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A. SEN'. Department of Chemistry, Bose Institute, 9$/l Acharya Prqfulla Chandra Road, Calcutta 700 009, India ... DAS, AND SEN .... dog, horse, guinea pig,.
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 1, October 1, pp. 459-470, 1981

Purification and Properties of D-Galactose-Binding Lectins from Some Erythrina Species: Comparison of Properties of Lectins from E. Indica, E. Arborescens, E. Suberosa, and E. Lithosperma LOKESH Department

BHATTACHARYYA,

PRADIP

KUMAR

of Chemistry, Bose Institute, 9$/l Acharya Prqfulla Received

March

DAS,

AND

A. SEN’

Chandra Road, Calcutta 700 009, India

4, 1981

The lectins of the seeds of four species of the genus Elythrina, namely E. indica, E. arborescens, E. lithospe-rma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,600 and 33,006 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. or&rosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4-9%. In carbohydrate specificity all the lectins were Dgalactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. sub-rosa lectins showed higher activity toward the 0 group and E. arbwescens toward the B group. The results show the similarity of’ the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.

Lectins which are cell-agglutinating and carbohydrate-binding proteins occur in many plants, particularly legumes (1, 2) and also in bacteria, fungi, and animals (3). Owing to considerable specificity of their properties, they have found wide application in cell biology, serology, biochemical separation techniques, and other fields. A large number of lectins of plant and other origins have been identified and purified to homogeneity and studied in regard to their physicochemical properties ’ To whom all correspondence should be sent.

and reprint

requests

and carbohydrate specificity. Goldstein and Hayes (3) in a recent review have reported the properties of various lectins derived from plant and other sources, among which 25 have been classified according to their well-established carbohydrate specificity with 5 of them being D-galactose specific. In our survey (4) on lectins in Indian leguminous plants, we found that seed extracts of some of the species of the genus Ergthrina which are found throughout India and are botanically well characterized (5), nonspecifically agglutinated human erythrocytes. Although MakeRi (1) in 459

0003-9861/81/110459-12$02.00/O Copyright All rights

0 1981 hy Academic Press. Inc. of reproduction in any form reserved.

460

BHATTACHARYYA,

his studies on lectins from leguminous plants observed that the seed extracts of a number of species of a given genus showed hemagglutinating activity, results so far available on purified lectins from different species of a particular genus are not enough to indicate any correlation or otherwise between the taxonomic relatedness of the species and the carbohydrate specificity or immunologic properties of the lectins derived from them. In this communication, we describe the purification and properties of lectins isolated from four Erythrina species, namely, Erythrina indica Lamm. (Syn. E variegata Linn.), Erythrina arborescens Roxb., Erythrina lithosperma Blume (Syn. E. subambrans (Hassk) Merr.), and Erythrina suberosa Roxb. These lectins, particularly E. indica lectin, have been studied in respect of their physicochemical, carbohydrate binding and immunochemical properties and compared. While this work was in progress, Horejsi et al. (6) have reported some of the properties of the E. indica lectin. MATERIALS

AND

METHODS

Materials. The seeds of E. indica Lamm., E. arborescens Roxb., E. lithosperma Blume, and E. suberosa Roxb. were purchased from local suppliers. Sepharose 6B was purchased from Pharmacia and lactose from BDH Laboratory Chemicals Division. Ampholine solutions of different pH ranges were products of LKB Produckter. Ultrapure sucrose was purchased from Mann Research Laboratory. Ovalbumin, a-chymotrypsinogen A, D-galactose, and other simple sugars were products of Sigma Chemical Company, and pepsin and trypsin of Worthington Biochemical Corporation. Cow &lactoglobulin B and Abrus precattius lectin were prepared in this laboratory. [1-“C]Lactose (55.7 nCi/mmol) was obtained from the Radiochemical Centre. All other reagents were of analytical grade and used as received. Human blood of different groups was obtained from the Metropolitan Blood Bank, Calcutta. Blood of different animals was obtained from Bengal Veterinary College Hospital and Cholera Research Centre, Calcutta. Preparation of acid-treated EC@-Se&arose 6B. z Abbreviations used: ECD-Sepharose 6B, ethylene chlorohydrin cross linked desulfated Sepharose 6B, SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetate (disodium salt); 2-Me, 2-mercaptoethanol; FDNB, 1-fluoro-2,4-dinitrobenzene.

DAS,

AND

SEN

This was prepared scribed by Porath

according

to the

procedure

de-

et al. (7) and Ersson et al. (8). Isolation and purification of leetins of Ergthrina species, The lectins of seeds of E. indica, E. arborestens, E. lithosperma, and E. at&rosa were extracted and purified essentially by the same procedure as described below for E. indica lectin and the fractions were designated in the same manner. All experiments were done at 2-4°C unless stated otherwise. Two hundred grams of finely divided seed kernels of E. indica were extracted in 1 liter of 5 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl (Buffer A) for 20 h. The suspension was filtered through cheese cloth and the residue reextracted with another 0.5 liter for 2 h. The combined filtrate was centrifuged at 10,000 rpm for 15 min and the supernatant was adjusted to pH 4.5 by dropwise addition of 2 N HCl with constant stirring. The precipitate formed was centrifuged off and the supernatant (fraction A) was saturated to 70% with (NH4)zS0,. The precipitate (fraction B) was dissolved in and dialyzed against Buffer A. Any undissolved residue was centrifuged off.

qffinity chromatography

012acid-treated ECD-Se-

pharose 6B. The affinity chromatography of fraction B was done at pH 7 at 2-4’C (for details of column size, buffer, etc., see legends to relevant figures). The fractions obtained during washing with Buffer A were discarded. Those eluted with 0.1 M lactose in Buffer A in a single peak (fraction C) contained the purified lectin, which were pooled and precipitated at 70% (NH4)zSOI saturation. The precipitate was either dissolved in Buffer A and stored frozen or an exhaustively dialyzed aqueous solution was freezedried. Protein concentrations. These were measured for the purified E. indica lectin by using the value of $,“, = 13.4 dl.g-’ at 280 nm. The value was determined with solutions of the protein in Buffer A, the concentrations of which were determined by drying at 108-110°C after making appropriate corrections for salt content. The concentrations of extracts, different fractions, and other purified lectins were determined according to Lowry et al. (9). Polyacr&xmide gel electrophoresis. This was carried out at pH 4.5 (10) and 8.3 (11).

SDS-polyacrylamide This was done Osborn (12).

in 7.5%

Immunodiffusion

gel (SDS-gel) electrophoresis. gel according

and

to Weber

and

immunoelectrophoresis.

These were done according to Hammarstrom and Kabat (13) with the antiserum to the purified lectin obtained by immunizing two rabbits subcutaneously with 2 mg of the protein emulsified in Freund’s complete adjuvant and a subsequent dose of 2 mg after 6 weeks. Animals were bled after 15 days. Determination of partial specific volume. This was done (14) using a 50-ml capacity pycnometer at 25.3”

D-GALACTOSE-BINDING + 0.05”C using a 2.15% stock solution of the protein which was diluted to five different concentrations. Measurements were made in 0.05 M sodium phosphate buffer containing 0.1 M NaCl, pH 6.7, ionic strength 0.2 (Buffer B). Vltracentr@& studies. Sedimentation velocity experiments were carried out in Spinco Model E analytical ultracentrifuge. The value of diffusion coefficient was measured from boundary spreading using a synthetic boundary cell at 8’7’76 rpm (15). Measurements of the patterns were done according to Ghose et al. (16). An Archibald experiment was done according to Schachman (15). Amino acid composition. Amino acid analyses were performed in a Beckman Multichrom liquid column chromatograph (Beckman Instruments, Munich, West Germany), according to Spackman et al. (17). Weighed samples of freeze-dried protein corrected for moisture content were hydrolyzed with 6 N HCl at 110°C for 24, 48, and 72 h in sealed evacuated tubes. Duplicate analyses were done with each hydrolysate. Tryptophan was estimated spectrophotometrically (18). N-Terminal amino acids were determined by FDNB method (19). Determination of neutral and amino sugars. Neutral sugar content was determined by the phenolsulfuric acid method (20) with D-glucose as standard. Amino sugars were determined according to Spiro (21) in the amino acid analyzer with samples (corrected for moisture content) hydrolyzed for 6 h with 4 N HCl in sealed evacuated tubes. Agglutination and sugar inhibition assays. These (22) were done at 25°C with 3% suspensions of human red blood cells of A, B, and 0 groups and of rabbit, sheep, cow, buffalo, dog, horse, guinea pig, duck, and hen. Some of the assays were done after trypsinization (23) of red cells. The inhibition assays (22) with simple sugars were carried out with human red cells of A, B. and 0 groups. Isoelectric focusing. This was done in the pH range 4-6 in a LKB Model 8101,110-ml electrofocusing column (LKB-Produkter AB) at 4°C according to manufacturer’s instructions. Ultrapure sucrose was used for preparation of the density gradient. Eight milligrams of the lectin was applied to the column. One and a half-milliliter fractions were collected and the pH of each fraction was determined in a Radiometer pH meter, PHM 26. Equilibrium dialyses. Pieces of Visking dialysis casing (8/32”) of 70-mm length boiled in 1% NaHCOs0.1% EDTA (disodium salt) and washed exhaustively with distilled water were tied to one end, inflated with nitrogen, and dried by hanging vertically (24). One milliliter of E. indica lectin solution equilibrated against 0.05 M phosphate-O.5 M NaCl buffer, pH 7.0, was taken in each casing. These were placed in Corning tubes (10 X 80 mm) containing 1 ml of the buffer and 1-15 ~1 of stock sugar solution which was pre-

LECTIN

461

PURIFICATION

pared by dissolving [1-“Cllactose in the same buffer containing 0.05 M unlabeled lactose to a specific count of 5.8 nCi/rmol of total sugar. The tubes were stoppered, placed in a rotating dialyzer inclined at an angle of 45” to the vertical which was rotated at 4 rpm for 40-44 h. The experiments were done at 8 rt 0.5”C, 15 ? 1°C. and 25 + 1°C. Radioactivity was measured on lOO-pl samples taken from inside and outside the dialysis bags. Absorbance at 280 nm was determined at the beginning and also at the end of the run, for making corrections for any change in volume (25). The values of K, the association constant, and n, the number of binding sites per mole, were obtained from the Scatchard equation (26),

r/c = -Kr + Kn, where c is the concentration of the free sugar and r the number of sugar molecules bound per protein molecule. Heat stability of the leetin. Solutions of E. indica lectin (100 pg/ml) in Buffer A were exposed to different temperatures in presence and in absence of simple sugars as described by Howard and Sage (27). Hemagglutinin titers were determined at different intervals and after dialysis when a sugar was present. DemetaUization bl/ EDTA. The lectin was subjected to demetallization treatment (28) with 1 M acetic acid followed by 0.1 M EDTA. Hemagglutinin titers of “native” and “demetallized” lectins were determined in Buffer A containing Caz+, Mn*+, and Ca*+ + Mn*+ (total molarity 0.1). RESULTS

Pur@ication

of Lectins

The precipitates obtained by adjusting the seed extracts of E. indica, E. arbwestens, and E. lithosperma to pH 4.5 did not possess any hemagglutinating activity. The extract of E. suberosa did not give any precipitate at pH 4.5. Results of affinity chromatography in an acid-treated ECDSepharose 6B column of fraction B from the four Ergthrina species are shown in Fig. 1. In each case, a large peak devoid of hemagglutinating activity eluted out during the washing of the column with Buffer A, and fraction C, which remained bound to the matrix and contained the lectin, was released by elution with 0.1 M lactose as a single peak whose hemagglutinin titers are also shown in the figure. It may be noted that the lectins of these Ergthrina species did not bind on Sepharose

462

BHATTACHARYYA,

DA&

,192 ,040

26

w. E F z z 5 Y I Y

AND

SEN

and from two lots of seeds collected at different times. It will be seen that with red cells of Group 0, E. indica lectin possesses the highest hemagglutinating activity, the activity of the other lectins being considerably less. The lectin content of the seeds other than E. suberosa in which it is 0.002%) is about the same being 0.04 % . Polyacrglamide

Gel Electrophoresis

Figure 2 shows the electrophoretic patterns of the purified lectins at pH values 4.5 and 8.3. Single sharp bands which show slight difference in distance of migration at both pH values may be noted for all lectin preparations.

2e

Immunodiffusion and Immunoelectrophoresis 0

20

40

,

-4 60 0

FRACTION

NUMBER

FIG. 1. Affinity chromatography of fraction B from the seeds of four Ergthrinu species in acid-treated ECD-Sepharose 6B column in Buffer A. (0) Absorbance at 280 nm; (A) hemagglutinin titer with human red cells of 0 group. (A) E. indict: 1500 mg of protein was applied to the column (20 X 2.7 cm). After elution with 240 ml of Buffer A, 0.1 M lactose in the same buffer was started (shown by arrow). Six-milliliter fractions were collected at a flow rate of 30 ml/h. Fraction numbers 51-59 were combined in a single pool (B) E. arboresceua 500 mg of protein was applied to the column (18 X 2.3 cm). After elution with 220 ml of Buffer A, 0.1 M lactose in the same buffer was started. Five-milliliter fractions were collected at the rate of 20 ml/h. Fraction numbers 50-58 were combined in a single pool. (C) E. lithospemmx 500 mg of protein was applied to the column (18 X 2.3 cm). After elution with 200 ml of Buffer A, 0.1 M lactose in the same buffer was started. Five-milliliter fractions were collected at a flow rate of 20 ml/h. Fraction numbers 41-53 were combined in a single pool. (D) E. suberosa: 60 mg of protein was applied to the column (18 X 2.3 cm). After elution with 140 ml of Buffer A, 0.1 M lactose in the same buffer was started. Fourmilliliter fractions were collected at a flow rate of 20 ml/h. Fraction numbers 48/56 were combined in a single pool.

but did so on acid-treated ECD-Sepharose 6B. Table I shows the results of recovery of the lectins. For E. indica, the figures are the average of several preparations

The results of Ouchterlony immunodiffusion tests (Fig. 3) show that the antiserum to the E. indica lectin cross-reacts with the purified lectin giving a single precipitin arc indicating homogeneity of the preparation. The antiserum also shows homologous cross-reaction with the lectins from E. aroborescens, E. lithosperma, and E. suberosa, there being no spurring. However, these antigens in addition to the main precipitin arc, also showed a second weak arc which is not reproduced in the photograph. The results show close immunochemical similarity of the lectins from the four Ergthrina species. The antiserum also gave a single precipitin arc against fraction B from E. indica indicating the absence of other crossreacting constituents in this fraction. In immunoelectrophoresis (Fig. 3) single arcs were obtained with the purified E. indica lectin as well as with fraction B from E. indica. SDS-Gel

Electrophoresis

In SDS-gel electrophoresis, E. indica and E. lithosperma lectins gave two bands whereas E. arboresescens and E. suberosa lectins gave single bands (Fig. 2). The position as well as the number of bands were the same in the presence and in the absence of 2-Me indicating the absence of

D-GALACTOSE-BINDING

LECTIN TABLE

463

PURIFICATION

I

PURIFICATION OF LEC~INS FROM FOUR ERYTHRINA SPECIES

Step E. indica Clarified Fraction Fraction Purified (Fraction

Weight of the seed kernel (%)

Minimum hemagglutinating dose“ k/ml)

Yield of protein (%I

Recovery of protein (%I

21.0 11.8 8.5 0.76

100 8.9

31.3 15.6 10.4 2.0

2.61 0.23

100 8.8

66.0 7.8

1.55 0.13

100 8.4

125.0 15.6

0.07 0.008

100 11.4

91.0 15.6

200 Crude Extract A B lectin C)

E. arborescens Fraction B Purified lectin (Fraction C)

60

E. lithosperma Fraction B Purified lectin (Fraction C)

30

E. suberosa Fraction B Purified lectin (Fraction C)

50

a Against 3% (v/v) human RBC of group 0 (22).

any interchain disulfide linkage. The two bands of E. indica lectin correspond to polypeptide chains of 30,000 and 33,000 molecular weights and those of E. Zithosperma. to 26,000 and 28,000 molecular weights. The polypeptide chain molecular weights corresponding to the single bands of E. arborescens and E. suberosa have been found as 28,000.

where s&,, is the sedimentation coefficient at zero protein concentration, k the slope of the line, and c the protein concentration.

Sedimentation Velocity E. indica lectin in Buffer B gave single symmetrical peaks at different protein concentrations in the range 0.5-2.4X. The least-squares plot of So,,+.at five different concentrations obeyed the relation s20w= s&J1 - kc) x lo-l3 s

Archibald

D@k3ion

Coefjjicient

The value of diffusion coefficient, D, of E. indica lectin at 0.88% concentration in Buffer B was calculated according to the Partial Spec$c Volume equation @I/H)2 = 4aDt, where A is the The value of partial specific volume, V area, H the maximum height, and t the of the pattern. The of E. indica lectin was found to be 0.732 time of phtograph ml/g (SE + 0.0004) from five determinavalue of Dao,wwas found to be 5.91 X 10m7 cm2 s-l. tions.

= 4.49 (1 - 0.105c) x lo-l3 5,

Experiment

An Archibald experiment with E. indica lectin at 0.88% protein concentration in Buffer B gave an weight average molecular weight of 69,100 calculated from four exposures at the cell meniscus and cell bottom taken at intervals of 24,32,40, and 48 min.

464

BHATTACHARYYA,

DAS,

AND

SEN

absence of cysteine and has a low methionine content, and is rich in aspartic acid, glutamic acid, serine, and threonine, which comprise about 40% of the total number of residues. The N-terminal amino acid of E. in&a lectin was found to be valine. Carbohydrate

Composition

The neutral sugar content of E. indica lectin was found to be 9.0%. Glucosamine, the only amino sugar present, was determined as 1.5%. The neutral sugar contents of other lectins were found as E. arborestens lectin, 5.7%, E. lithosperma lectin, 4.1%, and E. suberosa lectin, 6.8%.

FIG. 2. (Top) purified lectins

Polyacrylamide gel electrophoresis of at pH 4.5 (a-d) and 8.3 (e-h). Patterns a,e, E. indica; b,f, E. arborescens; c,g, E. lithospermw and d,h, E. suberosa. Direction of migration was from top to bottom. Fifty micrograms of each protein was applied. Gels were stained with amido black 10B in 7.5% acetic acid. (Bottom) SDS-gel electrophoresis of purified lectins(b-e) and standard proteins (a) in the presence of 2-mercaptoethanol. b-e, respectively, show the patterns of E, indica, E. arborescem, E. lithosperma, and E. mberosa. The standard proteins are, from top to bottom, ovalbumin, pepsin, a-chymotrypsinogen, and @-lactoglobulin B. Twenty-five micrograms of each protein was applied. The final acrylamide concentration was 7.5%. Staining was done with 0.1% Coomassic brilliant blue in 50% TCA. Direction of migration was from top to bottom.

Amino

Acid Composition

Table II shows the amino acid composition of E. indica lectin determined as average values from results of duplicate analyses of each hydrolysate. Concordant results were obtained with preparations from two different collections of seeds. In common with many lectins (3) it shows the

FIG. 3. (Top) Immunodiffusion patterns of purified lectin in 0.1 M Verona1 buffer, pH 8.6. Central well contained antiserum to E. indica lectin; top (left) well, E. indica lectin; top (right) well, E. adxmscew lectin; bottom (left) well, E. de-rosa lectin; bottom (right) well, E. Zithqnerma lectin. (Bottom) Immunoelectrophoresie of E. Mica lectin. The central slot contained antiserum to the purified lectin, the upper hole contained the lectin, and the lower hole fraction B. Electrophoresis was done at pH 9.0 at room tem_nerature at 5 V/cm for 2 h.

D-GALACTOSE-BINDING TABLE AMINO

Amino

Calculated residues per mole”

Lysine Histidine Arginine Aspartic acid Threonine* Serine* Glutamic acid Proline Glycine Alanine Half-cystine ValineC Methionine Isoleucine” Leucine Tyrosine Phenylalanine Tryptophand

18.2 10.3 10.9 63.3 43.7 50.9 60.6 34.3 37.8 40.4 0 42.4 6.0 28.7 37.2 21.6 28.7

Total

Elythrina

Hemagglutinating Nearest integer 18 10 11 63 44 51 61 34 38 40 0 42 6 29 37 22 29 13

Activity

TABLE

III

HEMAGGLUTINATION OF RED BLOOD CELLS FROM VARIOUS SPECIES BY E. indica LECPIN” Minimum

Red cells* Human

Weight

The molecular weight of E. indica lectin was found to be 68,200 by the Svedberg equation (14) using an s&,, value of 4.498, a V value of 0.732 ml/g, and a Dzo,w of 5.91 X 10e7 cm2 s-l. A value of 67,300 was obtained from amino acid and carbohydrate composition. From the above results and taking into account the Archibald molecular weight of 69,100, an average molecular weight of 68,200 (or 68,000) has been obtained. From the results of SDSgel electrophoresis, E. indica lectin appears to be composed of 33,000 and 30,000 molecular weight subunits. The somewhat low value of the subunit molecular weights obtained by this method might have been

content (29)

Table III shows the minimum hemagglutinating doses of E. indica lectin with normal and trypsinized red cells of human A, B, and 0 groups and of different animals. The results obtained with E. arobrescens, E. lithosperma, and E. suberosa lectins with normal human A, B, and 0 red cells are also shown in the same table (see footnote to Table III). It will be seen that E. indica lectin has the highest activity with human 0 group which decreases with B and A groups in that order. With animal cells its activity is much weaker except in the case of rabbit cells. Trypsinization considerably enhances activity in all cases and induces agglutination with dog and guinea pig red cells. Comparison of the values of minimum

548

’ Based on the assumption that the molecule contains six methionine residues per 67,390 molecular weight. * Obtained by extrapolation to zero time of hydrolysis. ’ Obtained in samples hydrolyzed for 72 h. d Determined spectrophotometrically (18).

Molecular

465

PURIFICATION

due to the high carbohydrate of the lectin.

II

ACID COMPOSITION OF PURIFIED indica LE~TIN

acid

LECTIN

Rabbit Duck Hen cow Dog Guinea

A B 0

pig

Normal 15.6 3.9 2.0 3.9 125 125 256 n.a.d n.a.d

hemagglutinating dosee (@g/ml) Trypsinized 0.25 0.06 0.03 0.24 62.5 4.9 125

’ The minimum hemagglutinating dose (pg/ml) with the other Ergthrina lectins with untrypsinized red cells of blood groups A, B, and 0 were found, respectively, as: E. arborescens, 15.6, 3.9, and 7.8, E. lithosperma, 15.6,15.6, and 15.6, and E. suberosa 62.5, 31.2, and 15.6. *Sheep, goat, horse, and mouse erythrocytes did not agglutinate whereas buffalo, turtle, and hamster erythrocytes showed very weak agglutination. ’ Average of three determinations. d Not agglutinated by 2% lectin solution.

466

BHATTACHARYYA,

hemagglutinating dose of the four Erythrina lectins shows that, among them the lectin from E. suberosa is the weakest hemagglutinin and E. indica the strongest with those from E. arborescens and E. lithosperma having intermediate values. While E. indica and E. suberosa lectins show higher activity toward blood group 0, E. arborescens lectin shows more specificity for blood group B, and E. lithosperma shows

equal activity

Carbohydrate

with

all the groups.

Specificity

The results of hemagglutination inhibition tests by simple sugars presented in Table IV show that all the four lectins are inhibited by sugars containing galactosyl moieties. For E. indica lectin, /? anomers of C-l-substituted galactosides appear to be more potent inhibitors than their cy anomers. On the other hand, for E. arborestens and E. lithosperma lectins, cr anomers may be seen to be more potent than their p anomers. The behavior of E. suberosa lectin is somewhat anomalous. While methyl-a-D-galactopyranoside is a stronger inhibitor than its /3 anomer, with pnitrophenyl derivatives, the p anomer is stronger than the (Y anomer. For all the four lectins, N-acetyl-D-galactosamine iS more potent than D-gdaCtOSe, being almost as potent as methyl-cY-D-galactopyranoside. D - Fucose(6 - deoxy - D - galactose) and 2-deoxy-D-galactose may be noted to be poor inhibitors compared to D-galactose.

Isoelectric

Focusing

The result of isoelectric focusing (Fig. 4) using ampholine (pH 4.0-6.0) indicates the presence of three components in the lectin preparation from E. indica having p1 values 4.83, 5.09, and 5.44. Hemagglutinin titers plotted in the same figure show their activities toward human blood groups A, B, and 0.

Equilibrium

Dialysis

Figure 5 shows the Scatchard plot of the equilibrium dialysis data of E. indica lectin in 0.05 M sodium phosphate buffer con-

DAS,

AND

SEN

taining 0.5 M NaCl, pH 7, with lactose at 8,15, and 25°C. A linear plot has been obtained at these temperatures with the values of n equal to 2. The binding constant K has been evaluated from the slope of the plots and from the plot of log K against l/T (Fig. 5), the value of standard enthalpy change AIYP has been obtained. Table V summarizes the results which include the values of standard free energy change AGO, standard entropy change A??‘, and heterogeneity index, a, calculated according to Karush (32). The value of a which is close to unity, indicates that the lectin contains two noninteracting identical binding sites.

Effect of Metal Ions “Demetallization treatment” did not bring about any change in the hemagglutinating activity of E. indica lectin, nor did incorporation of Ca2+ and Mn2+ ion in Buffer A have any effect, suggesting that metal ions did not play a role in the hemagglutinating property of the lectin.

Stability

of the Lectin

The results

of thermal

denaturation of that the lectin remained significantly stable for several days below 50°C as indicated by slight loss of hemagglutinin titer. Above 50°C the activity was gradually lost, and completely lost at 80°C. The presence of increasing concentrations (up to 0.1 M) of the inhibitory sugars, galactose and lactose, considerably protected the lectin from thermal denaturation. At 0.1 M concentration of the sugars the loss in activity at 70°C was about 25% in 90 min whereas, in the absence of sugars, there was total loss of activity in 30 min. The lectin was stable in the pH range 3-O-9.0. Above and below this range there was partial deactivation and above pH 12.0 the activity was totally lost.

E. indica lectin showed

DISCUSSION

It is of interest to note the close similarity in physicochemical properties and carbohydrate specificity of the lectins de-

D-GALACTOSE-BINDING

LECTIN TABLE

INHIBITION

OF HEMAGGLUTINATION

OF THE LECTINS Minimum

D-Galactose D-Fucose 2-Deoxy-D-galactose N-Acetyl-Dgalactosamine Methyl-a-Dgalactopyranoside Methyl-P-Dgalactopyranoside Phenyl-P-Dgalactopyranoside p-Nitrophenyl-a-Dgalactopyranoside p-Nitrophenyl+Dgalactopyranoside Lactose Melibiose Raffinose D-Galactosamine

L-Arabinose D-Ribose D-XylO.92

D-Sorbose D-Glucose D-Mannose L-Fucose a Determined according b The same values were

IV FROM FOUR

amount

completely doses”

Elythrina” inhibiting (rmol/ml)

SPECIES BY SIMPLE four

SUGARS

hemagglutinating

E. indica

E. arbwescens

E. lithosperma

12.5b 25b 100

12.5 50 25

6.25 25 25

6.25 25 25

E. suberosa

6.25b

3.12

3.12

3.12

6.25b

6.25

3.12

3.12

6.25

6.25

3.1P

The following

467

PURIFICATION

12.5

1.56

1.56

1.56

1.56

6.25

1.56

0.78

3.12

1.56 3.1P 6.25 12.5 100b

6.25 3.12 12.5 25 -

1.56 1.56 6.25 12.5 -

1.56 1.56 3.12 6.25 -

sugars

were

noninhibitory

at a concentration

N-acetyl-D-glucosamine N-acetyl-D-mannosamine Methyl-a-D-glucopyranoside Methyl-B-D-glucopyranoside Methyl-a-D-mannopyranoside Methyl-O-D-mannopyranoside D-Galacturonic acid to Osawa et al. (22). obtained with human

erythrocytes,

rived from the seeds of four species of plants of genus Erythrina. These plants have widely different habitats and are well distinguished (5). The lectins, all of which are D-ggk%CtOSe specific, were separable by the same procedure of affinity chromatography on acid-treated ECD-Sepharose 6B and not on Sepharose evidently because of their inability to complex with the internal galactosyl residues of the long linear galactan molecules of Sepharose (8). In polyacrylamide gel electrophoresis (Fig. 2) at pH 4.5 and 8.3, they gave single sharp bands migrating almost to the same positions. Their subunit molecular weights

of 0.2 M L-Rhamnose Maltose Sucrose Cellobiose Melizitose Gentiobiose Trehalose

A and B groups.

as determined by SDS-gel electrophoresis, both in the presence and absence of 2-Me, were within the range 26,000-33,000, with E. indica and E. lithosperma lectins each comprising two noncovalently bound subunits while E. arborescens and E. suberosa were single polypeptide chains. All the lectins have been found to be glycoproteins, their neutral sugar contents being in the range 4-9s. The close structural relationship of the lectins is also apparent from their homologous cross-reaction against anti-E. indica lectin serum by Ouchterlony immunodiffusion tests (Fig. 3). Although reports on immunologic cross-

468

BHATTACHARYYA, I

I

I

16

-5 ph

60 FRACTION

NUMBER

FIG. 4. Isoelectric focusing of E. indica lectin in the pH range 4-6. (0) Absorbance at 280 nm; (A) pH values. Hemagglutinin titers of different fractions against human erythrocytes of groups A (O), B (O), and 0 (X) are also shown.

reactivity of lectins derived from different species of plants of the same genus have not come to our notice, the same carbohydrate specificity of lectins derived from taxonomically related plants, with some exceptions, may be noted, e.g., Anguilla aiguilla (31) and Anguilla rostrata (32) are L-fucose specific, Lathyrus sativus (33) and Lathyrus odoratus (34) D-glucose (D-mannose) specific, Len culinaris (27) and Lens f?SCUk?nta (35) D-ghCOSe (D-mannose) specific, and Vicia faba (36), Vicia ervilia (3’7), Vicia sativa (38), and one lectin from Vicia Cracca (39) D-ghCOSe (D-mannOSe) specific. The trend appears to be toward similar carbohydrate specificity of lectins derived from the plants of the same genus but exceptions are known. For example, Vicia cracca (40) has also been shown to contain N- acetyl - D - galactosamine - spe cific lectins and Vicia graminea lectin (41) is specific for a complex carbohydrate. Further, Phaseolus lunatus lectin (42) is N-acetyl-D-galactosamine specific, and Phaseolus vulgaris lectin (43) is specific for a complex carbohydrate. Hence, no generalization can be made in the absence of more results on different well-identified plant species of the same genus. Results (Table IV) of sugar inhibition of agglutination of human erythrocytes show that C-l-substituted galactosides are more potent inhibitors than D-gahCtOSe

DAS,

AND

SEN

itself. Though the modification of C-2 hydroxyl causes substantial reduction of inhibition as in the cases of D-gak%CtOSaIIIine (for E. indica lectin) and 2-deoxy-D-galactose, there is considerable enhancement of inhibition with N-aCetyl-D-gdaCtOSamine suggesting a good steric fit with the lectin site. In fact, these Erythrina lectins might well be classed as N-aC&Yl-D-galactosamine specific rather than D-g&%tose specific, the former being a better inhibitor than the latter, but further tests with N-acetyl-D-galactosamine disaccharides are necessary to clarify this point. An unmodified C-6 hydroxymethyl group also appears important for binding, since galacturonic acid fails to inhibit the lectins and D-fUCOSe is a weak inhibitor. pNitrophenyl (Y- or P-galactosides have been found to be better inhibitors than the corresponding methyl galactosides. Such findings with p-nitrophenyl and methyl glycosides have also been noted in the cases of Con A (44), and lectins from Sophora japonica (45), peanut (46), and Bandeiraea simplictiolia (47, 48), and have been ascribed to hydrophobic interaction between the phenyl ring and the lectin site. In the case of E. suberosa lectin, methyl-@-D-galactoside is a weaker inhib-

r FIG. 5. Scatchard plot of the equilibrium dialysis data in 0.05 M sodium phosphate-O.5 M NaCl buffer, pH 7.0, for binding of lactose to E. indica lectin at 8°C (A), 15°C (0), and 25°C (0). Inset shows the Van% Hoff plot of K at different temperatures of lactose binding against reciprocal of absolute temperature.

D-GALACTOSE-BINDING

LECTIN TABLE

469

PURIFICATION

V

BINDING CONSTANT AND OTHER THERMODYNAMIC PARAMETERS OF INTERACTION BETWEEN LACTOSE AND E. indica LECTIN AT pH 7.0 Temperature (“C)

K x lo-’ (0

(kJ/mol)

15 * 1

5.63 4.47

-20.3 -20.2

25+1

2.20

-19.2

8 * 0.5

AG”

w

(kJ/mol)

Heterogeneity index” (a)

mol)

n

-0.068 -0.067

2.17

1.98

1.08 1.00

-0.068

2.24

1.04

-39.4 a Calculated according to Karush (30).

itor than the a! anomer, whereas the @anomer of the p-nitrophenyl derivative is stronger. A similar finding (49) reported with Phosphocarpus tetragonolobus has been explained as being due to stronger interaction with the phenyl ring in /3 configuration. E. indica lectin which has been studied in this work in greater detail, was found homogeneous in electrophoretic, sedimentation velocity, and immunochemical experiments and preparations from different collections gave concordant results. In isoelectric focusing, however, the presence of three isolectins has been indicated. Horejsi et al. (6) have reported some properties of E. indica lectin prepared by chromatography on 0-a-D-galactosyl polyacrylamide gels. Although our results in respect of molecular weights and N-terminal group are in accord with theirs, there are some differences, namely, in the polyacrylamide gel electrophoretic pattern at pH 8.3, amino acid composition, carbohydrate content, and inhibition by simple sugars. As the methods of purification were different, we are unable to comment on the differences observed. From equilibrium dialysis experiments the values of the association constants of E. indica lectin for lactose which is one of the potent inhibitors, have been found as 5.68 X lo3 M-l, 4.47 X lo3 M-l, and 2.20 X lo3 M-’ at 8, 15, and 25”C, respectively. These values are of a similar order of magnitude as found in the case of other lectins (3). Two non interacting binding sites have

been found for the dimeric molecule (Table V and Fig. 5) and it may be assumed that each subunit has one carbohydrate binding site. ACKNOWLEDGMENTS

We thank Dr. P. K. Sircar of the Department of Botany, Calcutta University, for help in identification of the seeds and Mr. S. Som for his assistance in immunochemical experiments and amino acid analysis. REFERENCES

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