ISOLATION AND CHARACTERIZATION OF RHIZOBIAL POLYSACCHARIDE RECEPTOR

(CAJANUS CAJAN LECTIN)

ft RIZWAN HASAN KHAN

INTERDISCIPLINARY BIOTECHNOLOGY UNIT ALIGARH MUSLIM UNIVERSITY. ALIQARH

Ditt

Approved:

A. Salahuddln (Supm/tor)

9 SifHertottfn <nbmttteb in partial (nlfilment of tfit reqairemenw tat t |e begcee o( i^aAer of $t)ilo«opJbp in liiotcc^nolegp,

of tfie flitgarli iKufItm Unibertfttp. SSUgarti 1991

2 8 JUN 1394

4^ . ^ ^

#

DS2347

C E R T I F I C A T E

I certify that the work presented in the following

pages has been carried out by Mr. Rizwan Hasan Khan and

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

Biotechnology of the Aligarh Muslim University, Aligarh.

V ( A. SALAHUDDIN )

PROFEBBOR 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

Rhizobium legume symbiosis, an attempt has been made to

isolate and characterize a lectin from a Cajanus cajan

seed. Cajanus cajan seeds were soaked in PBS and

homogenized. The homogenate was subsequently acidified

with 0.3M CH3COOH to pH4.0. The acid precipitated

extraneous protein was removed by centrifugation. The

clear solution containing lectin was purified by

affinity chromatography and/or by affinity precipitation

method. In affinity chromatography IgM-Sepharose 6B

affinity media was prepared by first activation

Sepharose with CNBr and subsequent coupling of activated

Sepharose with IgM. The solution containig lectin was

passed through IgM-Sepharose column and the bound lectin

was eluted with 0.25M glucose in PBS. Alternatively the

lectin was precipitated with IgM and the precipitate

disolved in 0.5M glucose. The dissociated lectin was gel

filtered on a Biogel P-200 column. The elution volume of

the lectin from Biogel P-200 column was 126 ml, which

was consistant with a molecular weight of 38,000, and

Stoke's radius of 2.62nm. Earlier it has been shown,

-iii-

that the lectin migrated as a single band on SDS-PAGE

.pa with a relative mobility consistant with a molecular

weight of around •16kD. This result taken together with

the gel filtration data on lectin seems to indicate that

lectin may be a dimer. Lectin from Cajanus cajan was

able to agglutinate rabbit erythrocytes. The

hemagglutinating activity was specificaly inhibited by

mannose and to a lesser extent by glucose, but it

remained uninflunced by galactose, suggesting that

lectin is mannose/glucose specific. Lectin absorbed

maximally near H78nm in the ultraviolet region. The

excitation and emission maxima of lectin in lOmM PBS

were found to be 278 and 326nm respectively. Lectin was

found to be a glycoprotein in nature. The lectin was

devoid of sialic acid. The ability of lectin to

precipitate IgM was not influenced by lin++ or lig++.

However lectin-IgM precipitation was significantly

inhibited by C3i++. Further work on the characterization

of lectin is in progress.

-IV-

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

I express my deep sense of gratitude to my

supervisor Prof. A. Salahuddin, Coordinator,

Interdisciplinary Biotechnology Unit, Aligarh Muslim

University, Aligarh. 1 am deeply indebted to him for

the monumental patience with which he has guided me

through the difficult and discouraging phases in the

fulfilment of this task.

I am thankful to Dr. Saad Tayyab for his help and

encouragement. I also thank Dr. Javed Mussarrat and

Dr. Arshad Rahman. 1 also take this opportunity to

thank my senior colleagues Dr. Parveen Salahuddin,

Dr. Najma Ali, Ms Seema Hasan, Ms Sadhna Sharma and

Ms Zoya Galzie for their valuable advice and academic

discussions.

I express my hearty thanks to my colleagues

namely Ms. Shama Siddiqui, Ms Sunita Warraich, Mr Aftab

Ahmad, Mr Mozaffarul Islam, Ms. Sakhra Shah, Mr B.T.

Ashok, Ms Seema Qamar and Ms. Sangeeta Saxena for their

help and cooperation during this study.

I also extend my thanks to the nonteaching

staff members Mr. Lai Mohammad khan, Mr Amir Ali, Mr

Syed Faisal Maqbool, Mr. Aqtedar Hussain Mr. Mohd Arif

for their cooperation.

-V-

1 am also grateful to the Council of

Scientific and Industrial Research for providing the

financial assistance in the form of J.R.F. during the

course of this study.

DATE : ^ijzj'^l (RIZWAN HASAN KHAN)

-VI-

C O N T E N T S

Page

ABSTRACT H i

ACKNOWLEDGEMENTS V

L I S T OF TABLES I X

L I S T OF FIGURES X

L I S T OF ABBREVATIONS X I I I

I. INTRODUCTION 1

II. EXPERIMENTAL 16

A. Materials 16

1.Proteins 16

2. Sugars 16

3. Chromatographic media 16

4. Miscellaneous reagents 16

B. Methods 17

•1. Measurment of pH 17

2. Optical measurements 17

a) Absorption measurements 17

b) Fluorescence measurements 17

3. Determination of lectin activity 18

(a) Hemagglutinating activity 18

<b) Lectin - IgM precipitin reaction 18

4. Determination of protein concentration 20

-vii-

5. Determination of neutral hexose content 22

6. Determination of sialic acid content 22

7. Gel chromatography 24

8. Isolation of lectin from Cajanus cajan 26

(I) Affinity chromatography on IgM-Sepha- 27 raose 6B column.

<II) Affinity precipitation method. 29

9. Effect of metal ions on the carbohydrate 29 binding property of the lectin.

III. RESULTS 30

1. Isolation of lectin 30

2. Optical properties 32

3. Hydrodynamic properties 35

4. Precipitin reaction 45

5. Effect of metal ions on carbohydrate 45 binding property of Cajanus cajan lectin.

IV. DISCUSSION 49

V. REFERENCES 50

VI. BIOGRAPHY 53

VII. LIST OF PRESENTATIONS 54

-Vlll-

L I S T O F T A B L E S

Table I: Origan and sugar specificity of purified

lectins from legume seeds.

Table 11: Various steps involved in purification of

Cajanus cajan lectin .

Tabel 111: Gel filtration data for marker proteins and

the lectin on Bio-gel P-200 column.

3-1

3<b

-ix-

L I S T O F F I G U R E S

Page No

Schematic representation of conA 11

tetramer binding sites.

Agglutination of cells by lectins.

Calibration curve for the

estimation of protein

concentration by the method of

Lowry et al . , •195-1 .

•13

•19

Calibration curve for the

estimation of carbohydrate

concentration by the method of

Dubois et al., 1956.

HI

Calibration curve for the

estimation of sialic acid

concentration by the method of

Warren et al., 1959.

23

Affinity chromatography of salt 28

-X-

fractionated fraction of Cajanus

cajan homogenate on IgM-Sepharose

6B column.

Ultraviolet absorption spectrum of

Cajanus cajan lectin in PBS on

double bean spectrophotometer

Fluorescence excitation and

emission spectra of Cajanus cajan

lectin in PBS.

33

34

Elution profile of Blue Dextran on

Biogel P-200 column.

37

Elution profile of BSA, ovalbumin

and chymotrypsinogen A on Biogel

P-200 column

38

E l u t i o n p r o f i l e of m y o g l o b i n and

c y t o c h r o m e C on B i o g e l P - 2 0 0

COlumn.

39

- X I -

12 . E l u t i o n p r o f i l e of d e x t r o s e and 40

t y r o s i n e on B i o g e l P - 2 0 0 c o l u m n .

1 3 . C h r o m a t o g r a p h y of a f f i n i t y 41

p u r i f i e d l e c t i n on B i o g e l P - 2 0 0

c o l u m n .

14. Calibration curve for the 42

determination of molecular weight

of priotein by standard plot of

Ve/Vo vs Log M

15. Calibration curve for the 44

determination of Stokes radius of

protein by standard plot of Stokes

radius vs erf <1-kd).

16. Precipitin titration curve of 46

Cajanus cajan lectin with goat

IgM.

'17. Curve to show effect of metal ions 47

on Cajanus cajan lectin mediated

precipitation of goat IgM.

-xii-

L I S T O F A B B R E V I A T I O N S U S E D

Con A • C o n c a n v a l i n A

Ca ' Calcium

D-Gal/Gal '• D - Galactose

EDTA : Ethylene diamine tetra

acetic acid

Gal NAc ! N-Acetyl-D-Galactosamine

IgM ! Immunoglobulin M

mRNA : messenger- Ribonucleic

Acid

PHA : Phytohemagglutinin from

Red kidney Bean

PBS : Phosphate buffer saline

-xiii-

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

OCCURANCE

Lectins can be defined as a multivalent

carbohydrate binding proteins or glycoproteins of non­

immune origin which agglutinate cells and / or

precipitate glycocojugates (Liener et al., 1986).

Lectins are widely distributed among living organism :

in plants, fungi and bacteria; in vertebrates such as

snails, horse shoe crabs and sponges; in fish; and in

mammals. <Sharon and Lis, 1989; and Liener et al.,

1986). Lectins were first discovered in plant seeds.

They have also been identified and purified from roots,

stems, leaves and bark. (Etsler 1986, Mc Pherson et al.,

1987 and Hiroshi Harada et al., 1990). In general plant

tissue contain one type of lectin. However some plant

tissues may contain lectins with distinctly different

molecular and carbohydrate binding property.

ISOLATION AND PURIFICATION OF LECTIN

Isolation of lectin generally begins with a buffer

Table 1

Origin and Sugar specificity of purified lectins from legume

seeds

Specificity Suborder & tribe

Speci es

(I) Mannose/

(glucose)

Vi cieae

II) Galactose/

N-acetyl

galactosamine

Carageae

Diocleae

Hedysareae

Vi cieae

Trifoli eae

Sophoreae

Phaseoleae

Pisum sativum

Vicia cracca

Vicia faba

Lens culinaris

Lathyrus ochrus

Coragana arborescaus

Canavalia ensiformis

Dioclea grandiflora

Onobrychis viciifolia

Vicia villosa

Vicia cracca

Medicago sativa

Sophora japonica

Vigna radiata

Psophocarpus tetragonolobus

Glycine max

Phaseolus lunatus limensis

I l l ) N - a c e t y l

g lucosamin t

(IV) L fucose

Caesalpinaceae

Caesalpinacese

Geni steae

Genisteae

Loteae

Amphicarpa hracteals.

Erythrina corallodendron

Dolichos bifloru5

Bauhinia purpurea

Griffonia simplicifolia

Griffonia simplicifolia

Cytisus sessifolius

Ulex europaeus.

Ulex europaeus

Lotus tetragonolobus

-4-

extraction of finely ground seed meal. Virtually all

lectin purification protocols employ affinity

chromatography based on the ability of lectins to bind

sugar specifically and reversibly. A few examples of

adsorbent utilized for affinity chromatography are

Sepharose <polymer of D galactose), Sephadex (polymer

of D- gulcose), or chitin (polymer of (i, 1-4 linked

N-acetyl D-glucosamine). In many cases, the purified

lectin do not occur singly, but as a group of closely

related proteins, called isolectins, with similar

biochemical and biological properties.

.Sugars specificities of some of the legume lectin 3.re

summarized in Table I.

PHYSICOCHEMICAL PROPERTIES OF LECTIN

<a) Molecular weight and subunit structure

The molecular weight of plant lectins varies from

20kD to 269 kD (Allen, 1979 & Galbraith A Goldstein,

1972). The plant lectins contain subunit structure.

-5-

Most subunits are identical but plant lectins may

contain nonidentical subunits. Each subunit may have

identical sugar binding specificities. Thus concanavalin

A possesses four binding sites per tetramer (Goldstein

et al., 1986) This is also true for soybean agglutinin

and pea nut agglutinin. (Neurohr et al., 1980 and De

Boeck 1964). In contrast, the lentil lectin and fava

bean lectin are heterodimer containing one sugar

binding site per dimer. Wheat germ agglutinin possesses

two binding sites per subunit. Isolectins with the same

sugar binding specificity but different electrophoretic

mobility and association constant have been identified

in plants,

(b) AMINOACID COMPOSITION

The aminoacid composition of large number of plant

lectins is known. The distinguishing feature is the

presence of higher concentration of aspartic acid serine

and threonine. Together these residues may comprise as

much as 307. of the total aminoacid content. Plant lectin

usally have little or no sulfur containing aminoacids.

However wheat germ, potato and pokeweed lectin are

-6-

rich in cysteine containing between 12 to 20% of the

aminoacid residues.

< c) NON PROTEIN MOIETY

The legume lectins are glycoprotein

containing N-acetyle glucosamine, mannose, xylose and

fucose sugars. Although c«ncanavalin A does not contain

carbohydrate, it is synthysized as glycosylated

precursor which on maturation yield conA. Maturation

involves proteolytic removal of some aminoacids as well

as N-linked glycan from the glycosylated preceursor,

with the result mature conA is devoid of carbohydrate.

The carbohydrate moiety of legume lectins is not

required for its carbohydrate binding activity because

unglycosylated L-PHA, synthesized in E.coli had the same

leuckoagglutinating and mitogenic activity as the native

glycosylated lectin (Lis and Sharon, 1981 and Hoffman

1978).

(d) STRUCTURAL PROPERTIES

A number of legume lectin have been sequenced

-7-

either by chemical or by rriolecular genetic technique

(Sharon and Lis, 1990). These include concanavalin A

(Cunningham, 1975), and fava bean lectin (Hopp et al.

1982) and Erythrina corallodendron lectin (Adar et al..

1989) etc. Comparison of the sequences of legume lectins

establishes what we called circular homology. Inspite of

significant sequence homology between legume lectins

they may or may not show similar carbohydrate binding

properties. The carbohydrate binding residues in legume

lectins are poorly conserved. However, the residues

involved in metal binding are conserved in many legume

lectins. Structures of legume lectins have been solved

tiy X~''sy diffraction. In addition to concanavalin A,

high resolution ^-ray crystal lographi c data s.re

available for the lectins of pea and fava bean (Reeke

and Becker, 1988), and of Erythrina corallodendron,

(Sharon et al., 1989) as well as Griffonia

simiplicifolia lectin (Vondonselaar, et al., 1987,

Nikrad et al., 1989). Several other lectins have been

investigated only at low resolution. Availavle data on

crystal structures of legume lectins show common

structural features.

-8-

These lectins contain very little AC - helical

structures and most of the aminoacid from ft - pleated

structu re.

FUNCTIONAL PROPERTIES

Since the uniqueness of lectins relate to their

sugar - binding properties, precise knowledge of the

nature of their specific combining sites is important

to understand their reactivity with cell surface

receptors and with soluble glycoproteins and

glycolipids (Pereira and Kabat 1979). The carbohydrate

binding specificity of lectin (Sharon and Lis 1989

Goldstein and Poretz; 1986) is usally established by

hapten inhibition technique, in which different sugars

are tested for their ability to inhibit either

hemagglutination or glycoprotein (or polysaccharide)

precipitation by the lectin. The lectin combining site

is considered to be most complementary to the sugar

that inhibited at the lowest concentration. Most

lectins are inhibited by monosaccharides,

although some lectins bind only to oligosaccharide

(Lis and sharon 1986). In such oligosaccharides, the

-9-

monosaccharide for which the lectin is specific is

present, usually at the non-reducing end, although it

may also be located internally. Moreover, some lectins

react almost exclusively with oligosaccharides.

Oligosaccharides are flexible molecules that may assume

different shapes owing to the freedom of rotation

around the glycosidic bonds. Molecular modeling as well

as high resolution nuclear magnetic resonance studies

of oligosaccharides in solution, have shown that the

shape of an oligosaccharide dictate its ability to

combine with a lectin < Romans et al., 1987 and Carver

et al . , -1989). Most lectins interact with more than one

monosaccharide for instance, lectins that exhibit a

primary specificity for mannose also bind glucose to a

lesser extent N-acetyl D-glucosamine. Lectins have

sites that seems to vary in size from a lower

limit of a monosaccharide unit plus a glycosidic

-10-

linkage to that of at least a tetrasaccharide. Some

lectins are specific for only one anomeric

configuration, whereas others interact equally well

with both anomeric configuration. There Are no cases of

lectins specific for <5-glycosid i c linkage.

METAL REQUIREMENTS OF LECTINS

Most legume lectins contain metal ions. Generally,

2+ 2+ lectins contain Ca and Mn . The role of metal ions

has been investigated in detail for concanavalin A

(Kalb and Levitzki., 1968). Concanavalin A combines

with a saccharide primarily by hydrogen bonds, and also

by hydrophobic interaction and Vander Waals contacts

<Sharon and Lis,1990). Treatment of concanavalin A with

acid reversibly removes the metal ions and destroys the

ability to bind carbohydrate. To convert it to an

active form, the metal ions must be added in a

2+ prescribed order : first Mn (or another divalent

P+ transition metal ions) and then Ca . <Fig. 1).

FIG.l: SCHEI>IATIC REPRESENTATION OF CON A TETRAMER Ca, Mn AND S INDICATE THE POSITIONS OF THE Ca2+ , Mn2+ AND CARBOHYDRATE-SPECIFIC BINDING SITES RESPECTIVELY. I LOCALIZES THE POSITION OF THE HYDROPHOBIC BINDING SITE PRESENT IN CRYSTALS OF THE 1222 SPACE GROUP (BECKER et.al 1976).

-12-

HYDROPHOBIC BINDING SITES

Legume lectins frequently possess three types of

hydrophobic binding sites. One of these is adjacwnl to

the carbohydrate binding sites, as evidenced by the

finding that hydrophobic glycosides, or other

hydrophobic derivatives of monosaccharides, bind more

strongly (upto 10 to 50 fold ) to the lectin than the

analogous nonhyd rophobi c compound (Sharon and Li?i.,

1989? Goldstein and Poretse, 1986) Legume lectins also

contain hydrophobic binding site that are remote from

the carbohydrate binding site. Some of these nydrophohir

compounds are phytohormones, which are believed lo play

an important role in plant growth.

AGGLUTINATING PROPERTY

The ability to agglutinate cells distinguishes

lectins from other sugar binding macromolecules such as

gycosidases and glycosyltransferases . For agglutination

to occur the bound lectin must form multiple cross

— • Glycocongugote receptor

£ 3 Lectin

FIG. 2 .: AGGLUTINATION OF CELLS BY LECTINS.

-Mr-

bridge between opposing cells (Fig.2). Cases are even

known where considerable amount of lectins are bound to

cells without causing agglutination. Agglutination is

affected by many factors, such as molecular properties

of lectin (number of saccharide binding sites, molecular

size), cell surface properties (number and accessibility

of receptor sites, memberane fluidity ) and metabolic

state of cells.

BIOSYNTHESIS

Plant lectin are synthesized in nearly the same

way as animal glycoproteins. Lectins are synthesized in

the form of prolectins in the endoplasmic reticulum,

were the leader (or signal) sequence, containing EO-30

aminoacids, is removed. Co-translational modification of

the carbohyrate units takes place in the golgi apparatus

(Chrispeels and Tague; 1990). Frequently post

translational proteolytic cleavage occur as woll. Cross

- hybridization of lectin cDNA was observed (Imbrie et

al., 1989, and Kaminski et al.,1986), strengthening the

idea that the legume lectin genes evolved from a common

-15-

ancestor (Foriers et al., 1977). It has also been found

that although concanvalin A is not a glycoprotein. It

is synthesized as a glycosylated precursor <Herman et

al., -1965). There is a high degree of homology between

the mRNA coding regions of the two genes, suggesting

that they are derived from the duplication of an

ancestral gene. Multiple lectins genes have also been

found in several other plants.

OBJECTIVE

There are several lines of evidence which

suggest the involvement of lectin in specific

recognition of Rhizobium by legume root. Cajanus cajan

is one of the important legume crop of India. It was,

therefore, thought desirable to isolate and

characterize lectin from Cajanus cajan seed.

EXPERIMENTAL

M A T E R I A L S

P r o t e i n s

Proteins used in this study were obtained from Sigma

Chemical Company, USA. The names of the proteins are:

bovine serum albumin ; ovalbumin ? chymotrypsinogen A;

trypsin (lot No 1-2395); myoglobin; and cytochrome C.

Sugars

Sugars used in these study weres dextrose; galactose;

lactose; mannose; and N-acetylneuraminic acid.

Chromatographic media

Biogel P-200 was obtained from Bio-Rad lab Richmond

California U.S.A. Sepharose 66 was the product of Sigma

Chemical Co USA.

Miscellaneous reagents

Acetic acid glacial, ammonium sulphate. Blue

Dextran 2000, calcium chloride, chloroform , copper

sulphate, eyelohexanone, dimethyldichlorosilane,

glycine, manganese chloride, phenol, sodium

bicarbonate, sodium carbonate, sodium molybdate,

sulphuric acid and tris ( hydroxy methyl) amino methane

were of reagent grade.

M E T H O D S

Measurement of pH

pH measurements were made at room temperature on an

Elico-digital pH meter, model LI-ISS, in conjunction

with combination electrode type CL- 51. The pH meter

was standardized with 0.05M potassium hydrogen pthalate

buffer, pH 4.0 or with O.OIM sodoium tetraborate buffer,

pH 9.2.

Optical measurements!

i) Absorption measurements

Light absorption measurements in the

ultraviolet region were made on a single beam Cecil

spectrophotometer (model CE-E02) or on a Cecil double

beam spectrophotometre ( model CE - 594) using silica

cells of 1 cm pathlength. Light absorption measurements

were also made in the visible region on AIMIL

photochem-8 colorimeter.

ii Fluorescence measurements

Fluorescence measurements were made on a Shimadzu

spectrofluorophotometer model RF - 540 in conjunction

with a Shimadzu Data recorder, model DR-3 at room

temperature. The slit width was 10nm through out these

measurements.

-18-

Detarmination of lectin activity

a) Hemagglutinating activity

The hemagglutinating activity was determined by using

rabbit erythrocytes. Four millititres of blood in 0.5ml

of 2.87. EDTA in phosphate buffered saline was

centrifuged at 2000 rpm for 30 minutes. The supernatant

was discarded and the pellets were washed three times

with phosphate buffered saline.

For hemagglutinating activity O.i ml of rabbit

4 erythrocytes (100 X 10 cells) was taken on a glass

slide. To this was added 0.1ml of protein solution

(containing 50 - 1000 pg protein). Agglutination was

checked visually and the time for onset of

agglutination was recorded. The hemagglutinating

activity was measured both, in the presence and absence

of specific sugar.

b) Lectin - IgM precipitin reaction

Fixed amount of lectin <H5^g) was mixed with

varying amounts of goat IgM (90-720 fig). The final

volume was made upto 2ml with lOmM sodium phosphate

buffer pH 7.0 containing 0.5 NaCl and it was incubated

6 c o o c •4->

o >. ij) c •a

"o u Q.

o

0.03 0.06 0.09 0.12 0.15 0.18 0.21 0.24 0.27

Protein concentration (mg)

FIG.3: CALIBRATION CURVE FOR THE ESTIMATION OF PROTEIN CONCENTRATION BY THE METHOD OF LOWRY et. al., (1951). BSA WAS USED AS THE STANDARD PROTEIN. THE STRAIGHT LINE DRAV7N BY THE METHOD OF LEAST SQUARES F0LL0V7S THE EQUATION:-.

(O.D).,nn = 2.05 (mg protein) + 0.07. /uunm

-20-

at 30'C for 1 hour. The mixture was left for overnight

at room temperature. The precipitated protein was washed

four times with normal saline and finally the

precipitate was dissolved in 0.1M NaOH. Protein

concentration in the precipitate was determined by the

method of lowry et al . , (•195-1).

Determination of protein concentration

Protein concentration was routinely determined by the

method of Lowry et al., (WSI) using bovine serum

albumin as the standard protein to Iml of protein

solution, 5ml of freshly prepared copper reagent (4X

sodium carbonate (w/v), A'/, sodium potassium tartarate

(w/v) in the ratio of lOOM:!) was added. After 10

minutes incubation at room temperature, Iml of Folin-

phenol reagent was added to it and left for 30 minutes

at room temperature. The absorbance was read at 700nm

against the blank prepared in the same manner. A

straight line was obtained between optical density and

protein concentration by least square analysis <Fig 3)

and was found to fit the equation.

(O.D.)yQQ^jj^ = 2.05 X (mg protein) + 0.071 ( 1 )

E c o

a

io c (b

XJ

"o u

+-»

o

Dextrose{jjg)

FIG.4: CALIBRATION CURVE FOR THE ESTIMATION OF NEUTRAL HEXOSE BY THE METHOD OF DUBOIS et. al. , (1956). DEXTROSE WAS USED AS A STANDARD. THE STRAIGHT LINE DRAWN BY THE METHOD OF LEAST SQUARE FOLLOVrS THE EQUATION:- • •

;o.D) 490nm = 3.21 (mg dextrose) + 0.17.

-22-

Determination of neutral hexose content

The neutral hexose content of the lectin was

determined by the method of Dubois et at (1956) using

glucose as the standard sugar.

To Iml of lectin solution was added Iml of 5"/.(w/v)

phenol solution. Then 5ml of concentrated sulphuric acid

was added gently by broken tip,pipette. After 20 minutes

the colour intensity was measured at 490nm against an

appropriate blank. A curve was drawn between optical

density and glucose concentration. The straight line

shown in Fig 4 was found to obey in the equation:-

<0.D. ) 9Qj j = 0.00321 X (mg glucose)+ 0.-174 <2)

Determination of sialic acid content

Sialic acid content of the lectin was determined

by the method of Warren et al., (1959) using N-acetyl

neuraminic acid as standard sugar. To 0.2 ml o€ a

protein solution was added 0.1ml of 0.2M sodium

metaperiodate solution. The tubes ware shaken and

allowed to stand at room temperature for 20 minutes.

After addition of 1 ml of the arsenite solution, these

tubes were shaken until! the yellow- brown colour first

o

Q.

o

0.28-

0.24-

e c o

-M 0.20 -a >-

c T3 0.16-

0.12-

0.08

0.04-

4 5

Sialic acid(jjg)

FIG.5: CALIBRATION CURVE FOR THE ESTIMATION SIALIC ACID BY THE METHOD OF WARREN et.al.,{1959). N-ACETYL NEURAMINIC ACID WAS USED AS STANDARD. THE STRAIGHT LINE OBTAINED BY THE METHOD OF LEAST SQUARE FOLLOWS THE EQUATION:" (O.D.) 550 nm = 35.4 (milligram, sialic acid) +0.01.

-24-

formed disappears. This was followed by addition of Sml

of thiobarbutric acid reagent. The tubes were heated in

a boiling water bath for 15 minutes. After 5 minutes

cooling, 2ml of this cooled solution was added to an

equal volume of eyelohexanone. The clear cyclohexanone

phase was transferred to an appropriate cell and the

optical density was determined at550nm. The standard

curve is shown in Fig.5 and the straight line was found

to obey the following equation i-

(a.D.)550nm ~ ^^-^ ^ <"»g sialic acid)+ 0.012 (3)

Gel chromatography

Gel filtration experiments were performed on

columns of Sephacryl S-200 and Biogel P-200. Analytical

gel chromatography was performed on Bio—gel P-200

column for the determination of the hydrodynamic

parameters.

Following the method described by Ansari and

Salahuddin (1973) about 8 gm of Bio-gel P-200 was

allowed to swell in excess amount of water for 48hrs.

The fine particles present in the gel slurry were

removed by decantation. This process was repieated till

all the fines were removed. Before packing, a column of

uniform diameter was mounted in a vertical position with

-25-

the help of two clamps. The narrow bore outlet of the

column was connected with a small latex tubing attached

with a screw stop-cock for regulation of the flow

rate. A small amount of glass wool, previously boiled in

water, was placed at the bottom of the column with

the help of a glass rod. The slurry was poured into the

column in a single burst. A constant flow rate was

maintained by slowly increasing the hydrostatic head and

opeinig the stop-cock. The flow rate was finally

adjusted to about SOml/hr. In a similar manner Sephacryl

S-200 column was packed. The homogenity of the packing

of the column was checked by passing a band of a 0.27.

( w/v ) Blue Dextran 2000 solution. The elution

volume thus obtained gave the void volume of the column.

The column was calibrated using the following marker

proteins with their molecular weights given in

parentheses s BSA (68,000); ovalbumin (43,000);

chymotrpsinogen A ( 25,700 ); myoglobin ( 17,200 ); and

cytochrome c ( 11,700 ). The inner volume; Vi , of the

column was determined by passing glucose and

tyrosine. The total volume: Vti°^ column was

determined by eluting a 3cm height of water in the

column and by weighing it. The column was filled

with distilled water. Three pieces of graph paper

each of 3cm height were pasted at different places

-26-

along the length of the column. The volume of water

corresponding to 3cm height was collected in preweighed

weighing bottles and its weight was determined. The

volume of water, v, is related to its weight, (MI, ^nd

density,d, by the equation.

v = w / d = TTr 1

Where r is the radius of the column in centimeters and 1

is the length of graph paper strip <i.e. 3cm). The

average of three weights of water corresponding to the

three place along the column length was found to be

13.529 gms. The density of water at 19'C was taken to

be 0.9973, With these value, the volume of *•;»*,r

3 collected was found to be 13.5469cm Thus, the d i ;• m pt e r

of the gel filtration column was 2.4cm.

Isolation of lectin from Cajanus cajan

Twenty grams of Cajanus cajan pluse were soaked in 100ml

of 10mM sodium phosphate buffer pH 7.0 with 0.5M NaCl

(PBS), containing 0.027. sodium azide. The soaked pulse

was homogenized in a blender for 5 minutes. The

homogenate was filtered through muslin cloth. The pH of

the filtrate was lowered to pH 4.0 by addition of 0.3M

-27-

acetic acid. The acid precipitate protein was removed

by centrifugation. The supernatant showing positive

hemagglutinating activity was made 307. with respect to

ammonium sulphate and the salt concentration was raised

to 50%. The protein thus precipitated was dissolved in

minimal volume of PBS and dialyzed extensively against

PBS, pH 7.0, to remove ammonium sulphate. The dialyzate

was tested for the absence of precipitation with a

solution of barium chloride. The salt fractionated

lectin was further purified either by affinity

precipitation or by affinity chromatography.

(i) Affinity chromatography on IgM - Sepharose 6B column

a) Preparation of affinity column

IgM Sepharose 6B column was prepared by t ^ method

of Cuatrecasas (1970). Twenty millilitres of Sepharose

6B gel was first washed with distilled water and then

suspeneded for overnight in normal saline. To decrease

the temperature, ice cubes prepared from distilled

water were added to the gel. Then 3.3gm (330mg/ml) of

solid cyanogen bromide was added and the pH was

adjusted to 11 by adding 4M NaOH. The activated gel

was washed immediately with chilled distilled water

followed by chilled 0.2M sodium carbonate buffer, pH

9.2 containing 0.15m NaCl. IgM (145 mgs) in carbonate

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-29-

buffer was mixed with activated matrix and left for

overnight at 7*C. Gel was washed several times with

carbonate buffer. The gel was incubated with 0.2M

sodium carbonate buffer, pH 9.H containing 0.2M glycine

for two hours, to block the free activated groups. The

affinity matrix was packed in a glass column and

equilibrated with the operating buffer.

b) Affinity Chromatography

About 32 mgs of salt fractioned protein was applied on

IgM - Sepharose 6B column. The column was washed with

the operating buffer. After washing the column three

times its volume, the bound protein was specifically

eluted with 0.25M glucose as shown in Fig 6.

II Affinity Precipitaion method

In affinity precipitaion method, lectin

was specifically precipitated with goat IgM. The

precipitate was dissloved in 0.5 M glucose, and thus

the clear supernatant obtained was passed on Bio-gel

P-200 column as shown in Fig 13.

Effect of metal ions on the carbohydrate binding

property of the lectin

Effects of metal ions on the Cajanus cajan lectin was

investigated by precipitin reaction. Gel filtered

lectin <50 g) and IgM (270 pg) were taken in Tris HCL

buffer pH 7.4 with CaClgr MgClg and MnClg- The protein

content of precipitate waas determined by Lowry's

method.

RESUL TS

R E S U L T S

Out of the twenty different samples of Cajanus cajan

pulse, positive hemagglutinating activity against

untrypsinized rabbit erythrocytes was observed only with

two samples of the pulse. These were used for the

isolation of lectin from Cajanus cajan. When the pulse

homogenate in PBS pH 7.0 was incubated for overnight at

7'C, 27.5"C and 37°C, the hemagglutinating activity

against unmodified rabbit erythrocyte was found to be

independent of temperature. The hemagglutinating

activity was specifically inhibited by glucose, N-

acetyl- glucosamine and mannose; the latter being most

potent inhibitor. Galactose and lactose had virtually no

effect on lectin mediated hemagglutinating activity.

Isolation of lectin

Different steps in isolation and purification of lectin

Are listed in Table II. Out of twenty grams of pulse 600

milligrams of protein was obtained in the acid

homogenate; nearly 33% of the extraneous protein was

precipitated with 507. ammonium sulphate, and the

precipitated protein showed hemagglutination activity

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-32-

against rabbit erythrocytes. The salt fractionated

lectin was further purified by affinity precipitation

with goat IgM followed by gel filtration of the

dissociated precipitate on Bio-gel P-EOO column. About

6.67. purified lectin was thus obtained. When the salt

fractionated lectin was purified by affinity

chromatography on IgM- Sepharose 6B column the lectin

yield was 3.3*/. (see Table I D .

Optical properties

The native lectin from Cajanus cajan absorbed maximally

near 278 nm as shown in Fig 7.. The excitation and

emission spectra of lectin were measured and the result

are depicted in Fig 8, where it can be seen that maxima

in excitation and emission spectra of the lectin

occured near 278, and 326nm respectively. The

carbohydrate content of lectin determined by Dubois

method was low. No sialic acid could be detected in

lectin by Warren method (Warren., -1959).

0.45

O.AO

u C

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0.35-

240 260 280 300

Wavelength (nm)

320

FIG. 7 :ULTRA VIOLET ABSORPTION SPECTRUM OF CAJANUS CAJAN LECTIN . THE SOLUTION CONTAINS 0.30 mg/ml OF LECTIN IN 10 luM SODIUM PHOSPHATE BUFFER, pH 7.0, CONTAINING 0.5 M NaCl.

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-35-

Hydrodynamic properties

Hydrodynamic properties of lectin were determined by

analytical gel chromatography on a Bio-gel P-200

column(54X2.4cm) in 10mM sodium phosphate buffer and

0.027. sodium azide. A band of Blue Dextran was passed

through the column four times during the gel filtration

experiments to ensure uniform packing as well as to

determine the void volume. Vo, of the column. The void

volume of the column was found to be 76ml. The inner

volume, Vi, of the gel was determind from the elution

volume of tyrosine and glucose for which the value of k.

is 1. The value of V^ thus determind was 236ml. The

total vloume, V^, was found to be 254ml. The marker

proteind listed in Table III were gel filtered under

identical conditions. The elution profiles are shown in

Figs 9-13.

A plot of Ve/Vo versus log M, was obtained according to

Andrews (1970). The straight line shown in Fig.14 fits

the equation :-

Ve/Vo = -0.978 LogM + 6.14 (4)

The ratio Ve/Vo for Cajanus cajan lectin was 1.66, which

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Elution volume (ml) FIG. 9 :ELUTION PROFILE OF BLUE DEXTRAN ON A BIO - GEL P-200

COLUMN (2.4 X 54 Cms) EQUILBRATED IN 10 mM SODIUM PHOSPHATE BUFFER CONTAINING 0.5 M NaCl.

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FIG. 11. ELUTION PROFILES OF (1) MYOGLOBIN AND (2) CYTOCHROME C ON THE BIO-GEL P-200 COLUMN EQUILIBRATED IN 10m MPBS pH 7.0.

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FIG. 13: CHROMATOGRAPHY OF AFFINITY PRECIPITATED LECTIN ON THE BIO-GEL P-200 COLUMN (2.4 x 54 Cm). ABOUT 25 mg PRECIPITATED LECTIN WAS APPLIED ON THE COLUMN EQUILIBRATED WITH 10 mM SODIUM PHOSPHATE BUFFER pH 7.0 CONTAINING 0. 5M NaCl. PROTEIN WAS ELUTED IN 3 ml FRACTIONS AT A FLOW RATE OF 20 ml/hr. THE PROTEIN CONCENTRATION V?AS DETERMINED BY LOWRY' S METHOD, (1951).

3 >

Log molecular weight

FIG. 14: PLOT OF GEL FILTRATION DATA ACCORDING TO ANDREV7S (1970). FOR CALIBRATION. THE MARKER PROTEINS USED WERE (1) BSA (M) (2) OVALBUMIN (3) CHYMOTRYPSINOGEN A (4) MYOGLOBIN AND (5) CYTOCHROME C. THE STRAIGHT LINE FITS THE EQUATION:V

V / VQ = -0.9781ogM + 6.14

-43-

according to equation 4, would correspond to a molecular

weight of 38000. The gel filtration data were analyzed

according to Ackers (1967). A linear curve between

stokes radius, a, and erf (l-k^j) was obtained by the

method of least squares. The stright line shown in Fig.

15 fits the equation :-

a(n^) = 3.45 erf"^ <1-Kd^ " •"'' <^^

The value of erf (l-kj) for the lectin was computed to

be 0.706 which according to equation 5 would correspond

to stokes radius of 2-62nm.

It is possible to calculate the diffusion coefficient

D, and frictional ratio; f/fo, of the lectin with the

help of following equations.

D = K T / 6 7 t n a (6 )

f / f o = a / (3 Mv7/4? j r N) " - ( 7 )

Where n is the coefficient of viscosity of the buffer

which was measured to be 0.0083 poise at 32'C. The

partial specific volume was taken to be 0.7E5ml/gm. K is

the Boltzman constant and is 1.386 X 10~ ergs per

degree and N is Avogadro's number (6.02 X 10 per

mole). With the stokes radius of 2.62nm and molecular

weight of 38,000 the values of D and f/fo for the lectin

were determined to be 1.008 X lO'^^cm^/sec and 1.23,

respectively.

E c

o \_ W Of

o CO

0.6 0.7

erf~''(1-kd]

FIG. 15 : CALIBRATION CURVE FOR THE DETERI IINATION OF STOCKES RADIUS OF PROTEINS. (ACKERS, 1970)MARKER PROTEINS USED WERE (1) BSA (M) (2) OVALBUMIN (3) CHYMOTRY-PSINOGEN A (4) MYOGLOBIN AND (5) CYTOCHROME C. THE STRAIGHT LINE FITS THE EQUATION :-

(nm) = 3.45 erf -1 (1-k^) + 0.19.

-45-

Precipitin reaction

For precipitin rection fixed amount .of gel purified

lectin (SO^g) was mixed with varying amounts of IgM (90-

720 uq). A precipitin curve thus obtain showed maximun

precipitation at l!6 ratio as can be seen in Fig 16.

Effect of metal ion on carbohydrate binding by

Cajanu* cajan lectin.

The effect of MnClg, ligClg and CaClg on the interaction

of Cajanus cajan lectin with IgM was investigated in

•10mM Tris HC1 buffer, pH 7. A, and at room

temperature i.e. 32"C. When 50 micrograms of the lectin

was incubated with IgM in Tris HC1 buffer at a ratio of

5.4 by weight for overnight, precipitation took place.

The protein content of the precipitate was determind by

Lowrys method was O.H5mg/ml. On incubation of lectin

with IgM in the presence of increasing concentrations of

MnClg from 2.5mM to 15mM, the amounts of IgM

precipitated by the lectin was the same i.e. 0.25mg/ml.

Identical results were obtained In the presence of

increasing concentrations of MgClg from 2.5 to 15mM.

Howere, in the presence of CaClg (2.5--13mM) the amount

of IgM precipitated by the lectin decereased by 127,.

O

0;

o. c

o a.

6 0 0 -

5 0 0 -

4 0 0 -

3 0 0 -

2 0 0 -

3 4 5 6 7 8

Ratio of lectin to IgM

FIG. 16 : IN 2 ml OF 10 mM PBS pH 7.0 CONTAINING 0.5 M Nacl WERE TAKEN 90 TO 720 ug OF IgM AND FIXED AMOUNT OF 80 ug LECTIN. THE MIXTURE WAS INCUBATED OVER NIGHT AND THE PRECIPITATED PROTEIN WERE COLLECTED BY CENTRIFUGATION AND ANALYSED FOR PROTEIN CONTENT BY THE METHOD OF LOWRY et. al., (1951) .

0; • • - •

O

o

n.

=1

2^0

230

220

210-

2.5 5 7.5 10 12.5 15

Concentration of metal ions,mM

FIG. 17 : EFFECT OF METAL IONS ON CARBOHY­DRATE BINDING PROPERTIES OF CAJANUS CAJAN LECTIN. IN 2ml OF lOmM Tris HCI BUFFER pH 7.4 CONTAINING 0.5 M NaCl WERE TAKEN 270 ug IgM, 50 ug OF LECTIN AND VARYING CONCENTRATION OF METAL IONS (2.5 TO 15mM) THE PRECIPITATED PROTEIN WERE COLLECTED BY CENTRIFUGATION AND ANALYZED FOR PROTEIN CONTENT BY THE METHOD OF LOWRY et. al., (1951).

-A6-

These result suggest a small but significant inhibitory

effect of Ca on the precipitation of IgM by Cajanus

cajan lectin (see Fig 17).

DISCUSSION

D I S C U S S I O N

The lectin from Cajanus cajan pulse obtained by

specific affinity precipitation with goat IgM was

homogenous with respect to size as indicated by a

single symmetrical peak on a Bio gel P-200 column. The

elution profile of the lectin from Cajanus cajan on a

Bio gel P-200 column was consistant with a molecular

weight of 38,000. Optical properties of purified lectin

suggested the presence of tryptophan residues in the

lectin. The Cajanus cajan lectin was found to be a

glycoprotein.The frictional ratio of the native protein

molecule was determined to be 1.23, which is

significantly higher for a globular protein. It is

possible that the lectin may exist in an assymetric

conformation. The lectin from Cajanus cajan was found

to be mannose/glucose specific and was able to

precipitate out IgM. The lectin induced precipitation

.2+ was adversly influenced by Ca

2+ 2+ uninfluenced by Mg or lin

but remained

R E F E R E N C E S

Ackers, G. K. (1967) J.Biol. Chem. 242, 3237-3238

Adar, R., Richardson, M., Lis, H. and Sharon, N. (1989) FEBS Lett. 257, 81-85.

Allen, A.K. (1979) Biochem. J., 183, 133-137

Andrvews, P. (1970) in "Methods of Biochemical Analysis" (Click, D., ed.), John Wiley and Sons, New York, Vol 18, pp 1-53.

Ansari, A.A. and Salahuddin, A.(1973). Biochem. J.135, 705-711.

Ashford, D, Desai, N.N., Allen, A.K., Neuberger, A., O'Neill, M.A. and Selvendran, R.R. (1982) Biochem. J.201, 57.

Cautrecasas, p. (1970).J. Biol. Chem. 245, 3059-3065

Chrispeels, M.J., and Tague, B.W. (1990) Recent Advances in Development and Germination of seeds (Taylorson, R.B.ed.), Plenum, New York.

Cunningham, B.A. (1975) Pure Appl . Chem. 41,, 31-34.

De Boeck, H., Lis, H., Van. Tilbeurgh, H., Sharon, N. and Loontiens, F.G. (1984) J. Biol. Chem. 259, 7067-7074.

Dubois, M., Gilles, A.K., Hamilton, J.K., Rebers, P.A. and Smith, F.(1956) Anal. Chem. 28, 350-356.

Etzler, M.E. (1986) in "The Lectins"; Properties Functions and Application in Biology and Medicine pp 371-435, Academic Press, Orlando, Fla.

Galbraith, W. and Goldstein, I.J. (1972) Biochemistry 11, 3976-3984.

Goldstein, I.J. and Hayes, C.E. (1978) Adv. Carbohydrate, Chem. Biochem. 35, 127-340.

-51-

Goldstein, I.J. and Porets, R.D. (1986). In "The Lectins" (Liener, I.E., Sharon,N. and Goldstein, I. J.,eds), Academic Press, Orlando, Florida, pp 33-243. Hasan, S. (1990) Ph.D. thesis.

Hoffman, L.M., and Donaldson, D.D. (1985) EMBO. 4, 883-889.

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Kalb, A.J. and Levitski, A. (1968) Biochem. J. 109,669-680

Khan, li.I. ( 198C ) Ph.D. thesis,

Liener,I.E., Sharon, N., and Goldstein, I.J. (1986). In "The Lectins" Properties, Functions and Application in Biology and Medicine". Academic Press. Inc. Orlando, Florida.

Lis, H. and Sharon, N. (1984) in Biology of Carbohydrates (Ginsburg, V. and Robbins, P.W. eds). Vol2 pp 1-85, Wiley Inter Science, New York.

Lis, H. and Sharon, IM. , (1981). In "Biochemistry of Plants". (Marcus, A. ed), Academic Press, New York, Vol 6,pp 371-447.

Lowry, O.H., Rose brough, N.J.,Farr, A.K. and Randall,R.J. (1951) J. Biol.Chem. 193, 265-275.

Maliarik, M. , Plessas, N.R., Goldstein-, J.J., Musci, G. and Barliner, L-J. (1989) Biochemistry 28, 912-917.

Nachbar, M.S., Oppenheim, J.D.and Thomas, J.O. (1980) J.Biol. Chem. 255, 2056-2061.

Neurohr, K.J., Young, N.M. and Mantsch,H.H.(1980). J.Biol.Chem.255,9205-9209

Reeke,G.N.Jr.,and Becker,J.W.(1988).Curr.Top.Microbiol. Immunol.139,35-58.

Sharon, N., and Lis, H. (1990) FASED, J.4, 3198-3208.

Sharon, N., and Lis,H (1989) Science 246, 227-234.

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Vandonselaar, h., Del. baeve, L. T. J., Spohr, V., and

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B I O G R A P H Y

Rizwan Hasan was born at Partapgarh on December 25,

1965. He passed his High school Examiniation WSI from

A.l.l.C. Lucknow. He recevied his B.Sc degree in 1986

from Lucknow University and M.Sc degree in Biotechnology

in 1989 from Aligarh Muslim University, Aligarh. He was

enrolled as MPhil student in November, 1989 in the

Interdisciplinary Biotechnolgy uit, Aligarh Muslim

University, Aligarh, He is a member of Society of

Biological chemists <India).

L I S T O F P R E S E N T A T I O N S

"Influence of metal ions on the activity of

mannose specific lectin from Cajanus cajan"

R.H.KHAN.

Proc. Diamond. Jubilee Ann. Gen. Body

Meeting Soc. Biol. Chemists (India) held at

Calcutta on December E6-30, 1991.