ISOLATION OF POLYSACCHARIDE RECEPTORS FROM TISSUES OF LEGUMINOUS PLANTS

(PIGEON PEA LECTIN)

6j,

SHAMA SIDDIQUI INTERDISCIPLJNARY BIOTECHNOLOGY UNIT

AUGARH MUSLIM UNIVERSITY. ALIGARH

Date

Approved :

A. SALAHUDDIN (Supervisor)

A DlssertaUory submirted In partial fuiflimcnr of the requlrcmenrs tor the degree of Master of Philosophy In Biotechnology,

of the Ailqarh Muslim University. Ailgarh 1991

DS1945

C E R T I F I C A T E

I certify that the wotk presented in the following

pages has been carried out by Miss. Shama Siddiqul and

that it is suitable for the award of M.Phil degree in

Biotechnology of the Aligarh Muslim University, Allgarh.

( A. Salahuddin )

Professor and Coordinator,

Interdisciplinary Biotechnology Unit

Aligarh Muslim University, Aligarh

ii

A B S T R A C T

In view of the suspected Importance of legume lectins in

specific recognition of Rhizobium by legume it was thought

desirable to isolate lectin from pigeon pea(Ca1anas ca1an).

one of the important legume crop of India.

Pigeon pea lectin was isolated from pulse which was

solubilised in 10 mM sodium phosphate buffer pH 7.0

containing 0.5 M NaC1 (PBS). The lectin activity was

determined by its ability to agglutinate rabbit erythrocytes.

Pulse was soaked overnight in PBS and homogenised. The

horaogenate was acidified with 0.3 M acetic acid pH 4.0. After

removing the acid precipitated protein which was devoid of

lectin activity, lectin was salt fractionated from the

supernatant with 50% ammonium sulfate. It was further

purified by affinity chromatography on IgM Sepharose 6B

column equilibrated with PBS. Lectin was specifically eluted

with 0.25 M glucose in PBS.

The affinity purified lectin was eluted from a

calibrated Bio-gel P-200 column in a single symmetrical peak

with an elution volume is to void volume ratio of 1.65 which

was consistent with a molecular weight of 38,000. The neutral

hexose content of affinity purified lectin was determined by

phenol sulfuric acid method to be 7%(w/w). The specific

-1 -1 extinction coefficient was found to be 12.35(g/ml) cm at

278 nm.

ill

The lectin mediated hemagglutination of rabbit

erythrocytes was specifically inhibited by 0.25 M glucose.

Likewise the lectin IgM precipitation was also inhibited

specifically by 0.25 M glucose. The stability of lectin was

investigated at various temperatures. When stored in a

refrigerator it retained its hemagg1utinating and

precipitating activity for two weeks. However, prolonged

storage in a refrigerator caused aggregation and partial

inactivation of lectin. When heated for 30 minutes in the

temperature range of 10 - 70 C the hemagglutinating and

precipitating activity of the lectin increased upon

increasing the temperature from 20 C to 40 C. However,

* o increase in temperature beyond 40 C produced decrease in

lectin activity probably due to heat denaturatlon.

iv -

A C K N O W L E D G E H E N T S

It gives me extreme pleasure to express my deep

gratitude to my honourable supervisor, Professor A.

Salahuddin, Coordinator Interdisciplinary Biotechnology Unit,

Aligarh Muslim University, Aligarh. His able guidance,

regular encouragement and intelligent criticism helped me a

lot in the fulfilment of this task. I am extremely grateful

to him for giving me an oppurtunity to work under his

excellent supervision and providing the necesssary

faci1ities.

My special thanks are due to Dr. Saad Tayyab for his

help and encouragement. 1 also thank Dr. Javed Mussarrat and

Dr. Arshad Rehman.

I thank Dr. Parveen Salahuddin, Dr. Najma All, Ms. Seema

Hasan, Ms. Shadhna Sharma and Ms. Zoya Galzie for their

cooperation and kind help.

I owe my special thanks to Mr. Aftab Ahmad, Mr. Rizwan

Hasan Khan and Miss Sunita Warraich for their help and

discussion throughout this study.

My affectionate thanks are due to Miss Sakhra Shah , Mr.

Mozaffarul Islam , Mr. Ashok B.T. and Miss Seema Qamar.

I would also like to acknowledge my younger brother Lt.

Shamsul Siddiqul for his moral support and constant

encouragement.

V -

I also extend my thanks to Mr. Lai Mohammad, Mr. Amir

Ali, Mr. Aqtedar, Mr. Faisal and other non teaching staff

members of the department for their cooperation.

In this research project , financial assistance as

Junior Research Fellowship from UGC is greately acknowledged.

Date (SHAMA SIDDIQUI)

- vi -

Dedicated to

Mq Parents

C O N T E N T S

Page

ABSTRACT 111

ACKNOWLEDGEMENTS v

DEDICATION vii

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF ABBREVIATIONS xlii

I. INTRODUCTION 1

II. EXPERIMENTAL 24

A. Materials 24

i. Proteins 24

2. Chemicals used in the isolation of lectin 24

3. Miscellaneous reagents 24

B. Methods 26

1. pH measurements 26

2. Measurement of light absorption 26

3. Determination of lectin activity 26

a) Hemagglutination 26

b) Precipitin reaction 27

4. Determination of protein concentration 27

5. Determination of carbohydrate content 28

6. Isolation of lectin 28

a) Isolation of IgM rich fraction from 28 goat blood.

- viii -

b) Preparation of IgM Sepharose 6B 31

1) Activation of Seoharose 6B 31

ii) Coupling of IgM with activated 31 Sepharose 6B

c) Affinity chromatography 31

7. Determination of specific extinction 33 coeff icient.

8. Gel chromatography 33

9. Effect of temperature on carbohydrate 34 binding properties of lectin.

I I I . RESULTS 35

1. Screening of the pulse 35

2. Solubilization of the lectin 35

3. Stability of the lectin 37

4. Isolation of the lectin 37

5. Effect of ageing 40

6. Gel chromatography 40

7. Determination of carbohydrate content 40

8. Precipitin reaction 45

9. Effect of temperature on carbohydrate 45 binding properties of lectin.

10. Optical properties 47

11. Determination of specific extinction 47 coefficient of pigeon pea lectin.

IV. DISCUSSION 56

V. REFERENCES 57

VI. BIOGRAPHY 61

- ix

LIST OF TABLES

Page No.

Table I: Origin and sugar specificity of purified 2 lectins from legume seeds

Table II: Common molecular properties of legume 4 lectins

Table III: Lectins with pronounced specificity for 12 oligosaccharides

Table IV: Molecular properties of some mannose 15 specific legume lectins

Table V: Protein yield in various stages of 30 purification of pigeon pea lectin

Table VI: Gel filtration data of marker proteins 44

Table VII: Calibration data of the double beam 51 spectrophotometer

- X -

LIST OF FIGURES Page

1. Amino acid sequences of leguminous 18 1 act ins

2. Schematic representation of the 19 circularly permuted sequence homology that relates Con A to other legume 1ectins.

3. A view of saccharide binding site of conA 21

4. Calibration curve for the estimation of 29 protein concentration by the method of Lowry et al., 1951.

5. Calibration curve for the estimation of 30 carbohydrate concentration by the method of Dubois et al., 1956.

6. Curve to show effect of NaC1 36 concentration on solubilization of pigeon pea lectin.

7. Affinity chromatography of 50% ammonium 39 sulphate fraction of pigeon pea homogenate on IgM Sepharose 6B column.

8. Calibration curve for the determination 41 of molecular weight of protein by standard curve of VQ/VQ VS log M.

9. Gel Chromatography of Blue Dextran on 42 Biogel P-200 column.

10. Gel filtration profile of BSA, myo- 43 globin and pigeon pea lectin on Biogel P-200 column,

11. Precipitin titration curve of pigeon pea 46 lectin with goat IgM.

12. Curve to show effect of temperature on 48 hemagglutinating activity of pigeon pea lectin.

13. Curve to show effect of temperature on 49 pigeon pea lectin mediated precipitation of goat IgM.

- xi

14. Curve to show percent residual activity 50 of pigeon pea lectin after the temperature treatment.

15. Absorption spectrum of pigeon pea lectin 52 in PBS on double beam spectrophotometer.

16. Scan of absorption spectrum of pigeon pea 53 lectin on BD 40 Klpp & Zonen chart recorder attached to double beam spectro­photometer .

17. Calibration curve for the estimation 55 of specific extinction coefficient of pigeon pea lectin.

- xii -

List of Abbreviations

BSA Bovine serum albumin

Con A Concanavalin A

Ca Calcium

EDTA Ethylene diamine tetra acetic acid

Gal Galactose

Glc Glucose

Glc NAc N-acetyl glucosamine

IgM Immunoglobulin M

Man Mannose

Mn Manganese

Mg Magnesium

PBS Sodium phosphate buffered saline

PHA Phytohemagglutinin.

xiii -

I N T R O D U C T I O N

I N T R O D U C T I O N

O c c u r a n c e

Lectins which were first discovered about" a century

ago by Stillmark (1888) from castor beans are defined as a

multivalent carbohydrate binding protein or glycoprotein of

non immune origin which agglutinate cells and /or precipitate

g1ycoconjugates (Leiner et a)., 1986).Lectins are widely

distributed in nature, being found in animals, insects,

plants and microorganisms (Leiner et al., 1986 and Sharon and

Lis, 1989). The richest source of lectin in plants is the

leguminosae family (Tom and Western, 1971). Lectins have

also been discovered in other families such as Euphorbiaceae,

Verbenaceae, Malvaceae, Polygonaceae, Solanaceae,

Acanthaceae, Ranunculaceae, Caprifo1iaceae. Cucurbitaceae and

Cactaceae etc. (Sharon and Lis, 1990).Many of them could be

grouped into families with sequence homologies and common

structural properties. The largest and best characterized

family is that of leguminosae lectins. Lectins from

Graminaceae (cereals) and Solanaceae (e.g., potato and

tomato) have also been characterized (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 (Rudiger, 1988).

Sugar specificities of some of these lectins are summarized

in' Table 1.The molecular properties of the legume lectins

are listed in Table II (Sharon and Lis, 1990). In

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TABLE I I

Common molecular properties of legume lectins

Subunit a Mol wt

Number Combining Sites

Number , sped f Icl ty

Metal ions. Amino acids

Hydroxy and carboxy Sulfur containing

Sequence homology N- Glycosylation 3 Dimensional structure

ex he 1 ix p sheet

25 - 30 kDa. 2 or 4

i per sub unit usually identical

Ca , Mn

High low or absent Extensive

common

low or absent Main structural e1ement.

a. In some lectin subunit consists of two polypeptides.

b. Lima bean lectin occurs also in a form consisting of eight subunits.

c. Do 1chos bif1orus and Abrus precator ius have 2 s.u. only one of which binds sugars

d. PhaseoIus vulgar is and Vicia vi11osa Contain 2 type of sub unit with different specificities.

e. Absent in con A , peanut and some of viciae lectins (Sharon and Lis,1990)

- 5 -

leguminous plants the main source is mature seeds where

lectins may constitute as much as 10% of the total protein.

Bulk of lectin is located in cotyledons in organelles known

as protein bodies (Etzler, 1986; and Chrispeels and Tague,

1990).

Importance:

Lectins are currently attracting much interest,

primarily because they serve as invaluable tools in diverse

areas of biological and medical research. Because of their

unique carbohydrate binding properties, lectins are useful

for the separation and characterization of glycoproteins and

glycopeptides and in study of glycolipids. They are useful in

following changes that occur on cell surface during

physiological and pathological processes i.e., from cell

differentiation to cancer. They can also be used in

histochemical studies of cells and tissues in typing blood

cells and bacteria, and for fractionation of lymphocytes of

bone marrow cells for bone marrow transplantation . They are

also used to stimulate lymphocytes to assess the immune state

of patients and for chromosome analysis in human

cytogenetics, as well as for the production of cytokines. In

addition lectins are excellent models for examining the

molecular basis of specific reactions that occur between

proteins and other types of molecules both of low or high

molecular weight, such as that of binding of antigens to

antibody, of substrate to enzyme, of drug to proteins and

- 6 -

of hormones and growth factors to cells (Sharon and Lis,

1990).

Possible role:

Most plant tissues contain one lectin, but in some

cases two (or more) lectins that differ in their sugar

specificities and other properties are present. The existence

of homologous lectin in different plant families demonstrate

that these proteins have been conserved through out evolution

and argues strongly that they must have an important

function (or functions) in nature. Although this function is

not known with certainity it is generally beleived that

lectins serve as cell recognition molecules (Sharon and Lis,

1990). Lectins may serve as mediators of the symbiosis

between nitrogen fixing microorganisms (primarily rhizobia)

and leguminous plants (Hamblin and Kent, 1973). This view is

based mainly on findings that a lectin from a particular

legume binds in specific manner to the corresponding

rhizobial species and not to bacteria that are symbionts of

other plants (Dazzo et a]., 1986). The most convincing

evidence for the symbiotic role of lectins is present for

the clove, Rhizobium trifo11i1 interaction only (Dazzo et

al., 1981). Cell surface carbohydrates of rhizobia, which

include extracellular polysaccharides, capsular

polysaccharides, 1ipopolysaccharides and periplasmic glucans

have been found to play an important role in plant infection

process leading to nitrogen fixation (Guerinot and Chelm,

1987; Long, 1989; Hollingsworth et aJ., 1989; Geremla et al.,

1987; Miller et a/., 1990). Lectins from leguminous tissues

have been extensively investigated for their molecular

properties and carbohydrate binding activities (Goldstein

and Poretz, 1986) .

Role of lectins in the defence of plants against

bacterial, viral and fungal pathogens have been proposed

(Barondes, 1981; Etzler, 1981; Leach et aJ., 1982; Mishkind

et al., 1982).

Presence of some lectins in the protein bodies suggest

the storage function of lectin (Manen and Meige, 1981; Manen

and Pusztai, 1982).

Biosynthesis:

Legume lectins are synthesized in the form of

prolectins in the endoplasmic reticulum, where the leader

(or signal) sequence containing 20-30 amino acids is

removed. Cotrans1ational N- glycosylation by dolichol

phosphate pathway and further posttranslational modification

of the carbohydrate units take place in the golgi apparatus

(Chrispeels and Tague, 1990). Frequently, post translational

proteolytic cleavages occur as well. The primary translation

products of the fava bean and pea lectin mRNAs are

polypeptides of Mr 29,000 and 25,000 respectively. In these

polypeptides the NH2 - terminal signal sequence of 29

residues is followed by the B chain and then the (X chain to

yield the mature favabean and pea lectins, occurs in the

- 8 -

protein bodies, the last point of their biosynthetic pathway,

where they are also stored. Processing of pea lectin

appears to be accompanied-by the additional removal of four

and six amino acids from the carboxyl terminal of the a

chain and p chain respectively (Higgins et aJ., 1983). These

posttranslationa1 modifications occur on the surface of the

molecule, after folding of the peptide chain has taken

place. The two subunits of Polichos biflorus lectin,

although different in size, appear to be the product of a

single gene, with subunits II resulting from proteolytic

cleavage of a 10 amino acid segment from the carboxyl

terminal of subunit I (Quinn and Etzler, 1989). This lectin

is also unusual in that the processing of its subunits

seems to occur before the arrival of the precursor to

protein bodies.

Complementary DNA of concanavalin A has been sequenced

and the amino acid sequence has been deduced,(Carrington et

al., 1988). Although con A is not a glycoprotein ,

experiments have shown that it is synthesized as a

glycosylated precursor of 290 amino acids,with the same

amino acid sequence as the deduced one (Bowles and Pappin,

1988). The carbohydrate of the precursor is first removed,

followed by excision of a peptide of 15 amino acid , to

which the carbohydrate was linked. The two fragments

formed are then covalently linked, presumably by

transpeptidation, to form the mature polypeptide in which

- 9 -

the alignment of residues 1-118 and 119-237 is reversed from

that of the precursor. The new covalent bond is between amino

acids 118 and 119, that is the cleavage position in the

naturally occuring fragmented subunits of con A. The two

fragments are thus unligated peptides rather than

proteolytic degradation products of the polypeptide chain

Both the deglycosy1 at ion of the con A precursor and the

subsequent processing steps most likely occur after the

protein has folded into a conformation similar to the

mature lectin, in which residues 118 and 119 protrude from

the surface of each of its subunits (Sharon and Lis, 1990).

Cloned lectin cDNAs and genes have been expressed in

bacteria, yeast and heterologous plants (Stubbs et aJ.,

1986; Prasthofer et aJ., 1989; Okamuro et ai.,1986; Tague and

Chrispeels, 1987; Sturm et al., 1988; Yamauchi et al., 1990).

The amino acid sequence of con A expressed in E.co1i was

reported to be the same as that of native lectin, indicating

that all the unusual processing steps required for the

formation of this lectin can be carried out by the bacteria

(Yamauchi et al., 1990). Although the recombinant pea lectin

was obtained from the bacteria as a single polypeptide chain

and was not processed as in the plant into ex and p chains,

it possessed the same hemagg1utinating activity and

carbohydrate specificity as the native lectin (Stubbs, et al

1986; Prasthofer et al., 1989). More over, crystals of the

polypeptide had a unit size similar to that of native lectin

- 10 -

suggesting the overall structure of the native and

recombinant protein is identical (Prasthofer et a7.,i989).

Carbohydrate binding specificity of legume lectins

The carbohydrate binding specificity of lectins (Sharon

and Lis 1989; Goldstein and Poretz, 1988) is customarily

examined by the hapten inhibition technique, in which

different monosaccharides, oligosaccharides or glycopeptides

are tested for their ability to inhibit either

hemagglutination or polysaccharide (or glycoprotein )

precipitation by the lectin. Binding of saccharides to

lectins can also be examined by physicochemical methods such

as equilibrium dialysis, spectrophotometry fluorimetry and

nuclear magnetic resonance. By these methods, the association

constant and other thermodynamic and kinetic parameters of

the reaction can be measured , thus affording information

on the binding process and giving a deeper insight into the

nature of combining sites of lectins. The binding constants 3

between lectins and monosaccharides are typically 10 to

4 - 1 4

5x10 M and between lectins and oligosaccharides 10 to

10 M . These values are in the same range as those for the

binding of haptens to antibodies or of substrate to enzymes.

The association constants for the interaction of a lectin

with a series of carbohydrate correlate well with the

relative inhibitory activity of the same sugars. Legume

lectins are classified into five specificity groups

according to the monosaccharide that is the best hapten

11 -

inhibitor of the lectin. These Include the mannose /glucose

binding lectins, the N- acetyl glucosamine binding lectins,

the N-acetyl galactosamine/galactose binding lectins, the L-

fucose binding lectins and sialic acid binding lectins

(Llener et al., 1986). Members of each group may differ in

their affinity for various analogs and derivatives of the

monosaccharides. Some lectins exhibit anomerlc specificity,

reacting preferentially with either the a or the p anomer of

the respective monosaccharides. Many lectins tolerate

variations at the C-2 position of the sugar to which they

bind; for example those specific for mannose usually also

react with glucose, and to some extent, with N-acetyl

glucosamine. Substitution at C-3 is tolerated by certain

lectins but not by others. The configuration of the C-4

hydroxyl group however. Is critical, as galactose specific

lectins do not react with glucose, nor do lectins specific

for glucose react with galactose.

Certain lectins interact more strongly with

oligosaccharides than with monosaccharides (Lis and Sharon,

1986) Table III. In such oligosaccharides, the

monosaccharide for which the lectin is specific is present,

usually at the nonreduclng end, although It may also be

located Internally. Moreover, some lectins react almost

exclusively with oligosaccharides. The lectins of Vlcia

gramineae. and those of Vlcia vlI Iosa bind glycopeptldes

better than the corresponding o1igosaccharides.They appear

TABLE ill

Lectins with pronounced specificity for oligosaccharides

Source of lectin

Datura stranoniun

Dotichos biflorus

Erythrina crystagalli

Arachis hypoxaea

Phaseotus vuli;aris E-PHA

Phased us vulgaris L-PHA

Vicia graainea

Vicia villosa

yheat gero

Monosacch­arides

Glc. NAc

Gal NAc

Gal

Gal

~

Gal NAc

Glc NAc

Oligosaccharides Structure

Glc.NAc 84 Glc.NAc M Glc NAc

Gal NAc a 3 Gal NAc

Gai & 4 Glc NAc

Gal & 3 Gal NAc

Gal & 4 Glc NAc £ 2 Han a 6 \ Glc NAc S 4 — H a n S 4R

Glc NAc B 2 Han a 3 / Gal fi 4 Glc NAc t 2

Han Gal & 4 Glc NAc & 6

HH2-Leu Gal £-3 Gal NAc a — ^ e r Gal £-3 Glc NAc a — T h r Gal 8-3 Gal NAc a — T h r

diu-CGOH NH2 - Set - (Pro)2 - Gly - (Ala)2 -

(Gal NAc a) (Gal

Glc NAc 8-4 Glc NAc 8-4 Glc NAc

Thr COOH

NAc a)

Relative inhibition

550

36

30-50

50

120

3000

- 13 -

to recognize carbohydrate sequence together with the amino

acid or peptide to which the latter are linked.

There are several blood group specific lectins that

agglutinate human erythrocytes of a particular blood type

(Goldstein and Hayes, 1978; Shibata et aJ., 1982). For

example A type cells are agglutinated by lectins from

Phaseolus lunatus. Vicla cracca and PolIchos blflorus

(Goldstein and Hayes, 1978), B type cells are agglutinated

by Griffonia simplicifolia (Hayes and Goldstein, 1975), 0

type cells are agglutinated by lectin from Ulex europeus and

Lotus tetragonolobus (Makela, 1957; Bird, 1978; Judd, 1980).

Lectin from Sophora laponica agglutinates both A and B blood

type cells ( Poretz et al., 1974).

Physicochemical properties of mannose binding legume lectins:

a)Molecular weight and subunit structure;

Generally, molecular weights of legume lectins range

between 38,000 and 289, 000. Molecular weight of pigeon

pea lectin is 38,000 (Hasan, 1990), whereas that of lima bean

lectin is 269,000 (Galbraith and Goldstein 1972). These

lectin consist of subunits (or protomers) that are usually

identical or very similar, each containing a single sugar

binding site with the same specificity. The subunits of the

legume lectins are made up of single polypeptide chains, but

in some cases they consist of two polypeptides. Concanavalin

A is composed of a mixture of intact subunits made up of a

single polypeptide chain containing 237 amino acids and of

- lA -

fragmented subunits in which the same polypeptide chain is

split into two fragments between residues 118 and 119 (Sharon

and Lis, 1990).The molecular weight, subunlt structure and

the number of sugar binding sites of some mannose binding

legume lectins are summarized in the Table IV.

b)Metals in lectins:

Some lectins require metal Ions for their biological

+ + + +

activity like concanava1in-A requires Mn and Ca

(Goldstein and Hayes, 1978). Effect of metal Ion on the

biological activity of lectins is best studied for

concanavalin A. Kalb and Levitzki (1968), found that con A

binds bivalent metals at two different sites Sj and S2 . Sj 2+ 2 +

site binds Mn while S2 site bind Ca . It was found that 2 + site was occupied first and then S2 could bind Ca and

occupation of both these sites was necessary for the binding

of carbohydrate residues. Removal of metal Ions in con A

results in unlocked state or conformation which is devoid of

any activity towards saccharides. When both metal binding

sites are occupied, there is a conformational transistion

from unlocked state to locked state. The metal ions seem to

function similarly in all other legume lectins.

c) Carbohydrate Contents

Lectins are generally glycoproteins with varying amounts

of carbohydrate content. Legume lectins contain upto 10%

carbohydrate in the form of a small number of N - linked

TABLE IV lolecular properties of some mannose SDecific leaume lectins

; M a I e c u 1 a i-- w e i q l-s t

Canavaila ensiformi

0 n o b r y c h i is v i c i i f a 1 i

Lens culinaris

F' i 5um sa t ivum

'icia fab a

53,000

46,500

49-53,000

52,000

a» Goldstein and Hayes. 197Ei; Lis and Share

buDun1 Mai.wt

25,500

26„509

5,700 17,500

5,800 12-18,000

5, 500 20,700

:m, 1974

Structure numbe^r

- 16 -

carbohydrate units (Sharon and Lis, 1990). These can be of

two type^ , one consists of N-acetyl glucosamine and mannose,

and is identical to the oligomannose units found, for example

in animals, indicating a common biosynthetic pathway . The

other consists of a structure unique to plants with B1-2

linked xylose and with (or without) «i-3 linked L-fucose in

the core pentasaccharide. Indicating that the processing of

N- linked Oligosaccharides in plants can differ from that in

other organisms (Sharon and Lis , 1990)Phaseolus vulgar is and

the lectin from Abrus precatorius contain oligosaccharides of

both types in the same molecule (Sturm and Chrispeels, 1986;

Klmura et al,, 1988). The carbohydrate does not appear to be

required for the sugar binding or biological activities of

the glycoprotlen lectins (Lis and Sharon , 1981). The most

convincing proof comes from the finding that the

unglycosylated L-PHA (1eukoagglutinatlng Phaseoulus

vulgarls) synthesized in Escherichia col 1, had the same

1eukoagglutinatlng activity as the native glycosylated lectin

(Hoffman and Donaldson , 1978). Lectins like concanava1in-A

(Olson and Liener, 1967) and wheat germ agglutinin (Allen,

et al., 1973) are few exceptions as they are devoid of

carbohydrate residues.

Sequence homology in mannose binding legume lectins:

Some 15 complete amino acid sequences of legume lectins

have by now established chemically or by molecular genetic

techniques (Sharon and Lis, 1990). Both the one and two chain

17

legume lectins exhibit extensive homologies when properly

aligned, i.e., by the p- chains of the two chain lectins

along the NH2 - terminal sequences of the one chain lectins,

followed by the <x chain and assuming deletions at selected

positions (Fig 1). Con A, either from Canavalia enslformi s

or from Canava1 la gladlata. and the lectin from Dioclea

grandiflora, all from plants belonging to the Dioclea tribe

occupy a special position. Homlogy of these lectins with the

other lectins 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 the

Dioclea lectins and continuing along their NH2 - terminal

regions (Fig 2). This unusual type of homology is referred

to as circular homology. The homologies are particularly

striking among lectins of the same tribe. The Vicleae lectins

exhibit more than 60% identities and Phaseoleae lectins

nearly 30%. Approximately 10% of the amino acids are

Invariant in all legume lectins and a similar proportion is

identical in atleast 75% of them (Sharon and Lis, 1990).

Further more , many of the replacements are conservative

one. N- g1ycosy1 at ion triplets or sequons (Asn -X- Ser/Thr)

when present are located at different positions of the

primary sequence and are not always occupied. The extensive

homology observed between different lectins suggests that

these are conserved protlens and have a common genetic

origin.

I 20 40 60 FBL TUEITSFSIPKFRPDQPNLIFQGGGYT—TKEKLTLTKAV K-NTVGRAL-YSLPIHIWDSETGNVADFTT LCL TE-TTSFSITKFSPDQQNLIFQGDGYT—GKEGLTLTKVS K-ETGGRAL-YSTPIHIWDRDTVNVANFVT LOL TE-TTSFSITKFGPDQQNLTFQGDGYT—TKEKLTLTKAV RNTVGRAL-YSSPIHIV7DSKTGNVANFVT PSL TE-TTSFLITKFSPDQQNLIFQGDGYT—TKEKLTLTKAV K-NTVGRAL-YSSPIHIWDRETGNVANFVT DBL AN-IQSFSFKNFNSPS—FILQGDATV—SSGKLQLTKVKENGFPL-RFPSGRA-FYSSPIQIYDKFTGAVASV7AT ECorL VE-TISFSFSEFEPGNDNLTLQGDSLP-ETSGVLQLTKINQNGMPA-WDSTGRTL-YAKPVHIWDMTTGTVASFET LBL AE—LFFNFQTFNA—ANLTLQGNAVS—SKCIILLLTNVTHNGEPS-VASSGRAL-YSAPIQIRD-STGNASSTPT PHA-L SN-DIYFNFQRFNET—NLILQUDASV-SSSGQLRLTNLNGNGEPR-VGSLGRA-FYSAPIQIVJDNTTGTVASFAT SEA AE-TVSFSWNKFVPKQPNMILQGDAIV-TSSGKLQLNKVDENGTP-KPSSLGRAL-YSTPIHIVJDKETGSVASFAA

SL AENTVSFDFSKFLSGOENLILQGDTVTDDSNRCLVLTR-ENNGRPV-ODS GRVL-YOTPJHLV7DK0IDKEASFET

LTA vs F YTEFKDDGS-LILQGDAKIWTDGR--LAMPTDPLVNNPKTTR5AGRAL-YATPVPIV;DSATGUVASFVT

conA TN-ALHFMFNQFSKDQKDLXLQGDATT-GTEGNLRLTRVSSNGSPE-GSSVGRALFY-APVHIVJDESSAVVASFEA 123 140 160 180

80 100 120 FBL T F I F V I D - A P N G Y N V A D G F T - F F I A P V D T K P O T G ~ G G Y L G V F - N G K D Y D K T - A O T V A V E F D T F Y N A A V 7 - D P - S N LCL ^ N G S O V F R E S P N G Y N V A D G F T - F F I A P V D T K P Q T G — G G Y L G V F Y N G K E Y D K T - S O T V A V E F D T F Y N A A H - D P - S N LOL S F T F V I U - A P N S Y N V A D G F T - F F I A P V D T K P Q T G — G G Y L G V F - N S K D Y D K T - S Q T V A V E F D T F Y N T A W - D P - S N PSL S F T F V I N - A P N S Y N V A D G F T - F F I A P V D T K P Q T G ~ G G Y L G V F - N S A E Y D K T - T Q T V A V E F D T F Y M A A V 7 - D P - S N

DBL SFTVKTS-APSKASFAnGTA-FAT.VPVG.SEPRRM — GnYT;GVF-nF;DVYNNS-A0TVAVEFDTI„StISnV7-DP-SM ECoi'L nFSl'S£V:QPYTlU'U'AOGLV-ri'MGP'i'K[Ui]'AOG — YCYLGlh'h'NSKQDNSY—OTJ.GVEFD'i'FSNP-W-DP-PQ LBL SllSYTLQ-QlFONVTDUPAWLFALVPVDSQPKKK — CKLLGLF-NKSEhJUIMALT-VAVErOTCHNLDW-D PHA-L SFTFNIQ-VPNNAGPADGLA-FALVPVGSQPKDK—GGFLGLFDGS-NSNFUT VAVEFDTLYTJKDW-DP SBA SFNFTFY-APDTKRLADGLA-FFLAPIDTKPQTH—AGYLGLF-N—ENESG-DQVVAVEFDTFRNS-W-DP-PN

SL r.F'ri'FTY--Kl';NINKCGlJGJT--KFLM"J'lJ'J'OPK:;(; • CGYLG lFKDAK":';ilKT~--VVAVl';i''»TFliUK-V?-lJl'-AU LTA SFNFLFVrRELKYTPTDGLV-FFLAPVGTEIPSGSTGGFLGIFKDGS-NNKN-GQRVAVEFDSYHN-IVJ-DPKSL

ConA TFAFLIK-SPDS-HPADGIA-FFISNIDSSIPSGSTG1U.LGLFPDAN AD-T IVAVELDTYPNTDIGDP-SY 2 0 0 220 K- I I X K -X- K -X- 22

o a o

140 IGO 180 FBL .GKRHIGIDVN-TIKSISTKSWNLQNGEEAHV-A-ISFNATTNVLSVTLLYPN LTGYRLSEVVPLKDW LCL KERHIGIDVN-SIKSVNTKSWNLQNG VTSYTLNEVVPLKDW LOL GDRIIIGIDVN-SIKSINTKSWKLQNGKEANV-V-IAFNA-TNVLTVSLTYPN • ETSYTLNEVVPLKEFV PSL RDRHIGIDVN-SIKSVNTKSWKLQNGEEANV-V-IAFNAATNVLTVSLTYPNSLEEEN VTSYTLSDWSLKDW DBL —KIIIGIDVN-SIKSTATVSWDLANGENAET — LITYNAATSLLVVSLVIIPSRR TSYTLSERVDITN'EL ECorL -VPHIGIDVN-SIRSIKTQPFQLDNGQVANV-V-IKYDASGKILHAVLVYPSSG AIYTIAEIVDVKQVL LBL KNS lAVNLGIGSVP WNFRDYDGQNADVLITYDSSTKFLAVSLFYPITG KRNNVSANVELEKVL PHA-L TERHIGIDVN-SIRSIKTTRWDFVNGENAEV—LITYDSSTKLLVASLVYPSOK TSFIVSDTVDLKSVL SBA —PHIGINVN-SIRSIKTTSWDLANNKVAKV—LITYDASTSLLVASLVYPSQR TSrJILSDVVDLKTSL

SL --r.IlTG.IMVN--r.VK,'lK I •J'TPWCI-KIinVl' -TV'J'"-JTYl)A'l'Kr>"L.';vn.'"iFYl«tlKl'0 Dl fTVKASVll I,I(DAI. LTA RSSHVGIDVN-SIMSLKAVNWNRVSGSLEKAT-II-YUSQTNILSVVMTSQNGQI TTIYGT IDLKTVL ConA —PHIGIDI-KSVRSKKTAKV7NMQDGKVGTAH-II-YNSVDKRLSAVVSYPN ADSATVSYDVDLDNVL

•X- -X- -X- 40 60 80

200 220 P i - --• --FBL PEWVRIGFS-ATTGAEYATHE VLSWTfc'LSELTGPSM

LCL PEWVRIGFS-ATTGAEFAAQE V H S W S F N S O L G H T S K S LOL PEWVRIGFS-ATTGAEFAAHE VLSWFFHSELAG'rsSM PSL PEWVRIGFS-ATTGAEYAAHE VLSWSFHSELSGTSSSKQAADA

DBL PEYVGVGFS-ATTGLSEGYIETHDVLSWSFASRLPDDST-A-EPLDLA-RYLVRNVL ECorL P E W V D V G L S G A T - G A Q R D A A E T H D V Y S W S F Q A S L P E T N D - A - V I P T S N - H N T F A I LBL D D W V S V G F S - A T S G A - Y ETHDVLSSSFASKL—SS-LDECPTGENLLNKIL

PHA-L PEVTVSVGFS-ATTGINKGNVETNDVLSWSFASKLSDGTTSEGLNLANLVLNKIL SBA PEWVRIGFSAAT-G-LDIPGESHDVLSWSFASNLPHASSNIDPLDLTSFVLIiEAI SL PQWVRIGLS-AATG-DLVEQHRL—YSWSFKSVLPLDSST

LTA PEKVSVGFS-ATTG-NPEREK-HDIYSWSFTSTLKEPEEOA

ConA P E W V R V G L S - A S T G - L Y K E T N T — ILSV)SFTSKLKSNSTIIE 100 120 • o

FIG.I ; AMINO ACID SEQUENCES OF LEGUME LECTINS

F(G.2: SCHEMATIC REPRESENTATION OF CIRCULARLY PERMUTED SEQUENCE HOMOLOGY THAT RELATES CONCANAVALIN A TOOTHER LEGUME LECTINS (OLSEN 1983).

- 20 -

Three dimensional structure of a representative mannose

binding legume lectin:

Concanavalin A was one of the first lectin for which

the three dimensional structure was solved by high resolution

X-ray crystallography each of its subunits is in the shape

of a dome or gumdrop, approximately 42x40x30 A in size

(Reeke and Becker , 1988). The predominant structural element

of the subunit, which accounts for almost half of its amino

acid residues , is the arrangement of the polypeptide chains

in two anti parallel B-pleated sheets (Reeke and Beeker,

1986).

Two dome shaped protomers pair to from an ellipsoidal dimer

more than 80 A in length.

a) Metal and carbohydrate binding site

2+ 2 + The binding sites for Mn and Ca In con A are situated

5 A apart and are located at the top of each protomer. The

carbohydrate binding site is located 12 A from the

Mn binding site and 7 A from the Ca site. As with other

saccharide binding protiens , con A combines with a

saccharide primarily by hydrogen bonds, and also by

hydrophobic interaction and Vander Uaals contacts.

(Sharon and Lis, 1990). There are seven hydrogen bonds

between the glycoside and the protien (Fig 3). The

hydrophobic patch , which is formed by C-5 and C-6 of the

saccharide , is in Vander Waals contact with the ring atoms

of Tyr-12 and Tyr- 100.

Asp^O

Tyrl2 / 3

01

02 "

,«H20

FI6.3: 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 . Z . ETAL 1985 ) .

- 22 -

In addition to con A, high resolution x-ray

crystal 1ographic data are available for the lectins of pea

and fava bean (Reeke and Becker, 1988), Erythrina

corallodendron (Sharon et al 1989) and Griffonia

simplicifolla (Van donselaar, et al., 1987; Nikrad et a}.,

1989).

b) Hydrophobic binding site

In addition to carbohydrate binding sites lectins

frequently posses hydrophobic sites of three types. One of

these is adjacent to the carbohydrate binding site as is

evidenced by the finding that hydrophobic derivatives of

monosaccharides bind more strongly (upto 10-50 fold) to the

lectin than the analogous non hydrophobic compounds (Sharon

and Lis, 1989; Goldstein and Poretz, 1986). In addition most

legume lectins examined contain binding site or pockets for

hydrophobic ligands that are remote from the carbohydrate

binding site (about 30 A in case of con A). Ligands that

bind at this site include indole acetic acid and 1 - 8

3 4 - 1 ani1inonapthelene sulphonic acid (ka=10 -10 M ) and there

is one such binding site per subunit. Several of the legume

lectins also cntain a specific high affinity binding site

for adenine and its N-6 derivatives with association

5 6 -1 constants 10 -10 M . This binding site is unusual in that

there is only one such site per lectin molecule (and not per

subunit) (Maliarik and Goldstein, 1988). Thus, lectins not

only function as carbohydrate binding proteins but also

- 23 -

serve as binding proteins for biologically active hydrophobic

llgands (e.g. phyto hormones)

Objectives

Further research on lectins from legumes as well as from

other sources should advance our insight into precise

molecular details of the interaction between proteins and

carbohydrates. It will also lead to a better understanding

of many fundamental biological processs In which cell

recognition is involved. There are several lines of evidence

that suggest that lectins mediate rhizobium legume

interaction and hence are important for nitrogen fixation

(Dazzoet a/., 1984). In view of these considerations we have

studied a lectin from Ca.janas calan (pigeon pea) which is one

of the important legume crops of India.

E X P E R I M E N T A L

M A T E R I A L S

The following chemicals were used in this study:

Proteins

Bovine serum albumin (Lot no.lOOF-0249); ovalbumin (Lot

No.105 C-8022); chymotrypsinogen A (Lot No. 40F-8050);

cytochrome c (Lot No. 09C- 0088)} myoglobin; and trypsin (Lot

No.l- 2395) were obtained from Sigma Chemical Company,

St.Louis, Mo., USA.

I gM was isolated from goat blood by the method given

be 1ow.

Chemicals used in isolation of lectin:

Chemicals used in isolation of lectin from pigeon pea

pulse with their sources in parentheses were :

acetic acid glacial (Qualigens Fine Chemicals, India );

ammonium sulfate (S.D.Fine Chemicals, India); disodium

hydrogen phosphate (Sarabhai M Chemicals, India); monosodium

hydrogen phosphate (Sarabhai M Chemicals, India ); sodium

chloride (BDH, India ); Sepharose 6B (Sigma Chemical Co.

USA); sodium hydroxide (BDH,India); sodium carbonate and

sodium azide (Fluka, Switzerland); cyanogen bromide (SRL Pvt.

Ltd., India).

Miscellaneous reagents

Bio gel P-200 (Bio-Rad Lab, Richmond, California); blue

dextran (Pharmacia Fine Chemicals, Uppsala, Sweden); copper

- 25 -

sulfate (E. Merck, India ); lithium sulfate (BDH, India);

orthophosphoric acid <BDH,India); sodium molybdate,

sodium,potassium tartarate, sodium tungstate, - trisodium

citrate; potassium dichromate were obtained from BDH, India;

phenol (S.D. Fine Chemicals, India); ethylene di-amine

tetraacetic acid (Quallgens Fine Chemicals, India); potassium

hydrogen phthalate (BDH, India); methanol (S.D. Fine

Chemicals, India ); dextrose (Glaxo Laboratories, India);

glycerol (E.Merck, India ); glycine (Qualigens Fine

Chemicals, India); guanidine hydrochloride (Heico, USA) and

chloroform (E. Merck, India)

pH paper was obtained from E. Merck Germany and Whatman

filter paper No. 1 from W & R Balston Ltd. England.

M E T H O D S

Measurement of pH

Elico digital pH meter, model Ll-122 was used for pH

measurements in conjunction with Elico combined electrode

type CL-5i at room temperature.The pH meter was standardized

with 0.05 M potassium hydrogen pthalate buffer , pH 4.0 in

the acidic range or with 0.01 M sodium tetraborate buffer,

pH 9.2, in the basic range (both solutions were provided ).

Measurement of light absorption

In the ultraviolet region, light absorption measurements

were performed on Cecil single beam spectrophotometer model

CE 202 or on Cecil double beam spectrophotometer, model CE594

using silica cells of 1 cm path length. The spectrophotometer

was calibrated with 0.0303 g /It. solution of K2Cr207 in

0.05 M KOH (see Crelghton T.E.,1990)

Determination of lectin activity

a) Hemagglutination

Heraagg1utinating activity of the lectin was measured

using rabbit erythrocytes. Rabbit blood was collected in the

presence of 2.8 % EDTA in phosphate buffered saline (PBS) and

centrifuged at 2000 rpm for 20 mins. The supernatant was

discarded and the pellet of red blood cells was washed four

times with 10 mM sodium phosphate buffered saline , pH 7.0.

To check hemagglutinating activity of lectin 0.1 ml of

protein solution (containing 0.1 - 0.2 mgs of protein) was

27 -

taken on a glass slide and to this 0.1 ml of rabbit

erythrocytes (200 x 10 cells ) were added and agglutination

was checked visually.

b) Precipitin reaction

For precipitin reaction , varying concentration of IgM

(20 -120 mg) were incubated with fixed amount of lectin (5

mg) In a total volume of 2 ml in 10 mli sodium phosphate

buffer pH 7-0 containing 0.5 M NaCl, The mixture was

incubated at room temperature for overnight. The

precipitate obtained was washed three times with 0.15 M NaCl

by centrifugation at 2000 rpm for 30 minutes. The washed

precipitate was dissolved in 0.1 M NaOH and protein

concentration in the precipitate was determined by the method

of Lowry et aJ (1951).

Determination of protein concentration:

Protein concentration was estimated by the method of

Lowry et a] (1951) using bovine serum albumin as the standard

protein . To 1 ml of protein solution ,5 ml of freshly

prepared copper reagent (4 % sodium carbonate (w/v), 4X

sodium potassium tartarate (w/v) in the ratio of 100:1:1)

was added . It was incubated for 10 minutes at room

temperature. Then 1 ml of diluted (1:4) Folin phenol reagent,

prepared by the method of Folin and Ciocalteau (1927) was

added . The solution was allowed to stand at room temperature

for 30 minutes. The colour Intensity was read at 700 nm

- 28 -

against the blank prepared in the same manner on AIMIL

photochem -8 colorimeter. A straight line was obtained by

least square analysis (Fig. 4) which fits the equation.

(O.D.)7oonm = ^ • " ^ ^"^6 protein) + 0.034 ... (1)

Determination of carbohydrate content:

The neutral hexose content was determined by the method

of Dubois et al (1956) using glucose as standard .

To one ml of protein solution, 1 ml of 5% (w/v) phenol

solution was added . Then 5 mi of concentrated sulfuric acid

was mixed with it and the solution was allowed to cool at

room temperature for 20 minutes. The colour intensity was

measured at 490 nm against an appropriate blank . The

straight line obtained by the method of least squares (Fig.

5) fits the equation .

(O.D.) 490nm ~ O-OO- SG x(mg glucose) +0.33... (2)

Isolation of the lectin:

For the isolation of lectin, 40 gms of pigeon pea seeds

were soaked in 200 ml of 10 mM sodium phosphate buffer pH

7.0 containing 0.5 M NaCl (PBS) for 6hrs. Then it was

homogenized in a blender for 5 minutes and the suspension

was filtered through a muslin cloth. The pH of the solution

was lowered to pH 4.0 by addition of 0.3 M acetic acid and

It was kept overnight in a refrigerator. The precipitate

was removed by centrifugation on RC5C centrifuge at 8000

6 c o o t>

o

c o>

T3

"a o

••J a. O

0.2 0.3 0.4 0.5

Protein concentration (mg )

0.7

F(G.4 :CALIBRAT(ON CURVE FOR THE ESriMAf lON OF PROTEfN CONCENTRATION BY THE METHOD OF LOWRY et. a l . , 1951 USING BSA AS THE STANDARD PROTEIN . THE STRAIGHT LINE WAS DRAWN BY THE METHOD OF LEAST SQUARES AND FITS THE EQUATION :(0.0) 700 nm = 1.7 x (mg protein )+0.034.

£ c o en >» a >»

••J

VA C Q> X)

"5 U

Q. O

60 80

Jug of glucose

FIG.5.'CALIBRATION CURVE FOR THE ESTIMATION OF CARBOHYDRATE CONCENTRATION BY THE METHOD OF DUBOIS et al ., 1956 USING GLUCOSE AS THE STANDARD. THE STRAIGHT LINE WAS DRAWN BY THE METHOD OF LEAST SQUARES AND FITS THE EQUATION : (O.D) 490nm =0.00436 x (jug glucose) +0.033

- 31 -

rpm for 15 minutes . The supernatant was made 30% with

respect to ammonium sulfate. The protein precipitated was

discarded by centrifugation and the supernatant made 50 %

with respect to ammonium sulfate. The precipitate was

dissolved in minimal volume of PBS and dialyzed extensively

to remove ammonium sulfate. The dialyzed 50 % ammonium

sulfate fraction was further purified by affinity

chromatography on IgM Sepharose 6B column which involved the

fo11 owing steps.

a) Isolation of 1gM rich fraction from goat blood

In order to prepare IgM, freshly prepared goat

plasma was diluted 10 times with cold distilled water and

the solution kept in the refigerator for overnight to allow

precipitation of IgM rich fraction. The precipitate was

collected by centrifugation and washed three times with

cold distilled water. The precipitated protein was dissolved

in PBS for subsequent experiements.

b) Preparation of IgM Sepharose 6B

1) Activation of Sepharose 6B

Twenty millilitres of Sepharose 6B gel slurry was

washed with distilled water and then with 0.15 M NaC1. The

pH of the slurry was adjusted to 10.8 with 4 M NaOH. To this

slurry , solid CNBr was added (0.3 g of CNBr per ml of gel

slurry) while stirring. The temperature was kept low by

keeping the beaker in vessel of ice and the pH was

- 32 -

maintained at 11.0 with 4 M NaOH on addition of Cyanogen

bromide. The activated gel slurry was washed extensively

with chilled distilled water and then with coupling buffer.

(0.2 M sodium bicarbonate buffer, pH 9.2 containing 0.15 M

NaCl).

ii) Coupling of IgM with activated Sepharose 6B

The IgM rich fraction of the goat blood was dialysed

extensively against the coupling buffer. About thirty

millilitre of IgM (14.6 mg/ml) in coupling buffer was mixed

with the activated gel slurry. The mixture was incubated at

room temperature for overnight . The affinity gel was washed

with distilled water and the unreacted activated groups were

blocked with 0.2 M glycine in coupling buffer . The affinity

gel was washed extenively with distilled water and packed

in a glass column (2.2 x 6.0 cms).

c) Affinity chromatography

The salt fractionated lectin from pigeon pea was

applied on the IgM Sepharose 6B column equilibrated with 10

mM sodium phosphate buffer pH 7.0 containing 0.5 M NaCI. The

column was washed with two column volumes of PBS. The lectin

was eluted with 0.25 M glucose in PBS. Lectin activity was

measured by hemagglutination assay in presence and absence

of glucose.

- 33 -

Determination of specific extinction coefficient of pigeon

pea lectin:

For the determination of specific extinction

coefficient of pigeon pea. lectin, the concentration of the

lectin was determined by dry weight method . Affinity

purified pigeon pea lectin solution was taken and dlalyzed

extensively against 10 mM sodium phosphate buffer pH7.0

containing 0.15 M NaCl. Clean dried empty weighing bottle',

were taken and weighed to constant weight . A given volume

( 10 ml ) of the lectin solution was heated to constant

weight near 105 C. The bottle containing equal volume of

dialyzate (PBS) was heated similarly and served as control.

The difference in the weights of the two sets of bottles gave

protein concentration. Solutions containing different amounts

of the protein were prepared from the stock solution and

their optical densities were recorded at 280 nm. Six

experimental points were taken in the plot of protein

concentration versus optical density and least square

analysis was done.

Gel chromatography:

Gel chromatography was carried out on Bio gel P-200

column (2.4 x 54 cm) equilibrated with 10 mM sodium

phosphate buffer pH 7.0 containing 0.5 M NaCl. The column

was packed by R.H. Khan of this laboratory . The column was

calibrated with five marker proteins including bovine serum

albumin (68,000 D),ovalbumin (43,000 D) chymotrypsinogen

- 34 -

(25,700 D) myoglobin (17,200 D) and cytochrome c (11,700 D).

BSA and myoglobin were gel filtered in this study wheras

the rest of the proteins were eluted from the same column by

R.H.Khan of this laboratory .The flow rate of the column

was 20 ml/hr. Blue dextran was passed through the column to

determine the void volume (76 ml) and to check the uniform

packing of the column (Fig. 9) . About 15 mgs of protein

in a volume of about 5 ml was applied on the column and 3 ml

fractions were collected at a flow rate of 20 ml/hr.

Effect of temperature on the carbohydrate binding property of

the lectin:.

Effect of temperature on the pigeon pea lectin was

observed by hemagglutination assay as well as by precipitin

reaction. Gel filtered pigeon pea lectin (0.195 rag/ml) was

taken in PBS and exposed to a temperature range of 10 C to

70 C in an incubator for 30 minutes. The activity of heat

treated lectin was determined by hemagglutination assay

4 using rabbit erythrocytes(327 x 10 cells) and by precipitin

reaction of lectin (97.5 ug) with IgM (585 ug).

R E S U L T S

R E S U L T S

Screening of the pulse samples:

We have screened 50 samples of pigeon pea pulse

obtained from different shops of the local market for

their hemagglutinating activity against rabbit erythrocytes.

Pulses were homogenized in 10 mM sodium phosphate buffer

pH 7.0 containing 0.5 M NaCl (PBS) and their

hemagglutinatlng activity determined against rabbit

erythrocytes, modified (trypsinized) as well as

unmodified.Three out of 50 samples were found to

agglutinate the unmodified cells within 15 sees.

Solubilization of the lectin:

Solubilization of lectin from seeds was carried out in

10 mM sodium phosphate buffer pH 7.0 containing varying

amounts of NaCl ranging from 0.2 M to i.O M. Increase in

hemagglutinatlng activity was taken to be a measure of lectin

solubilization. When NaCl concentration was increased from

0.2 M to 1.0 M In the phosphate buffer, the amount of lectin

solubillzed increased and attained an optimum value at a

sodium chloride concentration of 0.5 M as can be seen in

the Fig.6. The extent of agglutination increased with

increase in NaCl concentration from 0.4 M to 0.5 M, however,

further increase in sodium chloride concentration beyond 0.5

M caused decrease in hemagglutinatlng activity. Therefore,

0.5 M NaCl in 10 mM sodium phosphate buffer pH 7.0 (PBS) was

used in subsequent studies for the solubilization of lectin

Q» -•-» O C

cn C7) o c

(/>

O

CD

CC

y»

"S u o d

2 0 0 -

180-

NaCl concentration (Molar)

FIG.6:CURVE TOSHOWEFFECTOFNaCI CONCENTRATION IN 10 mM SODIUM PHOSPHATE BUFFER pH 7.0 ON SOLUBLIZATION WAS DETERMINED BY THE HEMAG6LUTINATIN6 ACTIVITY OF LECTIN AGAINST UNTRYPSINIZEO RABBIT ERYTHROCYTES

- 37 -

from pigeon pea pulse.

Stability of the lectin;.

The effects of pH and temperature on hemagg1utinating

activity of crude preparation of lectin were investigated

using rabbit erythrocytes. The hemagg1utinating activities

of lectin in the homogenate were determined in PBS of pH

values 6.5, 7.0 and 7.4. No detectable difference in the

hemagglutinating activity against rabbit erythrocytes was

observed at three different pH values.

Isolation of the lectin;

Forty grams pigeon pea pulse was homogenized in 170

ml sodium phosphate buffer pH 7.0 containing 0.5 M NaCl, to

obtain a suspension which on acidification with 0.3 M acetic

acid and subsequent centrifugation yielded a clear protein

solution containing 1800 mg protein. In hemagglutination

assay (described in the experimental section ) the onset

of hemagglutination was obtained within 26 sees. Salt

fractionation resulted in deduction of 70 % protein and

improvement in hemagglutinating activity ( see Table V).

The onset of hemagglutination with this preparation was

found to be 12 sees under similar condition.

About 45 mgs lectin was obtained by affinity

chromatography on IgM Sepharose 6B column (2.2 x 6.0cm)

equilibrated with PBS (Fig.7 ). Thus the final yield was

2.5% as compared to the total protein present in the acid

TABLE V

Protein yield in various stages of purification of pigeon pea ], Bc. t i ri

"EF

Acid homooenate

50% (NHi^)^SO^ p r e c i p i t a t o 3

A f f i n i t y c h r o m a t o g r a p l"i y

V o l u m e (ml>

450

20

50

F " ' r o t e i n C o n e ( m q / m l )

4

0. 9

T o t a l -' r o t Ei i n

( q m s )

1 .8

O.

0 . 045

F ' r o t e i n y i B1 d

28

HasiTitaQq l u t •~ i n a t i n q a c t i v i t y

+•• •1-

++ +

-i- -t-+

* d i S B i g n a t i o n o f h a m a g q l u t i n a t i n g a c t i \ ' i t y i . e t i m e o f orii^tii^t o f hsfiiagQ 1 u 1 1 n a t i o n o f r a b b i t o r y t ! - , r o c y t ( ^ s

1 • i i j s e e s ~ i'+i 1 5 - 3 0 s e e s = •+-i-ZQ 5 0CS - 6 0 5 e c H = + 6 0 s e c E o r n^ore ~ •

6 c o o ••J

o

w c (b TJ

"5 U

•4-* a. O

Elution volume (ml )

FIG. 7: AFFINITY CHROMATOGRAPHY OF 50% AMMONIUM SULPHATE FRACTION OF PIGEON PEA HOMOGENATEON IgM SEPHAROSE 6B COLUMN (2.2 x6.0 cms). THE UNBOUND PROTEIN CAME AS WASH THROUGH IN 10 mM SODIUM PHOSPHATE BUFFER CONTAINING 0.5M NaCI. THE BOUND PROTEIN WAS ELUTED WITH 0.25M GLUCOSE IN PBS.

- 40 -

homogenate (see Table V). With the affinity purified

protein the time of onset of hemagglutination was 7 sees.

Effect of ageing:

The affinity purified pigeon pea lectin was found

fairly stable at room temperature and at 37 C. It retained

its hemagglutlnating activity in refrigerator for about two

weeks, however prolonged storage in refrigerator caused

aggregation of the lectin and the aggregated lectin did not

show any hemagglutinating activity

Gel Chromatography:

The affinity purified lectin was eluted as a single

symmetrical peak from a calibrated Bio gel P-200 column

(2.4 X 54 cm ) see Fig.10. The gel filtration results for

marker proteins are listed in Table VI. A least square

analysis of the data between Vg/Vp and logM (Fig. 8)

yielded the following equation;

VQ/VO = -0.978 logM + 6.14 ( 3 ).

The value of Vg/Vg for lectin was measured to be 1.85

which according to equation 3 corresponded to a molecular

weight of 38,000.

Determination of carbohydrate content:

The pigeon pea lectin was determined to be a

glycoprotein using phenol sulphuric acid method of Dubois et

al (1956) . With one milligram of protein in 0.5 ml

TABLE '.'I

Gel filtration data

PTotein r; li o 1 e c: u 1 a r ;: L D D H

s weiqht -• (a) ^ Ve (mi)

: bovine sierum albumim

Qvailbu.mim *

Ch > iTtQ t r y p s :J. n og e n «•

Myoglobin

Cytochrome c *

68., 000 4 „ 83 106

Ve/Vo

1 „ 39

43 , OOCi

25,, 700

1 / , l:vo

1 1,700

"

„

" •

:; :

4,. 63

4 „ 4 4

4. 24

4„07

"

"

„

: :

120

148

149

1 „ 61

.

„

•

3

:

1 „ 53

1 ,.94

1. ?6

2.. 11

a--Valu.es of moiacular weight imre taf en from Wsbs:' And Qsborr', 1969)

•! -The data were obtained by F;..'i. ihan of this laboi atorv '1991)

o >

a* >

4.4 4.8

Log molecular weight

5.2

FIG.8 CALIBRATION CURVE FOR THE DETERMINATION OF MOLECULAR WEIGHT OF PROTEINS BY STANDARD CURVE OF Ve/Vo VS. LOG M MARKER PROTEINS U S E D : (1) BOVINE SERUM ALBUMIN (2) OVALBUMIN (3) CHYMOTRYPSINOGEN A U ) MYOGLOBIN (5) CYTOCHROME C . THE STRAIGHT LINE DRAWN BY THE METHOD OF LEAST SQUARE FITS THE EQUATION ! Ve/Vo = - 0 . 9 7 8 LOG M + 6.14

Elution volume (m l )

FIG.9:GEL CHROMATOGRAPHY OF BLUE DEXTRAN ON BIO GEL P- 200 COLUMN (2.4 x 54 cms) EQUILIBRATED IN lOmM SODIUM PHOSPHATE BUFFER CONTAINING 0.5 M NaCI .

e c o o

o

"iJ5 c 0)

•X3

"o u

••-'

a. O

130 130 150 -170 n o

Elution volume (ml )

FIG.10 : G E L F I L T R A T I O N P R O F I L E OF B O V I N E S E R U M A L B U M I N (1) , MYOGLOBIN (2) AND PIGEON PEA LECTIN (3) ON BIO GEL P-200 COLUMN ( 2.4 x 54 cms) EQUILIBRATED IN lOmM SODIUM PHOSPHATE BUFFER pH 7.0 CONTAINING 0.5 MNaCI . ( + + ) FRACTIONS SHOWING POSITIVE HEMAGGLUTINATION WITH UNTRYPSINIZED RABBIT ERYTHROCYTES AT SAME PROTEIN CONCENTRATION.

- 45 -

solution the colour yield of phenol sulfuric acid solution

was 0.19 O.D. which according to the calibration curve

corresponded to 72 ug of neutral hexose i.e 7% (w/w).

Precipitin reaction:

Goat IgM was specifically precipitated with lectin.

Results on the precipitin titration curve obtained in lOmM

sodium phosphate buffer pH 7.0 containing 0.5 M NaC1 (PBS)

are shown in Fig.il, where it can be seen that maximum

precipitation of proteins occurred when the ratio of

lectin to plasma glycoprotein was six. Five mgs of lectin

(50% (NH/^)2S04 precipitate) when added to 30 mgs of plasma

glycoprotein (40% (NH4)2S04 plasma pecipitate) in a final

volume of 2ml , resulted in precipitation of 4.3 mgs of

protein, accounting for 12% of total protein added. Out of

this total protein precipitated about 90% is specific , as

it dissolves in 0.25 M glucose and 10% is non specific. The

observation that the precipitate dissolved completly in 9 M

urea suggests that the interaction is noncovalent in

nature. Hence using the ratio 1:6 of lectin to plasma

glycoprotein the precipitation reaction was caried out in

presence and in absence of glucose.

Effect of temperature on carbohydrate binding properties of

pigeon pea lectin:

Pigeon pea lectin ( purified by gel filtration )

(0.195 mg/ml) in 10 mM sodium phosphate buffer pH 7.0

6 10 U 18

Ratio of lectin to IgM

FIG.n : PRECIPITIN TITRATION CURVE OF PIGEON PEA LECTIN WITH GOAT IgM. VARYING CONCENTRATION OF Ig M (407. AMMONIUM SULPHATE PLASMA PRECIPITATE ) ( 20-120 mgs) WERE INCUBATED WITH FIXED AMOUNT OF LECTIN (50 V. AMMONIUM SULPHATE FRACTION) (Smg) IN A TOTAL VOLUME OF 2 ml IN 10 mM SODIUM PHOSPHATE BUFFER pH 7.0 CONTAINING 0.5 M NaCI.

- 47 -

containing 0.5 M NaC1 was taken and exposed for 30 minutes

in an incubator to a temperature range of 10° - 70°C. The

activity of heat treated lectin was determined by

4 hemagglutination assay using rabbit erythrocytes ( 327 x 10

cells ) (Fig. 12) and by precipitation reaction of lectin

(97.5 ug) with IgM (Gel purified 40% (NH4)^04 plasma

precipitate) ( 585 ug) (Fig. 13).

The lectin retained its normal activity in the

temperature range of 20 C to 40 C. However, on further

increase up to 70 C there was considerable loss in activity

(Fig. 14).

Optical properties:

For the optical measurements CECIL double beam

spectrophotometer model CE 594 was calibrated using 0.0303

gm/lit. of K2Cr207 in 0.05 M KOH. The calibration data is

given in the Table V1 I. The UV spectra of lectin from pigeon

pea was measured in 10 mM sodium phosphate buffer pH 7.0

containing 0.5 M NaCl in the wavelength region 200 nm to 360

nm. The lectin absorbed maximally near 278 nm , (Fig. 15)

however, when the absorption scan (Fig. 16) was taken on BD 40

Kipp ii Zonen chart recorder attached to the

spectrophotometer , a bifurcated peak having maximum

absorption at 278 nm and 282 nm was obtained, this may be

due to the aggregation of the lectin. There is a

characteristic shoulder peak at 290 nm , which may be due

to the heterogeneity of the solution or due to some

•o ^ 2 0 0 - - 100 o c

en o c

(/) O

CD

cr - 1 0 0 -

u « • -

o d

30 40 50

TEMPERATURE °C

IG.12 -.CURVES SHOWING THE EFFECT OF TEMPERATURE ON HEMAGGLUTINATING ACTIVITY OF PIGEON PEALECTIN WITH UNTRYPSINIZED RABBIT ERYTHROCYTES .

• O O NO. OFCELLS UNAGGLUTINATED. HEMAGGLUTINATION.

200

en

3 T3

U

.£ 100

O i_ Q.

Temperature °C

FIG.13: CURVE TO SHOW THE EFFECT OF TEMPERATURE ON PIGEON PEA LECTIN MEDIATED PRECIPITATION OF GOAT Ig M .

100-

> O

o

"in

0)

Temperature °C

FIG.U : CURVE TO SHOW PERCENT RESIDUAL ACTIVITY OF PIGEON PEA LECTIN AFTER THE TEMPERATURE TREATMENT. 0 - — O ON PRECIPITIN REACTION. • • ON HEMAGGLUTINATING REACTION.

TABLE V I I C a l i b r a t i o n o f t h e d o u b i e beam i s p a c t r o p h o t o m e t a r

Xnm

220

240

260

280

300

320

S t andai"d absat-'oance V a 1 u s D f 1 •'. 2. C f" 2 *••' 7

solution

0,. 446

0 „ 295

0.633

0,. 712

0. 149

0 „ 064

E;; P e i-- i aiBn t a i v a 1 u e

0 . 4 3 3

0 . 299

0 . 664

C». 716

0 . 153

0» 063

V a r i a t i o n

1: 3X

+ 1 '/••

•L 5X

+ 0„5%

+ 17.

240 250 260 270 280

Wavelength ( Anm)

290 300

F I G . 1 5 : A B S 0 R P T I 0 N S P E C T R U M O F P I G E O N P E A L E C T I N I N lOmM SODIUM P H O S P H A T E BUFFER pH 7.0 CONTAINING 0.5 M NaCI ON CECIL DOUBLE BEAM SPECTROPHOTOMETER CE 594

"^ - P i » ENRAPTN0NIU&4.t<<'-

360 200

FIG. 16 SCAN OF ABSORPTION SPECTRUM OF PIGEON PEA LECTIN ON BD-40 KIPP & ZONE:N CHART RECORDER ATTACHED TO DOUBLE BEAM SPECTROPHOTOMETER

- 54 -

intrinsic property of the lectin.

Determination of specific extinction coefficient of pigeon

pea lectin:

The lectin concentration was obtained by dry weight

method. Optical densities of solution of known concentration

prepared from the stock solution were determined at 280 nm

and 278 nm. The plot of absorbance versus protein

concentration (Fig.17) yielded the following equations on

least square analysis

(O.D.)280nm = 1.203 (mg protein ) + 0.034 ....(4)

(O.D) 278nm = 1.201_(mg protein) + 0.034 (5)

The absorbance of 1% protein solution at the above

wavelength was computed from the equation (4) and (5)

respectively. The value of E IX thus determined was 12.37

- 1 - 1 - 1 - 1 (g/ml) cm at 280 nm and 12.35 (g/ml) cm at 278 nm.

E c o 00

a >.

•4->

c 0) "O

"o u

o

Protein concentration (mg)

FIG.17:CALIBRATI0N CURVE FOR THE ESTIMATION OF EXTINCTION COEFFICIENT OF PIGEON PEA LECTIN IN lOmM SODIUM PHOSPHATE BUFFER pH 7.0 CONTAINING 0.5 M NaCI .

D I S C U S S I O N

D I S C U S S I O N

Studies on inhibition of lectin mediated

agglutination of intact rabbit erythrocytes have fully

established that the pigeon pea lectin is glucose / mannose

specific. Accordingly It could be isolated by affinity

chromatography on IgM Sepharose -6B column whence from where

it was specifically eluted with 0.25 M glucose. The isolated

lectin was stable for two weeks In a refrigerator. It was

homogenous with respect to size as indicated by gel

filtration experiment. Chemical analysis showed that the

lectin contained 7% (w/w) neutral hexose. Gel filtration

behaviour of the lectin from a calibrated Bio gel P 200

column was consistent with a molecular weight of 38, 000.

The specific extinction coefficient of the lectin was

measured to be 12.35 (g /ml) am at 278 mm; the value

being the same at 280mm.

The lectin is fairly stable at room temperature but

it underwent thermal inactivation above 40 c. Thus the

lectin which was heated above 40 c for 30 minutes showed

substantial decrease in activity both in hemagglutnation

assay as well as in lectin mediated precipitation of IgM.

Below 40 c the increase In temperature from 20 c- 40 c had

no detectable effect on the carbohydrate binding activity of

lectin . The observation that the heat treated lectin did

not regain its full activity after cooling at noon

temperature for 24 hrs suggests the irreversible nature of

thermal inactivation.

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B I O G R A P H Y

Shama Siddiqui was born at Lucknow on December 19, 1965.

She passed High School Examination in 1981 from Carrael

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 is a member of the

Society of Biological Chemists (India).