

- XVI1 -

FIGURE 15: Plot of Ve/Vo versus logM 49

FIGURE 16: Plot of relative mobility (Rm) versus 52 logM for marker proteins.

FIGURE 17: Ultraviolet absorption spectra of 54 Cajanus cajan lectin.

FIGURE 18: Determination of specific extinction 55 coefficient of Cajanus cajan lectin.

FIGURE 19: Specific interaction of the glyco 57 protein Cajanus cajan lectin with con A.

FIGURE 20: Elution profile of standard amino acid 58 sample in LKB Biochrom (4101) amino acid analyzer

FIGURE 21: Amino acid hydrolyzate of Cajanus 59 cajan lectin by LKB Biochrom 4101 amino acid analyzer.

FIGURE 22: Effect of saccharides on hemagglu- 63 tinating activity of Cajanus cajan lectin.

FIGURE 23: Effect of methyl oc mannoside on 64 hemagglutinating activity of Cajanus cajan lectin,

FIGURE 24: Effect of glucosamine on 65 hemagglutinating activity of Cajanus Ccijan lectin.

FIGURE 25: Effect of -N-acetyl glucosamine on 66 hemagglutinating activity of Cajanus cajan lectin.

FIGURE 26: Effect of fructose on hemagglutinating 67 activity of Cajanus cajan lectin.

FIGURE 27: Effect of sucrose on hemagglutinating 68 activity of Cajanus cajan lectin.

FIGURE 28: Effect of maltose on hemagglutinating 69 activity of Cajanus cajan lectin.

FIGURE 29: Effect of saccharides on the 72 precipitation of IgM with Cajanus cajan lectin.

- xviix -

FIGURE 30: Effect of methyl « mannoside on the 73 IgM precipitation of IgM with Cajanus cajan lectin.

FIGURE 31: Effect of N-acetyl glucosamine on the 74 precipitation of IgM with Cajanus cajan lectin.

FIGURE 32: Effect of glucosamine on the 75 precipitation of IgM with Cajanus cajan lectin.

FIGURE 33: Effect of fructose on the 76 precipitation of IgM with Cajanus cajan lectin.

FIGURE 34: Effect of sucrose on the 77 precipitation of IgM with Cajanus cajan lectin.

FIGURE 35: Effect of maltose on the precipitation 78 of IgM with Cajanus cajan lectin.

FIGURE 36: Effect of isomaltose on the 79 precipitation of IgM with Cajanus cajan lectin.

FIGURE 37: Effect of P-NO2 phenyl oc mannoside on 80 precipitation of IgM with Cajanus cajan lectin.

FIGURE 38: Effect of saccharides on hemagglu- 84 tinating activity of con A.

FIGURE 39: Effect of methyl oc mannoside on 85 hemagglutinating activity of con A.

FIGURE 40: Effect of fructose on hemagglutinating 86 activity of con A.

FIGURE 41: Effect of sucrose on hemagglutinating 87 activity of con A.

FIGURE 42: Effect of maltose on hemagglutinating 88 activity of con A.

FIGURE 43: Effect of saccharides on the 89 precipitation of IgM with con A.

FIGURE 44: Effect of methy] oc mannoside on the 90 precipitation of IgM with con A.

- xi:: -

FIGURE 45: Effect of fructose on the precipitation of IgM with con A.

FIGURE 46: Effect of sucrose on the precipitation of IgM with con A,

FIGURE 47: Effect of maltose on the precipitation of IgM with con A.

FIGURE 48: Effect of isomaltose on the precipitation of IgM with con A.

FIGURE 49: Interaction of dextran with Caianus cajan lectin and con A.

FIGURE 50: Interaction of dextran with Caianus cajan lectin and con A.

FIGURE 51: Schematic diagram of mannose.

91

92

93

94

97

98

104

- XK -

/. INTRODUCTION

I. INTRODUCTION

Lectins are carbohydrate binding proteins of non

immune origin which agglutinate cells and/or precipitate

polysaccharides or glycoconjugates (Goldstein ^t. al.,

1980 and Dixon, 1981).

1. Occurrence

Lectins are heterogenous molecules widely

distributed in nature and have been isolated from plants

(Etzler, 1986), fungi (Kretz et a^. , 1991; Licastro et

al. , 1993), bacteria (Sharon, 1986), viruses,

invertebrates (Kocourek, 1986) and vertebrates (Barondes,

1986; Licastro et aj.. , 1991). Family leguminoseae is the

richest source of lectin in plants (Tom and Western,

1971), although significant number of lectins have been

identified from members of Euphorbiaceae, Verbenaceae,

Malvaceae, Polygonaceae, Solanaceae, Cactaceae,

Cucurbitaceae etc. (Sharon and Lis, 1990). In leguminous

plants, lectins have been detected in more than 600

species and varieties. Nearly 70 legume lectins have

been isolated by affinity chromatography and about 40 are

commercially available. Some of the legume lectins with

their sugar specificities are listed in Table I (Sharon

and Lis,, 1990) .

The main source of legume lectins are mature seeds

and the bulk of lectin is located in the cotyledons in

- 2 -

TABLE - 1. Origin jmd Sugar specificity of purified lectins from legime seeds

Suborder and tribe Spec 1es Specificity

Caesalpinaceae Bauhinia purpurea

Griffonia simohcifolia

Papi honaceae

Abreae Abrus pre:atorius (jequirity bean)

Carageae Caragana arborescens (pea tree)

Dalbengeae Lonchocarpus capassa

galactose/N-acetyl galactosamine

galactose/N-acetyl galactosamine

N-acetyl glucosamine,

oligosaccharide

galactose/N-acetyl galactosamine

mannose (glucose)

galactose/M-acetyl galactosamine

Diocleae Canavalia ensiformis (Jack bean)

Canavalia galadiata (Japanese Jack bean)

Dioclea grandiflora

mannose (glucose)

inannose (glucose)

mannose (glucose)

Galageae Robina pseudoacacia (black locust)

Wistaria flonbunda (wistaria)

only oligosaccharides

galactose/N-acetyl galactosamine

and oligosaccharides

Genisteae Crotalaria juncea (sunnhemp)

Cystisus scopanus

Cyst 1sus sessifolius (broom)

Ulex europaeus (furze, gorse)

galactose/N-acetyl galactosamine

galactose/N-acetyl galactosamine

N-acetyl glucosamine

N-acetyl glucosamine, L. Fucose

Hedysareae Arachis hypogeae (peanut)

Oiobrychis vicnfolia (sainfoin)

galactose/N-acetyl galactosamine

mannose (glucose)

Loteae Lotus tetraqonolobus (asparagus pea) L. Fucose

Phaseoleae Amphicarpea bracteata (hog peanut)

Dolichos biflorus (horse gram)

Erythnna coral lodendron (coral tree)

GIyc1ne max (soybean)

Phaseolus lunatus limensis (limabean)

Phaseolus vulgaris (kidney bean)

galactose/N-acetyl galactosamine

galactose/N-acetyl galactosamine

galactose/N-acetyl galactosamine

galactose/N-acetyl galactosamine

galactose/N-acetyt galactosamine

oligosaccharides

Psophocarpus tetraqonolobus (winged bean) galactose/N-acetyl galactosamine

Viqna radiata (mung bean) galactose/N-acetyt galactosamine

Sophoreae Bownnqia mi Ibraedi i

Haackia amurensis

Sophora laponica (Japanese Pagoda Tree)

mannose (glucose)

Oligosaccharides

galactose/N-acetyl galactosamine

Tnfolieae Medicago sativa (alfalfa)

Tnfol uim repcns (white clover)

galactose/N-acetyl galactosamine

2. aeoxy glucose

- 3 -

V i c i e a e l a t hy rus ochrus

Lens cut m a r i s

Pi sum sai lvum

V i c i a eracca (cormion ve tch)

V i c i a fa te ( fava bean) * V i c i a qramineae

V i c i a VI Hosa ( h a i r y ve tch)

mannose (glucose)

mannose (glucose)

mannose (glucose)

mannose (glucose), galactose/

N-acetyl galactosamine

mannose (glucose)

oligosaccharides

glactose/N-acetyl galactosamine

bind more specifically with glycopeptides. Ref. Sharon and Lis, 1990.

- 4 -

organelles known as protein bodies (Etzler, 1985; Etzler,

1986; Clarke et al., 1975; Chrispeels and Tague, 1990).

Most plant tissues contain only one kind of lectin

but there may be two or more types of lectins in a given

plant with distinguishable molecular and sugar binding

properties (Law and Strijdom, 1984; Kaku et al. , 1990;

and Mach et al., 1991).

2. Carbohydrate binding specificity

The sugar specificity of a lectin is defined by the

monosaccharide, disaccharide, oligosaccharide or

glycoprotein which is required in minimal concentration

to inhibit the lectin mediated agglutination of cells or

precipitation of glycoconjugate (Goldstein and Hayes,

1978; Sharon and Lis, 1989; Goldstein and Poretz, 1986).

Binding of saccharides to lectin can be examined by

physicochemical methods such as equilibrium dialysis,

spectrophotometry, fluorometry and nuclear magnetic

resonance ( Liener 1986; Sharon and Lis, 1990;

Sharon and Lis, 1989). Sugar binding activity is also

measured by using biosensor technology which involves

binding of lectin to immobilized high affinity glycan

ligand to a sensor chip (Diaz and Kijne, 1992) . By these

methods the association constant and other thermodynamic

(Schwarz et. aJL. , 1993) and kinetic parameters of the

reaction can be measured. The binding constants between

lectins and monosaccharides ara typically 1x10 to 5 x

- 5 -

10'* M~- and between lectin and oligosaccharide lo' to lo'M"-'-,

The associated decrease in free energy for monosaccharide

is about 5 k cal/mole (at 25°C) and for oligosaccharide

the value is 7 k cal/mole (Salahuddin, 1992). These

values are in the same range as those found for the

binding of haptens to antibodies or of substrate to

enzymes.

Lectins are classified into five specificity groups

(Table II) according to the monosaccharide that is the

best hapten inhibitor of the lectin activity. Members of

each group may vary in their affinity for various analogs

and derivatives of the monosaccharide. Carbohydrate

binding specificity of some lectins from legume seeds is

shown in Table I.

Lectins differ markedly with respect to their

anomeric specificity. Some lectins exhibit pronounced

specificity (Pereira et ., 1974; van Wauve, et aJ. , 1975

and Koi-tt, 1984) whereas others do not show much

difference between oc and p anomers (Nicolson et al. ,

1974; Mazumdar, et al. , 1981 and Lis et al., 1970).

Variation at C-2 position may be tolerated by some

lectins, for example mannose specific lectins also react

with glucose and N-acetyl glucosamine. Some lectins

tolerate variation at C-3 position, but the configuration

at C-4 hydroxyl group is critical so that mannose/glucose

specific lectins do not combine with galactose and vice

- 6 -

Table II Classification of lectins into specifity groups according to the monosaccharide that is the best hapten inhibitor of the lectin

Specificity group Specific Sugar

I Mannose (glucose)

II galactose/N-acetyl-

galactosamine

III N-acetyl glucosamine

IV L fucose

V Sialic acid

X interact only with oligosaccharide

- 7 -

versa. The binding site of most legume lectins

accomodate a single saccharide residue. In a few cases

however, the nature of saccharide linkage plays a

significant role in determining the binding specificity.

Thus isomaltose and other oligosaccharides containing non

terminal <x 1-6 glucosidic bonds have a higher affinity

for con A than oligosaccharides having non reducing

oc 1 > 2, 1 > 3 or 1 > 4 glucosidic linkages

(Goldstein and Hayes, 1978). For some lectins it has

been shown that the carbohydrate binding site is

complementary to two or more sugar units i.e. they have

an extended binding site (Horejsi and Kocourek,1978a & b;

Decastel, et al. , 1982; De Boeck, et al.. , 1983). There

are lectins which interact more strongly with

oligosaccharides than with monosaccharides (Table III)

(Lis and Sharon, 1986). The specific monosaccharide in

the oligosaccharide is usually at the non reducing end

but the lectin will react even if the specific sugar is

internal. A few legume lectins interact only with

oligosaccharides. Lectins such as Vicia graminoseae and

Vicia villosa bind a glycopeptide more strongly than the

corresponding oligosaccharide implying that the peptide

to which the oligosaccharide is linked is also recognized

by the lectin. From a mixture of oligosaccharides, a

lectin prefers to form a homopolymeric complex as against

heteropolymeric ones (Bhattacharyya, et aj . , 1989; Mandal

- 8 -

T a b l e I I I . L e c t i n s w i t h p r o n o u n c e d s p e c i f i c i t y f o r o l i g o s a c c h a r i d e s

S o u r c e of l e c t i n M o n o - O l i g o s a c c h a r i d e

s a c c h a r i d e s s t r u c t u r e

R e l a t i v e

i n h i b i t i o n

D a t u r a s t r a m o n i u m G I c N A c

Dot i c h o s b i f I o r u s G a l N A c

E r y t h r i n a c r y s t a g a l I i G a I

A r a c h i s h y p o q e a e G a I

P h a s e o l u s v u l g a r i s

E . P H A

P h a s e o l u s v u l g a r i s

L . P H A .

V i c i a g r a m i n e a e

V i c i a v i I I o s a

W h e a t g e r m

M a c k i a a m u r e n s i s

Ga I N a c

G I c N A c

G l c . N A c . p . G l c . N A c p , -

G I c N A c

G a l N A c <x 3 G a l N A c

G a l p . G l c N A c

G a i p , G a l N A c

G a i p . G l c N A c p , M a n oc6

G I c N A c p M a n p 4 / R

G I c N A c p p M a n « 3

Ga ip , G I c N A c p p

Ga ip , G I c N A c p ,

N M a n

N H j - L e u

G I c N A c p ^ G I c N A c p ^ G I c N A c

N e u Ac « 2,3 Gal p 1,4 G l c

5 5 0

36

30-50

50

Gal P- GalNAc « - Ser

I Gal p , G a l N A c 0( - T h r

I Gal p , G a I N A c a - T h r

I G I u - C O O H

N H 2 - S e r - ( P r o ) 2 - G l y ( A l a ) 2 - T h r C O O H

I I G a l N A c a G a l N A c a 120

3000

- 9 -

and Brewer, 1992, Bhattacharyya, et al. , 1990). The

formation of homopolymeric oligosaccharide and

glycopeptide lectin cross linked complex represents a new

dimension of binding specificity between carbohydrates

and proteins (Mandal and Brewer, 1992; Bhattacharyya and

Brewer, 1992). The ability of an oligosaccharide to

combine with a lectin depends on its conformation (Romans

et al. . 1987: Carver et ai. , 1989). It is therefore

possible that two different oligosaccharides may adopt

similar conformational format and react with a lectin

similarly (Salahuddin, 1992).There are several blood

group specific lectins that agglutinate human

erythrocytes of a particular blood type (Goldstein and

Hayes, 1978; Bird,1978; Judd, 1980; Poretz et al., 1974).

3. Types of interactions involved in carbohydrate binding

The nature of forces involved in carbohydrate-lectin

interaction is a subject of considerable interest and has

attracted much attention in view of their role in a large

number of key events in cell biology. Consequently there

is a growing interest in the understanding of the

molecular basis for specificity and affinity in these

interactions (Solis, et aj,. , 1993). X-ray

crystallographic studies of several carbohydrate lectin

complexes have shown that hydrogen bonds together with

- 10 -

van der Waals contacts and stacking interactions (Lee,

1992) are the dominant forces that stabilize the

complexes (Vyas, 1991; Bundle and Young, 1992). Because

hydrogen bonds are highly directional, they ensure the

correct fit of the ligand and confer stereospecificity to

the binding. They also provide a stable solvation shell

for the bound sugars. In addition, hydrogen bond strength

is high enough to stabilize the complexe but low enough

to allow rapid ligand dissociation. All these factors

make hydrogen bonding the primary interaction in lectin

carbohydrate complex formation.

X-ray crystallographic studies have revealed that

the adjustable topological parameters such as the outline

of the B-turns and shape of the variable residues lining

the carbohydrate binding site along with the conserved

sequence of residues involved directly in hydrogen bonds

and van der Waals contacts, confer specificity to the

legume lectin. Stacking of aromatic aminoacid residues

against the face of hexapyranosides (the hydrophobic

patch formed by its C-5 and C-6) is a feature of lectin

carbohydrate interaction that has only recently been

appreciated (Vyas, 1991). It contributes to the

stability and specificity of the complexes formed.

Saccharide induced conformational changes in the

side chains involved in sugar binding in wheat germ-N

acetyl neuraminyl lactose complex were found to be small

- 11 -

but were repeatedly observed (Wright, 1990). No

significant conformational change was observed in regions

remote from the saccharide binding site nor were there

any movement of chain segments (Wright, 199 0).

4. Physicochemical properties

(a) Molecular properties

Despite marked differences in sugar specificity of

legume lectins they share many common molecular

properties (Sharon and Lis, 1990). The subunit molecular

weight, and the number of sugar binding sites of some

mannose binding legume lectins are summarized in the

Table IV and Table V.They are usually composed of 2(or 4)

subunits with molecular mass of 25-30 kD. Nearly all of

them are glycoproteins and metalloproteins containing

Ca "*" and Mn . These lectins consist of subunits (or

protomers) that are usually identical or very similar,

each containing a single saccharide binding site with the

same specificity. The subunits of the legume lectins are

made up of a single polypeptide chain but in some cases

they consist of two polypeptide chains. Con A is composed

of a mixture of intact and cleaved subunits. Its single

polypeptide chain contains 237 amino acids; the cleaved

subunit possesses the same amino acid sequence but a

nicked peptide between residues 118 and 119. Some lectins

exist in mora than one elsctroohoreticallv

- 12 -

Table IV. Common molecular properties of legixme lectins

Subunit Mol.mass^ Number

Combining Sites Number' specificity^

Metal ions

25 - 30 kD. 2 or 4

1 per sub unit usually identical

Ca ++ Mn ++

Amino acids Hydroxy and carboxy Sulfur containing

Sequence homology N-Glycosylation^ 3 Dimensional structure Of helix B sheet

High low or absent

Extensive common

low or absent Main structural element.

a. In some lectins subunit consists of two polypeptides. b. Lima bean lectin occurs also in a form consisting of

eight subunits. c. Dolichos biflorus and Abrus precatorius have 2 s.u.

only one of which binds sugar. d. Phaseolus vulgaris and Vicia villosa contain 2 types

of sub units with different specificities. e. Absent in con A, peanut and some of viciae lectins

(Sharon and Lis, 1990).

- 13 -

Table V. Molecular properties of some mannose specific legume lectins

Source Molecular Subunit Subunit

weight inol. wt. number

Canavalia ensiformis 102,000

Onobrychis viciifolia

Lens culinaris

Pisum sativum

Vicia faba

Bowrinqia milbraedii

102,000

53,000

46,500

49-53,000

52,000

38,000

25,500

26,509

5,700 17,500

5,800 12-18,000

5,500 20,700

16,000

4

2

2 2

2

2

2

14 -

distinguishable forms or isolectins. Thus Phaseolus

vulgaris lectin is a tetramer and can exist in five

different forms containing varying amounts of two types

of subunits E and L, which differ in carbohydrate binding

specificity and other biological properties. The

isolectin E^ for example is a potent hemagglutinin but a

poor mitogen whereas the L^ is a potent mitogen and

agglutinates lymphocytes (Miller et al., 1975).

(b) Amino acid composition

Legume lectins are characterized by their high

acidic and hydroxylic amino acid content and less or none

sulfur containing amino acids. Hydrophobic amino acids

like valine, leucine, alanine and phenylalanine are also

found in considerable amounts. The content of aromatic

amino acids like tyrosine and tryptophan is low and

glycine and proline are present in substantial amounts.

(c) Non protein moiety

Most legume lectins contain Mn and Ca'' which

appear to be essential for their activity. In con A,

b m d m g of both Mn and Ca is essential for generation

of saccharide binding site (Goldstein and Hayes, 1978).

The amino acids that coordinate the metal ions to the

proteins include an aspartic acid residue and an

asparagine that also form hydrogen bonds with the

monosaccharide in the combining site. Thus the metal ions

- 15 -

do not interact directly with the carbohydrate but help

to position amino acid residues for carbohydrate binding

(Sharon, 199 3),

Lectins are generally glycoproteins with varying

amounts of carbohydrate content. They may contain upto

10% carbohydrate in the foirm of a small number of N-

linked carbohydrate units (Fig. 1) (Sharon and Lis,

1990).

A variety of biological functions have been assigned

to N-linked oligosaccharides of glycoprotein. The sugar

chains of glycoprotein lectin play an important role as

the signal of cell to cell recognition. They are also

associated with the biosynthesis of glycoprotein lectins

facilitating the folding and assembly of nascent

polypeptides (Nagai and Yamaguchi 1993). The sugar

chains,, however, do not appear to be required for the

activity of the glycoprotein lectins (Lis and Sharon,

1981). The most convincing proof comes from the finding

that the unglycosylated L-PHA (leukoagglutinating

Phaseolus vulgaris) synthesized in E-coli had the same

leukoagglutinating activity as the native glycosylated

lectin (Hoffman and Donaldson, 1985). Lectins like

con A and wheat germ (Allen et ad. , 1973) are few

exceptions as they are devoid of carbohydrate residues.

Although con A is not a glycoprotein, it is synthesized

as a glycosylated precursor (Bowles and Pappin, 198S).

- 15 -

( Man ^ 1 )-—>4Glc Nac p i — > 4 G l c N a c - N Asn

{Oligomannose type)

Man a 1—> 6 \

Ma n a 1 > 3 /

Man p i > 4 Glc Nac p i >4Glc Nac-NAsn.

2 ^

Xy ^ 1

3

t Fuc a 1

(Fucosylated type)

Man a 1—>^

Man a 1 >3

\

/

Man p i >4-G lcNac-G lcNac-NAsn

t Xyipi

(Non fucosylated type)

Fig.l. Structure of oligosaccharide unit of legume lectins.

17 -

5. Primary structure and sequence homology

The primary structure of more than 20 legume

lectins have been established by chemical or molecular

genetic techniques. They exhibit remarkable sequence

homology with over 20% of invariant aminoacid residues.

Lectins of the same tribe may exhibit substantial (above

60% as in Vicieae lectins) to moderate (about 30% in the

Phaseoleae lectins) sequence homology. The conserved

residues include several of those involved in binding of

monosaccharides and almost all the residues that

coordinate the metal ions. The N-glycosylation sequons

Asn X Ser/Thr if present, are located at different

positions of primary structure and may or may not be

occupied (e.g. con A).

Sequence homology of the lectins from plants

belonging to the Dioclea tribe (for e.g. Canavalia

ensiformis, Canavalia gladiata and Dioclea grandiflora)

is obtained by aligning residue 123 of the Dioclea

lectins with the NH2-terminal amino acids of the other

lectins, proceeding to the carboxyl ends of Dioclea

lectins and continuing along their NH2 terminal regions.

Such unusual type of homology is referred to as circular

homology. The extensive homology between different

lectins suggests that these are conserved proteins and

have a common genetic origin.

- 18 -

6. Biosynthesis

Legume lectins are initially synthesized in

endoplasmic reticulum as prolectin comprising a single

polypeptide chain (Sharon and Lis, 1990). Two chain

lectins from the Vicieae tribe (pea, lentil, favin etc.)

are first synthesized as preprolectin which are

subsequently processed to give preforms. After removal of

2-3 0 amino acids the polypeptide chain is proteolytically

processed frequently yielding cleaved subunits (Bowles

and Pappin, 1988). Cotranslational N-glycosylation and

further post translational modifications of the

carbohydrate units takes place in the golgi apparatus.

More definitive information on molecular events occuring

during biosynthesis of lectins was provided by recent

studies on cloning of genomic and cDNAs of a number of

legume lectins (Arango, et al. , 1992; Yamamoto, et al. ,

1992; Edwards and Hepher, 1991; Schroeder et al. , 1992;

Arango, et aJ., 1990). Cloned cDNAs and genes of lectins

have been expressed in bacteria, yeast and heterologous

plants (Prasthofer, et aX-, 1989; Okamuro, et al.. , 1986;

Tague and Chrispeels, 1987; Sturm, et al., 1988; Yamauchi

and Minamikawa, 1990; Schroeder, et al.. , 1992; Edwards

and Hepher, 1991; Arango et al., 1992; Wales et al. ,

1992). Recombinant con A in E.coli had the same sequence

as the native lectin suggesting that the machinery for

post translational processing of the precursor con A also

- 19 -

exists in bacteria (Yamauchi and Minamikawa, 1990).

7. Three dimensional structure of a model mannose/glucose specific lectin (Concanavalin A)

Con A is the first lectin whose three dimensional

structure was solved by X-ray analysis (Reeke and Becker,

1988),. The lectin subunit (protomer) acquires a

compactly folded and dome (42 x 40 x 39 AQ) shaped

structure (Fig. 2). Two of the subunit polypeptides with

a structure based on antiparallel p pleated sheets are

joined in a functional ellipsoidal dimeric unit and two

of these are then paired to form the native tetrameric

lectin., The tetramer is mostly stabilized by hydrogen

bonds although some polar and hydrophobic interactions

are also present. The saccharide and metal ion binding

sites in con A occur on the seven chain front B sheets of

a con A monomer. The two metal binding sites are 5 A°

apart and are located at the top of the monomer. The

saccharide binding site is situated 12 A° from Mn from

binding site and 7 A° from the Ca " binding site (Fig.2).

The amino acid residues which form the seven hydrogen

bonds with the saccharide are Asp-14, Leu-99, Tyr-100,

Asp 208 and Arg 228 (Fig. 3) . Tyr-12 and Tyr-100 are

involved in hydrophobic interactions. The metal ions are

each bound to four amino acid side chains and two water

molecules are hydrogen bonded to the protein (Fig. 3) .

20 -

Fig.2. Schematic representation of the Structure of con A monomer.

++ CA and MN represent the Ca'' and Mn and CHO localizes the carbohydrate (Hardman et al., 1982)

++ binding site binding site

21 -

AsplO

.•H2O

Fig.3 A view of saccharide binding site of con A showing a network of hydrogen bonds that stabilize the interaction between the protein and the saccharide (Derewenda et al., 1989).

- 22 -

Out of the six residues of con A that interact with the

sugar, two (Asn-14 and Asp 208) are conserved in all

legume lectins.

8. Three Dimensional Structure of lectin carbohydrate complex

X-ray crystallographic studies have revealed that

structural similarities and homologies found in legume

lectins is better expressed in terms of similarities in

their three dimensional structure. High resolution X-ray

crystallographic structure of fava bean and pea (Reeke

and Becker, 1986), lentil lectin (Loris et al., 1992) and

peanut lectin (Banerjee, et aJ. , 1992) has been worked

out. X-ray crystallographic structure of legume lectin in

complex with their specific saccharide viz..Pisum sativum

(Rini, et ai., 1991), Lathyrus ochrus (Bourne, 1990),

Erythrina corallodendron (Shaanan, e^ aJ., 1991),

Griffonia simplicifolia (Delbaere, 19 90) and con A

(Derewenda, et al. , 1989) have also been reported. The

subunits of these lectins are in a shape of a half dome

(Fig. 2 ) having the sugar binding site as a shallow

depression at its apex. They are abundant in antiparallel

p sheets and <x helices are almost absent. Differences in

structure of lectins is confined primarily to the B

turns and loop regions. The Ca and Mn binding site

are located close to the carbohydrate binding site and

are identically positioned in all legume lectins

- 23 -

examined. The combining sites of legume lectins are quite

similar and partly superimposable. In each of the legume

lectins studied so far, seven hydrogen bonds (Fig.3) hold

the monosaccharide in the combining site. Strikingly, the

side chains of the two conserved residues, an aspartic

acid (Asp 81 in LOLI and Asp 89 in EcorL) and an

asparagine (Asn 125 in LOLI and Asn 13 3 in EcorL), that

form three key hydrogen bonds with the saccharide (and

also coordinate the metal ions) have an identical spatial

disposition in these lectins. The substitution of Asn 125

with Asp in pea lectin completely abolishes it saccharide

binding activity (Eijsden, et. al_-i 1992). Another

residue that is conserved is glycine (Gly 99 in LOLI and

Gly 107 in EcorL) . It forms a hydrogen bond with its

main chain amide and 4-OH (and 3-OH) of mannose or 3-OH

of galactose. In the combining site the adjacent amino

acid residues are not always identical, resulting in

different topographies of the combining site. Analysis of

sequence variation among legume lectins showed a ring of

hypervariable residues forming a perimeter of the

carbohydrate binding site (Young and Oowen, 1992).

Presence of a variable binding region in legume lectin

that determined their carbohydrate binding specificity

was for the first time demonstrated by Yamamoto, et al.

1992 who prepared a chimeric lectin gene of a galactose

specific lectin Bauhinia purpurea containing the sequence

- 24 -

corresponding to the binding site of Lens culinaris

lectin a mannose specific lectin. The chimeric lectin

expressed in E.coli was found to be specific for mannose.

It appears, therefore that the specificity of legume

lectins results from coupling adjustable topological

parameters such as the outline of the B turn and shape of

the variable residues lining the binding pocket, with a

conser^'ed constellation of residues involved directly in

hydrogen bonds and van der Waals contacts. The changes in

orientation and locations of these residues determines

the differential carbohydrate binding specificity in the

same type of lectin (Ayouba, et aj . , 1992).

Objective

Legume lectins are proteins in search of their

function. Several lines of evidence suggest that they may

serve as storage or reserve proteins, cell recognition

molecules and they are involved in the defence against

bacterial, viral and fungal pathogens. (Sharon and Lis,

1990; Barondes, 1981; Etzler, 1981; Leach, et al. , 1982;

Mishkind, et. aj.. , 1982 and Broekaert et. aj^. , 1989;

Pusztai, et al. , 1983; Etzler, 1986; Greenwood, et al. ,

198 6; Nsimba and Peumans, 198 6; Peumans, et al. , 1985;

Peumans and van Damme, 1992).

The involvement of lectins in rhizobium legume

interactions was indicated by the experiment on specific

agglutination of Rhizobium phaseoli (this infects

- 25 -

nodulous roots of Phaseolus vulgaris) by the seed

extracts of kidney bean (Phaseolus vulgaris) . However, a

convincing evidence was provided by Dazzo and co-workers

(1983) who eluted lectin from the roots of white clover

with 2-deoxy glucose. The lectin was able to agglutinate

Rhizobium trifolii whose capsular polysaccharides

contain 2-deoxyglucose. R. trifolii was found to bind

white clover roots and the binding was interestingly

inhibited by 2-deoxy glucose. Diaz, et aJ.. (1989)

introduced the pea lectin gene into white clover roots

using Agrobacterium rhizogenes as a vector. Rhizobium

leguminosarum was found to infect the transgenic clover

roots causing root hair curling. These results are

consistent with the lectin mediated rhizobium - legume

interaction.

In view of the above considerations it was thought

desirable to isolate and purify lectin from a hitherto

less investigated leguminous plant viz. Caianus caian.

This thesis describes the isolation, purification and

physicochemical characterization of a mannose/glucose

specific lectin from Cajanus cajan pulse. A detailed

study on its carbohydrate binding properties has also

been made.

II.EXPERIMENTAL

A,MATERIALS

1. Proteins

Bovine serum albumin (lot no. 10F.0331); ovalbumin

(lot no. 105C-8022) ; chymotrypsinogen A (lot no. 40F-

8050) ; cytochrome c (lot no. 09C-0088) ; myoglobin (lot

no. 85C-0138); and trypsin (lot no. 1-2395) were obtained

from Sigma Chemical Company, St. Louis, MO., USA.

IgM was isolated from goat blood by precipitation

at low ionic strength from goat plasma and con A was

isolated by affinity chromatography as reported earlier

from this laboratory ( Waseem and Salahuddin, 1983).

2, Sugcirs

Ccirbohydrates used in these studies with their

sources in parentheses were: dextran (lot no. 18F-0532

Sigma Chemical Company, St. Louis, MO., USA); glucosamine

(lot no. 108C-0197 Sigma Chemical Company, St. Louis,

MO., USA.); N-acetyl D-glucosamine (lot no. 90F-0348

Sigma Chemical Company, St. Louis, MO., USA.); methyl cc

D-mannopyranoside (lot no. 108C-5046 Sigma Chemical

Company, St. Louis., MO., USA.); p-nitro phenyl cx-D

mannoside (lot no. 29C-7804 Sigma Chemical Company, St.

Louis, MO., USA.); isomaltose (lot no. 99F-4031 Sigma

Chemical Company, St. Louis, MO., USA.); cellobiose (lot

no. 69F-0387 Sigma Chemical Company,. St. Lcuis. MO.,

- 27 -

USA,); glucose (Glaxo Laboratories, India); sucrose (BDH,

India); maltose (BDH, India); galactose (BDH, India);

fructose (Sarabhai M. Chemicals, India) and mannose

(Sarabhai M. Chemicals, India).

Sepharose 6B and Sephadex G-lOO were purchased from

Sigma Chemical Company, St. Louis, MO., USA. Cyanogen

bromide was obtained from SRL Pvt. Ltd. Bombay India

Other reagents were laboratory grade and were used

without further purification. Samples of Cajanus caian

pulse were purchased from the local market.

Glass distilled water was used throughout this

study.

B. METHODS

1. Measurement of pH

Elico digital pH meter, model Ll-122 in conjunction

with Elico combined glass electrode type CL-51 was used

for pH measurements at room temperature. The pH meter was

standardized with 0.05 M potassium hydrogen pthalate

buffer, pH 4.0 in the acidic range and with 0.01 M sodium

tetraborate buffer pH 9.2 in the basic range.

2. Measurement of light absorption

Light absorption measurements were made on CECIL

double beam spectrophotometer, model CE 594 in the ultra

violet region and on AIMIL photochem-8 colorimeter in the

visible range.

3. Determination of protein concentration

Protein concentration was routinely determined by

the method of Lowry et. aJL. (1951) using crystalline

bovine serum albumin as a standard. A calibration curve

between optical density and protein concentration was

obtained by the method of least squares and was found to

obey the equation:-

(O.D)7Q,3 ^^ = 1.7 ( mg protein) + 0.034 (1)

4. Determination of carbohydrate content

Neutral carbohydrate content was determined by the

- 29 -

method of Dubois, et, al.. (1956) using glucose as

standard. The colour intensity at 490 nm was plotted as a

function of glucose concentration. The straight line

(Fig. 4) obtained by the method of least squares fits the

equation:-

(O.D) 4gQ jjjj = 0.00436 ( Jug glucose ) + 0.033 (2)

5. Determination of amino acid composition

Amino acid composition was determined by Dr. Dhan

Prakash NBRI, Lucknow on LKB Biochrom 4101 automatic

amino acid analyser according to the method of Spackman

et al. (1958). Three buffer solutions, A, B and C of pH

3.25, 4.25 and 6.45 respectively were prepared from

sodium acetate, concentrated HCl, thiodiglycol and phenol

according to the standard procedure. Ninhydrin solution

was a commercial sample. Protein solution (5 ml) was

mixed with concentrated HCl (5 ml) to obtain a final

concentration of 100 nM in a glass tube. After adding a

few drops of 2 mercaptoethanol and phenol the sample was

flushed with nitrogen for 2 0 minutes and the tubes were

sealed. After acid hydrolysis the residue was dissolved

in 20 ml of the loading buffer and analysed by amino acid

analyzer. A calibration curve with known standard LKB

amino acid mixture was also obtained. The concentration

was calculated from the area relative to the area of the

standard amino acid (Fig. 5).

- 30 -

£ c o en

••-• O >. ••-•

*(/>

c

"5 o "• Q. O

40 60 60 100

Glucose concentration (ug /ml)

Fig. 4 Determination of carbohydrate concentration by the method of Dubois et al., 1956. The linear plot was obtained by the method of least squares and was found to obey the equation;-(O.D) ,nn =0.00436 X ijag glucose) + 0.033

- 31 -

Area= HxW where

H = net peak height W = width of the peak at 1, H

Fig. 5: Determination of amino acid concentration from the peak area.

- 32 -

6. Gel chromatography

Gel chromatography was performed on a Sepharcse 6B

column (1.8 X 77 cm) prepared by the standard procedure.

It was calibrated with marker proteins ,which, with their

molecular weights and Stokes radii in parentheses were:

bovine serum albumin (68,000; 3.55), ovalbumin (43,000;

3.0), chymotrypsinogen A (25,700; 2.2), myoglobin

(17,200; 1.9) and cytochrome c (11,700, 1.7).

7. Sodixim dodecyl sulfate polyacrylamide gel electro­phoresis

Sodium dodecyl sulfate polyacrylamide tube gel

electrophoresis was carried out by a method essentially

due to Laemmli (1970). For the preparation of small pore

gel (12.5% cross linking), 7.5 ml of 30% acrylamide

solution, and 0.8% N N' methylene bis acrylamide, 0.01 ml

of N, N, N', N' tetramethyl-ethylene-diamine tetra acetic

acid and 0.07 ml of 10% ammonium per sulfate were mixed.

Sodium dodecyl sulfate (0.1%) was also present in the

gel. A drop of water was carefully applied on top of the

gel and it was left to polymerize for some time. Stacking

gel (2% crosslinking) was layered on top of the small

pore gel and a drop of water was carefully placed over it

as before. After polymerization, 20-100 ul of protein

sample (containing 20-100 ug) prepared by heating the

sample with 5% 2-mercaptoethanol, 2 drops of glycerol, 1

drop of 0.01% bromophenol blue and 20% SDS was applied.

- 33

The protein sample was electrophoresed for 3-4 hours in

Tris-glycine buffer pH 8.3 using a current of 3-6 mA per

tube (0.5 X 9 cm). The gels were taken out of the gel

tubes and the protein bands were stained with 1%

Coomassie brilliant blue R-250 dye solution in a mixture

of 5% (v/v) methanol and 10% (v/v) acetic acid for one

hour. The gels were finally destained by washing with a

mixtiire of 5% (v/v) methanol and 7% (v/v) acetic acid.

8. Equilibriiom d i a l y s i s

Dialysis tubing 1 cm in diameter was used in making

the dialysis bag 8 cm in length. The protein solution was

placed in the dialysis bag which was suspended in varying

concentrations of mannose (0.05 - 50 mM) . After

equilibration, the concentration of mannose was measured

both in dialysis bag and dialyzate.

9. Measurement of lectin activity

The hemagglutinating activity of lectin was

determined against rabbit erythrocytes prepared from

fresh rabbit blood and the number of cells unagglutinated

counted in a hemocytometer.

The lectin activity was also determined from its

ability to precipitate IgM in 0.01 M Tris KCl buffer pH

7.0 containing 0.5 M NaCl and 1 mM each of CaCl2, MgSO^

and MnCl2 (metallized Tris buffer) . The lectin and IgM

mixture was incubated at 37°C for one hour and overnight

- 34 -

at room temperature and the precipitate obtained was

analyzed by the method of Lowry et al. (1951) (Fig.6 & 7),

10. Isolation of lectin

Cajanus cajan pulse (40 gm) was suspended either in

200 ml of 10 mM sodium phosphate buffer pH 7.0 containing

0.5 M sodium chloride (PBS) or in metallized Tris buffer

pH 7.0. After six hours, the contents were homogenized

and the homogenate filtered through a muslin cloth. The

mixture was acidified to pH 4.0 with 0.3 M acetic acid

and kept overnight in a refrigerator. The acid

precipitated extraneous proteins were removed and the

supernatant was raised to 50% with respect to ammonium

sulfate concentration. The salt fractionated crude

lectin thus obtained was further purified by affinity

chromcitography on IgM Sepharose 6B.

a) Affinity chromatography

Sepharose 63 gel slurry (20 ml) was washed with

distilled water and its pH adjusted to 10.8 with 4 M

NaOH. To this mixture was added solid cyanogen bromide

(0.3 g per ml of gel slurry) while stirring. The contents

were cooled by placing the beaker in a tray of ice and

the pH adjusted to 11 with 4 M NaOH. The activated gel

slurry was extensively washed with chilled distilled

water and then with the coupling buffer (0.2 M sodium

carbonate buffer pH 9.2 containing 0.15 M NaCl). The

- 35 -

C7>

73

° 200 -

o

o. c oi

+-> O l_ Q.

C 3 O E <

0 2 4 6 8 10

Ratio of IgM.'Cajanus cajan lectin f by weight)

Fig. 6: Precipitin reaction of Cajanus cajan lectin with IgM.

In 1 ml metallized Tris buffer pH 7.0 were taken 33-400 jug of IgM with 44 ug of lectin and the specific protein precipitated assayed by the method of Lowry et_ a2_. , (1951) as described in the Experimental Section .

- 36 -

en =1

T3

.2 100 -

u

Q. C

*a» 4-*

o L. Q.

C

O E <

2 4 6 8 10

Ratio of IgM! con A ( by weight)

Fig. 7: Precipitin reaction of con A with IgM.

In 1 ml of metallized Tris buffer pH 7.0 were taken 83 ug of the lectin with 83-830 pg of IgM and the specific protein precipitate analyzed for its protein content by the method of Lowrv et al. 1951.

- 37 -

solution of IgM (30 ml containing 438 mg protein in the

coupling buffer) was then added and the mixture incubated

overnight. After washing the uncoupled activated groups

were blocked with 0.2 M glycine in the coupling buffer.

The gel was washed and then packed in a glass column.

III.RESULTS

1. Isolation of lectin

Optimal conditions for solubilization of Caianus

cajan lectin from pulse were studied. Among the two

buffer systems used viz. PBS and metallized Tris buffer

pH 7,0, the latter was found to be more suitable. The

lectin yield in metallized Tris buffer pH 7.0 was almost

double the yield obtained in the phosphate buffer. The

scheme of purification of lectin is given in Table VI.

When forty gram pulse was homogenized in metallized Tris

buffer pH 7.0 and acidified at pH 4.0 the amount of

lectin present in the supernatant was 2.2 g out of which

17% was salt fractionated at 50% ammonium sulfate

saturation. Further purification of the salt fractionated

lectin by affinity chromatography yielded 75 mg pure

lectin which is about 3% of the total protein present in

the acid homogenate. The elution profile in affinity

chromatography of the salt fractionated lectin is shown

in Fig. 8, where the curve A represents wash through peak

obtained by elution with metallized Tris buffer pH 7.0

alone and contained 75% of the total protein (50 mg)

applied on the column. About 20% protein was specifically

eluted with 0.25M glucose in the operating buffer (Fig.8

curve B) . When affinity purified lectin was passed

through Sephadex-G 100 column (1.8 x 87 cm) equilibrated

with 0.25 M glucose in metallized Tris buffer pH 7.0, a

- 39 -

Table VI Purification of Ca j anus ca i an lectin

Step Total Hemaggluti- Specific Fold Puri- Percent

Protein (mg) nating units Activity fication Recovery

Acid homogenate 2185 185,169 85 1 . 0 1 00

50% Ammonium

sulfate fraction

371 165,625 446 5.27 16.9

Affinity Chroma- 75

t og r a phy

150,000 2,000 24 . 00 3.4

One unit is defined as the minimum dilution of protein

that gives visual hemagglutination of 1.0 x 10 rabbit

erythrocytes in metallized Tris buffer pH 7.0 at 29 C

after 1 hour of incubation.

- 40 -

£ c o o

o >. ••^ w c a» •a

"5 u

••-> a. o

20 30 40 50 60 70

Elution volume (ml)

90 110

Fig. 8: Purification of salt fractionated Cajanus ca jan lectin by affinity chromatography on IgM Sepharose 6 B column (2.2 x 6.0 cm).

Fifty milligram protein in 4 ml metallized Tris buffer pH 7.0 was applied on the column and eluted at a flow rate of 5 ml/hr with the operating buffer. The unbound protein under peak A was devoid of lectin activity. The protein fractions under peak B were obtained by specific elution with 0.25 M glucose in the operating buffer at a flow rate of 20 ml/hr. The column v/as monitored by Lowrv' s method by taking 0.2 ml and 0.5 ml aliquots of the fractions under peak A and peak B respectively.

- 41 -

single symmetrical peak was obtained ( Fig. 9). Further

the lectin moved as a single protein band both under

reducing and non-reducing conditions ( Fig. 10). These

results show that the affinity purified lectin was

homogenous with respect to size.

2. Molecular weight under native conditions

The molecular weight of the lectin was determined by

gel filtration on a Sepharose 6B column (1.8 x 78 cm)

equilibrated with the metallized Tris buffer pH 7.0. The

column was calibrated with five marker proteins listed

in Table VII. The elution profiles of proteins are shown

in Figs. 11, 12 & 13.The elution profile of Blue Dextran

for the determination of void volume is shown in Fig. 14.

The values of the ratio of elution volume to void volume

for each marker protein including the lectin were

calculated and are given in Table VII. A curve between

VQ/VQ and log M was drawn by the method of least squares

and the straight line thus obtained is shown in Fig. 15.

The linear plot obeys the equation:-

V^/VQ = - 0.69 log M + 5.102 (3)

The value of V^/VQ for the lectin was measured to be

1.93 which according to equation (3) corresponds to a

molecular weight of 39,500. The precision with which the

elution volume of proteins could be determined in this

study was generally better than 1%. An error of 1% will

42 -

E c o o ••-' o

>.

c Of

"o u

O.

o

130 150 170

Elution volume (ml)

190

Fig. 9: Gel filtration behaviour of affinity purified Cajanus cajan lectin on Sephadex G 100 column (1.8 X 87 cm).

Lectin (10 mg) in 4 ml metallized Tris buffer pH 7.0 containing 0.25 M glucose was applied on the column equilibrated with the metallised Tris buffer pH 7.0 containing 0.25M Qlucose. The lectin was eluted at a flow rate of 20 m,. /hr ana concentration of each fraction the method of Lov;ry et. aj^. , 1951

:he protein measured by

- 43 -

Fig. 10: SDS polyacrylamide gel electrophoresis of affinity purified Cajanus cajan lectin.

Twenty microgram of the lectin was electrophoresed in 0.025 M Tris 0.192 M glycine buffer, pH 8.3 containing 0.1% SDS in 12.5% polyacrylamide gel for 6 hrs. at 5mA current flow per tube. The gel was stained with 0-2% Coomassie brilliant blue R-250 and destained mechanically with 10% acetic acid as described in the Experimental section.

- 44 -

Table VII Gel filtration data

Protein

Bovine serum albumin

Ovalbumin

Chymotrypsi-nogen-A

Myoglobin

Cytochrome c

Molecular weight (a)

68,000

43,000

25,700

17,200

11,700

Log M

4.83

4.63

4.44

4.24

4.07

Ve (ml)

132

140

151

161

176

Ve/Vo

1.76

1.87

2.01

2.15

2.35

a. Values of molecular weight were taken from Weber and Osborn, (1969)

- 45 -

6 0 0 u H

0) -H (T3 i H OJ-P a (0 (0

CO

00

C B 3

to (0

O U

P

VD

0) m O

m x: (1) w c

0

CO

c •H <u -p o Q4

O

> i

(0

Ui o -p (0 6 0 M

o

0)

en •H

10 C o H +J O (0

e (U en

T3 CO <U

•H +J P 3

a) to

•H o

(0 U

e 4-1

rH C E - H

Q) r-< 4.)

O C VJ

•W 04

> i U

0

m 0

o s: 4-1 (U E Q)

£: -p

> i X5

-C (D > O

- P -H C o

CO

>

c E 3

i H 0 u

-p

c

^ T 3 en c E (0

o c

c o •H U (U '

4-> O Q)

Oi -P

x :

E rH m

O (Ti

>« - .,

V

iiiU00£ P-^Ijsuap p u i d c

- 45 -

UJU01S3-D A;isu3p iD3i;do

o rv j

O

o

a>

1

o

o i n

o m

O C7>

O C^

»—

£ ^ r f

o> E

_3

O

> c "•^ a liJ

to -H -H 0 0 U ^ T3 3 S O )

rO 0 0

0 '^

0 ^

0 - " OJ

OJ CD

0 0 -H

Uico 0 C r-.'O 0

E X tn 0 (C rM )-l 00 ? lO

£ • -P 0 ^ S (D

0 e H M

•4-1 •-* m ^

*W H 0 0 u

>1VD fl) • •c c x: cp Qj (U 0 4J -H "3 CQ ---I O t i

t p V4 (d ' 0 0 (0 ( vo -P •P £ -P in <^ a, C a\X! E 0) (D rH C 0 u3 U (U ^ C - cn u n u H H

r-l ' - (U OJ OJ 1^ x : -Mix:

O - ^ &H QJl-U

o

(N

en

O d d

^'UOOi |2 ' ^ ; ' ' suap !D3!jdo

47 -

E c o o r>. *-• O >. 4-; 'w C 0; "O

"o U

O. O

150 120

Elution volume (ml)

170

Fig. 13: Gel filtration behaviour of con A and Cajanus cajan lectin on Sepharose 6B column (1.8 x 78 cm) . For experimental conditions see legend to Fig. 11.

- 43 -

E c o o \o

a >.

_••-•

w c at •o "o o

o. O

80 90

Elution volume (ml)

Fig. 14: Elution profile of Blue Dextran on Sepharose 6B column (1.8 x 78 cm).

. Blue dextran (10 mg) in 1 ml metallized Tris buffer pH 7.0 was applied to the column and eluted with the operating buffer in 3 ml fractions at a flow rate of 20 ml hr. The colour intensity of each fraction was measured atSOO.nm.

- 49 -

o > 0; >

l o g f M )

F i g . 1 5 : P l o t of Ve/Vo vs log M.

The marker proteins were l-BSA {dimer) 2. BSA [monomer) 3. OvalbuKiin, 4. Chymotrypsinogen A 15. Myoglobin and 6. Cytochrome £.

The values of Ve/Vo and log M listed in Table VII were analyzed by the method of least squares and the linear plot between Ve/Vo and log M was found to fit the equation:-

Ve/Vo = -0.69 log M - 5.102

- 50 -

introduce a maximum uncertainity of around 13% in the

computed value of molecular weight, so that the measured

molecular weight of lectin would be 39,500+5141.

3. Molecular weight under denaturing conditions

Molecular weight of lectin under denaturing

conditions was determined by SDS-polyacrylamide gel

electrophoresis in the presence and absence of 0.7 M 2-

mercaptoethanol. As shown earlier in Fig.10 the lectin

moved as a single protein band with a relative mobility

of 0.79 which was compared with the relative mobilities

of marker proteins viz. bovine serum albumin, ovalbumin,

chymotrypsinogen A and cytochrome c (Table VIII). The

results obtained for marker proteins were analyzed by the

method of least squares in the form of a linear plot of

log M versus Rm (see Fig. 16) obeying the equation;-

Log M = - 1.189 Rm + 5.197 (4)

The measured relative mobility of the lectin (0.79)

corresponded to a molecular weight of 18,226. The

relative mobility of the lectin could be measured with a

maximum uncertainity of 2.6% in this study. This will

introduce an error of 7% in the computed value of

molecular weight. Thus the molecular weight of the lectin

under denaturing condition would be 18,226 + 1258.

4. Optical properties

The ultraviolet spectrum of affinity purified lectin

- 51 -

Table VIII. Relative mobility of marker proteins on SDS-PAGE

Marker Protein^^^ Log M Relative mobility

Bovine serum albumin 4.83 0.29

(68,000)

Ovalbumin (43,000) 4.63 0.49

Chymotrypsinogen A 4.44 0.66 (25,700) Cytochrome c 4.07 0.93 (14,700)

Lectin 0.79

a. Values of molecular weight were taken from Weber and Osborn (1969)

- 52 -

0.4 0.8

Relative mobility

Fig. 16: Plot of relative mobility (Rm) vs log M for marker proteins.

The marker protein were (1) bovine serum albumin (2) ovalbumin (3) chymotrypsinogen A (4) cytochrome c_. The Rm value of the lectin is indicated by an arrow. The straight line obtained by the method of least squares fits the eauation:-

Log M -1.189 X Rm + 5.197

- 53 -

was measured in PBS in the wavelength region 200-360 nm.

Results are graphically shown in Fig, 17. The protein

concentration was determined using a measured specific

extinction coefficient of 10.2 (g/ml)"-'- cm"-'- at 278 nm.

The main spectral features include a trough near 2 50 nm

and a peak near 280 nm. Measurement of absorbance at

different wavelengths show that the lectin absorbs

maximally at 278 nm.

In order to determine the specific extinction

coefficient of the purified lectin the protein

concentration was determined by dry weight method. The

lectin solution was dialyzed extensively against PBS and

equal volume of dialyzed lectin solution as well as

dialyzate were taken in weighing bottles and heated at

105°C to dryness. The protein concentration was

determined by difference. (Ahmad and Salahuddin 1974).

Lectin solutions of known protein concentration were

prepared and their absorbance measured at 278 nm or 280

nm. The absorbance was plotted against protein

concentration by the method of least squares and the

slope of the straight line (Fig. 18) representing

specific extinction coefficient was found to be 10.2

(g/ml)"-'- cm" at 278 nm. The precision with which the

specific extinction coefficient was calculated was better

than 4%. The specific extinction coefficient at 280 nm

was computed to be 10.1 (g/ml)~ cm" . Thus the molar

- 54 -

0.5

c

- 0.4 O o

' • * - ' o. O

0.2

220 260 300 340

X nm

Fig. 17: Ultraviolet absorption spectra of Cajanus cajan lectin.

The spectra of the lectin solution (0.57 mg/ml) in PBS was recorded in the wavelength region 240-350 nm.

- 55 -

E c

CO

rg ••-»

o ct u c a

Xi i -o U)

Xi <

0.15

Protein concentration (g/100ml)

Fig. 18: Determination of specific extinction coefficient of Cajanus cajan lectin.

The absorbance of the lectin solution in PBS was measured at 278 nm. A plot was obtained between absorbance and protein concentration by the method of least squares. The straight l:'.ne obeys the equation :-

(O.D.)2^g = 10.16 (protein g/lOOml) + 0.029

56 -

extinction coefficient of the lectin at 278 nm will be

39,780 M~^ cm"-'-.

5. Carbohydrate content

The carbohydrate concentration of the Cajanus cajan

lectin was determined by phenol sulfuric acid method

(Dubois et al., 1956). The incubation mixture containing

1.2 mg of protein yielded an optical density of 0.19

which according to the calibration curve (see Fig. 4)

would correspond to 30^g of neutral hexose per milligram

of lectin. This would mean that the lectin contains 3.0%

neutral carbohydrate.

In a separate experiment incubation of 1 mg of

lectin with 0.2-1 mg of con A in 2 ml metallized Tris

buffer pH 7.0 resulted in specific precipitation

(Fig. 19). The precipitate was completely solubilized in

0.25 M glucose. These results suggest that the

carbohydrate moiety of Cajanus cajan lectin would

contain mannose/glucose residues which were responsible

for specific precipitation of the lectin with con A.

6. Amino acid composition

Amino acid analysis of the 24 hours acid hydrolyzate

of the affinity purified Cajanus cajan lectin is depicted

in Fig. 21. The peak of a given amino acid was

identified by comparing its elution volume with that of

the standard (Fig, 20) . The concentration of the amino

- 57 -

Amount of con A (mg/ml)

Fig. 19: Specific interaction of the glycoprotein Cajanus cajan lectin with con A.

In 1 ml metallized Tris buffer incubated Cajanus cajan lectin varying concentrations of con

pH 7.0 was (1 mg) with A and the

precipitated protein estimated by the method of Lowry et. £i-' 1951.

- 58 -

Fig- 20: Elution profile of standard amino acid sample in LKB Biochrom (4101) amino acid analyzer.

The peak numbers corresponded to amino acids listed in Table IX, except peak number 18 which represents ammonia. For other details see text.

17 16 15

U 13

9 7

18

1211 10

u U v u I • I • • • • I I — I 1 — I — I — I — I > — I — ' — I — I

- 59 -

Fig. 21: Amino acid analysis of acid hydrolyzate of Cajanus cajan lectin by LKB Biochrom (4101) amino acid analyzer.

The peak numbers refer to the amino acids listed in Table IX, except peak number 18 which represents ammonia. For other details see text.

17

- 60 -

acid was determined from the peak area as explained in

the Experimental Section. The elution profiles A, B and

C in Fig. 21 do not correspond to any known amino acid

used in obtaining the calibration curve. The

concentration of amino acid in nanogram per ml was

converted into moles/litre by dividing it with the

molecular weight of the amino acid. The resulting value

was then divided by the molecular weight (39,000) of the

lectin to obtain the number of amino acid residues per

mole of the protein. The results are summarized in Table.

IX. It should be pointed out that the tryptophan content

of the lectin could not be determined by this method

since tryptophan is known to be acid labile (Creighton,

1980). Further, the cysteine and methionine were not

detectable in the acid hydrolyzate of the lectin. No

attempt has been made to identify the unknown peaks A, B

and C which did not correspond to any known amino acid

used in the analysis (Fig. 20) . It is conceivable that

they may represent small peptides containing acid

resistant bonds such as valine-valine and valine-

isoleucine.

7. Carbohydrate binding specificity

The carbohydrate binding specificity of Cajanus

caian lectin was studied in metallized Tris buffer pH 7.0

at room temperature by measuring the extent of lectin

mediated hemagglutination or glycoprotein precipitation

- 61

Table IX. Amino acid composition of Can anus cajan lectin

Amino acid Residues/molecule (M.wt: 39,000)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Asx

Thr

Ser

Glx

Pro

Gly

Ala

Cys

Val

Met

H e

Leu

Tyr

Phe

His

Lys

Arg

Trp

38

31,

19,

39.

18.

25.

27.

.5

.6

.6

.7

.4

,2

.3

too low

31. 2

too low

17.

17.

13.

16.

9.

21.

8.

N.D

5

9

2

2

8

2

1

1

N.D. = Not determined. * Cysteine and methionine contents of the lectin were too low to be measurable.

- 62 -

in the presence and absence of saccharides. The effect of

increasing concentrations of mannose, glucose and

galactose on lectin-mediated hemagglutination of rabbit

erythrocytes is shown in Fig.22. Except glucosamine and

N-acetyl glucosamine the concentration of all neutral

carbohydrates were determined by the method of Dubois et

al., 1956. For the preparation of the solution of

glucosamine and N-acetyl glucosamine the sugar crystals

were directly weighed without considering their moisture

content. Evidently the hemagglutinating activity is

substantially inhibited by the increasing concentration

of glucose or mannose but it remained unaltered by

galactose upto 0.35 M concentration. The concentration

of mannose or glucose corresponding to 50% inhibition

i.e., Cjj value was computed from the curve and was found

to be 9.5 and 19.5 mM respectively. Similar experiments

were performed with other sugars and the results are

graphically shown in Figs. 23-28. The Cj values for each

sugar was calculated with the help of these curves and

are listed in Table X. Inhibitory potency of a given

sugar was computed by dividing the Cjyj value of mannose by

the C^ value for that saccharide. The results on

relative inhibitory potencies are listed in the last

column of Table X. It can be seen that among the nine

saccharides listed in Table X, methyl « mannopyranoside

is the most potent inhibitor of hemagglutinating activity

- 63 -

>• ••-•

">

u O

20 40

Sugar concentration (mM)

350

Fig. 22: Effect of saccharides on hemagglutinating activity of Cajanus cajan lectin.

The lectin (80 ;ug) was ^incubated with rabbit erythrocytes (4 x 10 cells) in 2 ml Tris buffer pH 7.0 and the number of cells unagglutinated were counted as described in the Experimental Section. The hemagglutination in absence of sugar represented 100% lectin activity.

Glucose (#) Mannose (O)

Galactose (A)

- 64 -

2 4

Methyl a-mannoside (mM)

Fig. 23: Effect of methyl <x mannoside on hemagglutinating activity of Cajanus cajan lectin.

For other experimental details see the legend to Fig. 22.

- 65

10 20

Glucosamine (mM)

30

Fig. 24: Effect of glucosamine on hemagglutinating activity of Cajanus cajan lectin.

For other experimental details see the legend to Fig. 22.

- 65

10 20

N-ffcetyl glucosamine (mM)

Fig. 25: Effect of N-acetyl glucosamine on hemagglutinating activity of Cajanus cajan lectin.

For other experimental details see the legend to Fig. 22.

- 67 -

5 >.

•¥••

u a

u - I

Fructose concentration (nnM)

Fig. 26: Effect of fructose on hemagglutinating activity of Cajanus cajan lectin.

For other experimental details see the legend to Fig. 22.

68 -

••-•

!E u o c

20 ^0

Sucrose concentration (mM)

60

Fig. 27: Effect of sucrose on hemagglutinating activity of Cajanus cajan lectin.

For other experimental details see the legend to Fig. 22.

- 69 -

100-

u a

u a*

Maltose (mM)

Fig. 28: Effect of maltose on hemagglutinating activity of Cajanus cajan lectin.

For other experimental details see the legend to Fig. 22.

- 70 -

Table X. Effect of carbohydrates on the hemagglutinating activity of Cajanus cajan lectin

Carbohydrate

Mannose

Methyl oc mannopyranoside

Glucosamine

N-acetyl glucosamine

Glucose

Fructose

Galactose

Sucrose

Maltose

Cj, Value (mM)

9.0

0.85

17.5

17.0

19.5

17.0

>350

20.0

5.8

Relative Inhibitory Potency^

1.00

10.6

0.50

0.5

0.47

0.50

<0.04

0.45

1.55

a. Mannose is normalized to 1.0

- 71 -

of Cajanus cajan lectin followed by maltose and mannose.

The inhibitory potencies of glucosamine and N-acetyl

glucosamine were found to be similar. Although the

results indicate similar inhibitory effect by N-acetyl

glucosamine and glucose the comparison is difficult

because of the uncertainity in the determination of the

carbohydrate concentration of N-acetyl glucosamine.

Glucose and fructose possess similar inhibitory potency.

Among the two disaccharides viz. sucrose and maltose,

maltose was far more potent inhibitor than sucrose.

Galactose had virtually no effect on hemagglutinating

activity of Cajanus cajan lectin.

Similar observations were made on the inhibitory

potencies of these saccharides on IgM precipitating

activity of Cajanus cajan lectin. The results are

graphically shown in Figs. 29-37 and are summarized in

Table XI. The C^ value of each saccharide was calculated

from the curves (see Figs. 29-37) and are listed in

column 3 of Table XI. Here again methyl oc mannopyranoside

is a far better inhibitor of the precipitating activity

of lectin than mannose. However, p-nitrophenyl mannoside

whose effect on hemagglutinating activity was not

investigated, in this study, was found to be the most

potent inhibitor. The inhibitory potency of glucose,

fructose and sucrose were found to be similar.Galactose

and cellobiose showed no inhibitory activity. Although N-

- 72 -

100 -

o

20 40

Sugar concentration (mM)

Fig. 29: Effect of saccharides on the precipation of IgM with Cajanus cajan lectin.

- The lectin (75 jug) was incubated with IgM (350 /ug) in 1.5 ml, metallized Tris buffer pH 7.0 and the specific protein precipitated analysed by the method of Lowry et al., 1951. The glycoprotein precipitation in absence of sugar represented 100% glycoprotein precipitating lectin activity.

Glucose {#) Mannose (0)

Galactose (A)

- 73 -

2 4 6 6

Methyl a-mannoside(mM)

Fig. 30: Effect of methylxmannoside on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

- 74 -

100

r 50

X 10 20

N-flcetyl glucosamine (mM)

30

Fig. 31: Effect of N-acetyl glucosamine on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

- 75 -

"> ••-•

a ,c ' • » - •

u 0)

10 20

Glucosamine (mM)

Fig. 32: Effect of glucosamine on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

- 76 -

+^ '> •P u O

at

Fructose (mM)

Fig. 33: Effect of fructose on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

- 11 -

••-'

"> "+-u o

u

20 40

Sucrose (mM)

Fig. 34: Effect of sucrose on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig, 29.

f « ^

- 78 -

Maltose concentration

Pig. 35: Effect of maltose on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

79 -

Qt

4 8

Isomaltose(mM)

Fig. 36: Effect of isomaltose on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

- 80 ~

2 4

p-Nitrophenyl a mannoside(mM)

Fig. 37: Effect of p-nitrophenyl K mannoside on the precipitation of IgM with Cajanus cajan lectin.

For other experimental details see the legend to Fig. 29.

- 81 -

Table XI, Effect of carbohydrates on the IgM precipi­tating activity of Cajanus cajan lectin

Carbohydrate C ^ Value Relative

(mM) Inhibitory Potency

Mannose 13.0 1.0

Methyl oc mannopyranoside 1.3 10

p-Ni t rophenyl oc-mannoside 0.4 32.5

1.0

1.1

0.7

0.68

<0.04

0.8

2.4

4.3

a

Glucosamine

N-acetyl glucosamine

Glucose

Fructose

Galactose

Sucrose

Maltose

Isomaltose

Cellobiose

12.5

12.0

18.0

19.0

>330

16

5.5

3.0

>29

a. Mannose is normalized to 1.0

!2 -

acetyl glucosamine and glucosamine were similar to that

of mannose, the data should be taken with caution because

of the experimental error in the determination of the

concentration of glucosamine and N-acetyl glucosamine.

It should be noted, that among the disaccharides,

isomciltose was a far more potent inhibitor than maltose

for glycoprotein precipitating activity of the lectin.

Since Cajanus cajan lectin is a glucose/mannose

specific lectin, its carbohydrate binding specificity was

compared with that of con A. The effect of seven

carbohydrates on the hemagglutinating activity of con A

were investigated and the results are summarized in Table

XII and are graphically shown in Figs. 38-42. The order

of inhibitory potency of various sugars is nearly the

same as described in the literature. The hemaggluti­

nating activity of the lectin was not influenced by

galactose.

The effect of eight saccharides on the IgM

precipitating activity of con A was investigated as

described in the legend to Fig. 43. The results are

graphically shown in Figs. 43-48. The Cj values along

with relative inhibitory potency are listed in Table

XIII. Here again, the order of inhibitory potency of

various sugars for precipitating activity of con A was

the same as found earlier (Goldstein and Poretz 1986).

Comparison of the carbohydrate binding specificity

13 -

Table XII. Effect of carbohydrates on hemagglutinating activity of concanavalin A

Carbohydrate C^ Value Relative

(mM) Inhibitory Potency^

1.0

4.7

0.27

0.4

<0.01

0.3

1.17

a. Mannose is normalized to 1.0

Mannose

Methyl oc mannopyranos

Glucose

Fructose

Galactose

Sucrose

Maltose

ide

3.5

0.75

13.0

9.0

>350

11.5

3

- 84

100

1 50

c •f u a*

- J

0

6

I

% ^

A A

. •

\ «

sT_o o i 1

i A

1

// A // J

// ' 1 20 40

Sugar concentration (mM)

350

Fig. 38: Effect of saccharides on hemagglutinating activity of con A.

In 2 ml metallized Tris buffer pH 7.0 was

incubated the lectin(8 0 ,ug) with 4 x 10 rabit erythrocytes and number of unagglutinated cells counted as described in the Experimental Section.

Mannose ( O O ) ; Glucose ( ) ("Galactose ( A-A

- 85 -

u O

u at

0 2 4

Methyl a mannopyranoside (mM)

Fig. 39: Effect of methyl oc mannopyranoside on hemagglutinating activity of con A.

For other experimental details see the legend to Fig. 38.

- 86 -

100 -

">

u o

u 0*

10 20

Fructose concentration (mM)

Fig. 40: Effect of fructose on hemagglutinating ac t iv i ty of con A.

For other experimental de t a i l s see the legend to Fig. 38.

- 87 -

O

u Of

20 40

Sucrose concentration (m M)

Fig. 41: Effect of sucrose on hemagglutinating activity of con A.

For other experimental details see the legend to Fig. 38.

u O

Maltose concentration (mM)

Fig. 42: Effect of maltose on hemagglutinating activity of con A.

For other experimental details see the legend to Fig. 38.

- 89 -

100

">

u a

20 40

Sugar concentration (mM)

350

Fig. 43: Effect of saccharides on the precipitation of IgM with con A.

In 1.5 ml metallized Tris buffer was incubated lectin (75 ,ug) with 350 jag IgM. the specific protein precipitated was analyzed by the method of Lowry et_ a_l. , 1951. Mannose (O) Glucose (•) Galactose (A).

90 -

O

u

2 4

Methyl a mannopyranoslde (mM )

Fig. 44: Effect of methyl oc mannopyranoslde on the precipitation of IgM with con A.

For other experimental details see the legend to Fig. 43.

- 91 -

100-

U

a

20 40

Fructose concentration (nnM)

Fig. 45: Effect of fructose on the p rec ip i t a t ion of IgM with con A.

For other experimental de ta i l s see the legend to Fig. 4 3.

- 92 -

20 40

Sucrose concentration (mM)

Fig. 46: Effect of sucrose on the precipitation of IgM with con A.

For other experimental details see the legend to Fig. 4 3.

- 93 -

100-

u o

20 40

Maltose concentration (mM)

Fig. 47: Effect of maltose on the precipitation of IgM with con A.

For other experimental details see the legend to Fig. 4 3.

100

- 94 -

"•«-•

u a 50

0*

0 -

8

Isomaltose (mM)

Fig. 48: Effect of isomaltose on the precipitation of IgM with con A.

For other experimental details see the legend to Fig. 43.

- 95 -

Table XIII. Effect of carbohydrate on the IgM precipitating activity of concanavalin A

Carbohydrate

Mannose

Methyl « manno-pyranoside

Glucose

Fructose

Galactose

Sucrose

Maltose

Isomaltose

Cjyj Value (mM)

2.5

0.5

11

5.5

>330

10.5

3.0

1.4

Relat Publi

ive inhibit! shed Data'-'

1.0

7.33

0.22

0.5

<0.04

0.19

1.02

2.0

ory Th

po lis

tency— study

1.0

5.0

0.23

0.46

<0.01

0.24

0.83

1.79

a. Mannose is normalized to 1.0 b. Data taken from Goldstein and Poretz (1986)

- 96 -

of Cajanus cajan lectin and con A (Table XI & XIII) show,

that con A possesses far greater affinity for mannose

than Cajanus cajan lectin. Infact, for nearly all the

saccharides listed in Table XI and XIII the Cj values are

substantially higher for Cajanus cajan lectin than those

found for con A under identical conditions. However, the

inhibitory order of various sugars were found to be the

same for both the lectins.

8. Interaction of lectin with dextran

After comparing the carbohydrate binding specificity

of Cajanus cajan and con A, the interaction of these

lectins with a polysaccharide viz. dextran was studied in

metallized Tris buffer pH 7.0. The lectin was incubated

with increasing concentrations of dextran and the amount

of precipitate formed was analyzed both for protein and

carbohydrate contents. The results are graphically shown

in Fig. 49 and 50. With 25.6 micromoles of Cajanus cajan

lectin the maximum amount of protein precipitated was

0.165 mg. With equal concentration of con A dimer

(12.8 jiiM con A tetramer) the amount of protein

precipitated was substantially high i.e. 0.4 mg (see Fig.

49) .

9. Equilibrium dialysis

The interaction of mannose with Cajanus cajan lectin

was also studied by equilibrium dialysis in metallized

- 97 -

1 2 3 4

Dextran concentration (mg/ml)

Fig. 49: Interaction of dextran with Cajanus cajan lectin and con A.

In 1 ml metallised Tris buffer pH 7.0 was taken lectin (1 mg) and increasing concentration of dextran and the mixture incubated for 30 minutes at 37°cand than for 18 hrs at room temperature. The protein preciptated was estimated by the method of Lowry et_ £l. , 1951.

Cajanus cajan lectin ( i ); con A ( • ).

- 98 -

2 4

Dextran concentration (mg/ml)

Fig. 50: Interaction of dextran with Cajanus cajan lectin and con A.

The carbohydrate content of the precipitate obtained was estimated by the method of Dubois et_ al. , 1956. Other experimental conditions were same as described in the legend to Fig. 49.

Cajanus caian le ctin { A con A ( • )

- 99 -

Tris buffer pH 7.0 and at 20°C. No significant binding

of the ligand by dialysis bag was observed under

conditions of our experiment. Further, in the presence of

0.5 M NaCl Donnan effect would be negligible. When

32.05 )aM of lectin was placed inside the bag and

equilibrated against 117 pM mannose, after equilibration

the concentration of mannose outside and inside was

determined. The concentration of mannose outside was

91.7 pM whereas, the concentration of carbohydrate inside

was 3 09 pM.. The latter includes the contribution from

32.05 pM of lectin which contains 3% carbohydrate. After

correction of the lectin contribution to the carbohydrate

concentration the concentration of sugar inside turns out

to be 101 pM. Thus the actual difference between the

sugar concentration inside and outside the bag is very

small. This introduces error in the evaluation of the

binding constant. At 20°C these results were consistent

with a kd of 2.2 x 10" M. The experiments were carried

out at other sugar concentration but the difference in

the concentration of the sugar inside and outside

continued to be small. It was therefore not possible to

use these data to calculate the number of binding sites

as well as the association constant for the interaction

of mannose with the lectin.

IV.DISCUSSION

The procedure used in this study for the isolation

and purification of mannose/glucose specific lectin from

Caianus caian pulse yielded 75 mg affinity purified

lectin from 40 g pulse. Gel filtration and SDS

polyacrylamide gel electrophoresis showed size

homogeneity of the lectin. The molecular weight of the

lectin as determined by gel filtration under native

conditions in metallalized Tris buffer was 39,500. In

SDS-polyacrylamide gel electrophoresis, the lectin

moved as a single protein band of mass 18,000 kD both in

the presence and absence of 2 mercaptoethanol. These

results suggested that the Caianus cajan lectin is a

dimer and that the two subunits are linked together only

by non covalent interactions.

The specific precipitation of the glycoprotein

lectin with con A and its complete dissolution in 0.25 M

glucose solution suggested the presence of con A specific

saccharides in the carbohydrate moiety of the lectin.

The optical properties of the lectin were indicative of

the presence of tyrosine and tryptophan residues.

A comparison of the aminoacid composition of Caianus

cajan lectin with that of a typical mannose/glucose

specific lectin i.e., con A (Table XIV) shows some

interesting similarities. Thus both the lectin contain

comparable amounts of threonine, glycine and proline.

- 101 -

Table XIV. Comparison of amino acid composition of Cajanus cajan lectin with con A

Amino acid Con A/100,OOOg Cajanus cajan/100,OOOg

Asp 78 99 Asn 47 Glu 27,45 102 Gin 19.6 Thr 74.5 81 Ser 121.6 50 Pro 43 47 Gly 59 65 Ala 74.5 70 Val 66.7 80 ^ Met 7.8 too low H e 58.8 45 Leu 70.6 4 6 Tyr 27.5 34 Phe 43.1 42 His 23.5 25.5 Lys 47.0 55 Arg 23.5 21 Trp 16.0 10^

* Methionine and cysteine contents were too low to be measurable.

$ DeteLTnined spectrophotometrically (Edelhoch, 1967) by S.Hasan (1991) of this laboratory.

- 102 -

Further, the ratio of (Asx+Glx)/(Basic aminoacids) in

Caianus cajan lectin and con A was found to be close to

2. The aromatic amino acids residue contents of both the

lectins were also found to be similar. The ratio of the

aromatic aminoacid residues in con A and Caianus caian

lectin was close to 1.1. It should be pointed out that

the studies on hydrogen ion titration curve of the lectin

undertaken in this laboratory (R.H.Khan 1993 Personal

Communication) indicate the acidic nature of the lectin.

Legume lectins show considerable sequence homology. The

three dimensional structure of the seven legume lectins

have already been determined by X-ray crystallography.

The results shows that regardless of the carbohydrate

binding specificity , the structural organisation of

legume lectins are similar . It is therefore, expected

that they will possess similar physicochemical properties.

Infact earlier studies have shown that most of them

are glycoproteins containing 2-4 subunits of mass 25-30

kD . Like most legume lectins Caianus caian lectin was

found in this study to be a glycoprotein consisting of 2

subunits.However, the subunit molecular weight of 18,000

is somewhat smaller than the expected value . It should

be noted that studies on Phaseolus mungo lectin reported

recently (Sharma and Salahuddin 1993) suggested that the

lectin is a glycoprotein containing 2 subunits of

molecular weight 66,000 . It therefore, seems that the

- 103

size of the subunit of legume lectins may show

significiant variation.

The results presented here in this thesis on the

inhibition of lectin mediated hemagglutination of rabbit

erythrocytes and lectin IgM precipitation by a large

number of carbohydrates have conclusively shown that the

lectin is specific for glucose and mannose. Here it will

be instructive to compare the carbohydrate binding

property of this lectin with those of concanavalin A.

Pioneering studies carried out in the laboratory of

Goldstein have established that the hydroxyl groups in

mannose that are most critical for binding to the lectin

are at positions C-3, C-4 and C~6 (Fig. 51) of the

pyranosyl ring system. Considerable variation at C-2

position is tolerated. However, any modification at C-3,

C-4 and C-6 position results in substantial decrease or

complete abolition of the carbohydrate binding. After

modification of -OH group at C-2 position the affinity

of the lectin for carbohydrate is retained, so that

glucose and N-acetyl glucosamine are also able to bind

to the lectin. When hydrogen of -OH group at C-1

position was replaced by methyl group, the affinity of

the lectin increased substantially. However, the

increment was even higher when the hydrogen of C-1

hydroxyl group was replaced by p-nitrophenyl group. These

results are significant since they indicate the

- 104 -

CH20H

Fig. 51: Schematic diagram of D-mannose,

- 105 -

involvement of hydrophobic interactions during

carbohydrate-lectin interations. Higher affinity of

Cajanus cai an lectin for isomaltose as compared to

maltose clearly indicates the preference for oc-1,6

glycosidic bond. With more complex carbohydrates such as

dextran, con A showed a greater affinity than Cajanus

cajan lectin.

In conclusion the lectin from Cajanus cajan pulse was

found to be specific for mannose and glucose. The

carbohydrate binding by the lectin appears to be mediated

by hydrophobic interactions. However, the affinity of

this legume lectin for specific carbohydrates is

substantially less than the atypical mannose/glucose

specific legume lectin^con A.

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VI. BIOGRAPHY

Shama Siddiqui was born at Lucknow on December 19,

1965. She passed High School Examination in 1981 from

Carmel Convent High School, Lucknow. She received her

B.Sc. Degree in 1985 and M.Sc. degree in Biochemistry in

1988 from Lucknow University, Lucknow. She was enrolled

as Ph.D. student in October, 1989 in the

Interdisciplinary Biotechnology Unit, Aligarh Muslim

University, Aligarh. She received her M.Phil, degree in

Biotechnology in 1991. She is a member of the Society of

Biological Chemists, India.

VII.LIST OF PUBLICATIONS/PRESENTATIONS

1. "Some properties of lectin from pigeon pea"

Siddiqui, S. and Salahuddin, A.

Proceedings of the Diamond Jubilee Annual General Body

Meeting of the Society of Biological Chemists (India)

held at Calcutta from December 26-30, 1991.

2. "Comparison of carbohydrate binding specificity of

pigeon pea lectin with that of concanavalin A"

Siddiigui S.

Proceedings of 61st Annual General Body Meeting of the

Society of Biological Chemists (India) held at Hyderabad

from December 21-23, 1992.

3. " Carbohydrate binding specificity of Caianus caian

lectin-involvement of acidic groups in lectin

carbohydrate interaction." Khan, R. H and Siddiqui, S.

Proceedings of IXth Carbohydrate Conference of Association

of Carbohydrate Chemists and Technologists (India) held

at Lucknow from November 24-26, 1993.