Studies on the interaction of immobilized lectin from Ricinus communis with a simple sugar and a polysaccharide

Biochimica et Biophysica Acta, 371 (1974) 491-500

© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 36885 STUDIES ON T H E I N T E R A C T I O N OF I M M O B I L I Z E D L E C T I N F R O M R I C I N U S C O M M U N I S WITH A SIMPLE S U G A R A N D A P O L Y S A C C H A R I D E

AVADHESHA SUROLIA, ATEEQ AHMAD and B. K. BACHHAWAT Neurochemistry Laboratory, Department of Neurological Sciences, Christian Medical College Hospital, Vellore-632004 (India)

(Received May 30th, 1974)

SUMMARY A glycoprotein lectin from Ricinus communis (RCA~) has been immobilized using concanavalin A. The concanavalin A-immobilized RCA1 retains its sugar binding properties. The number of binding sites (n) and the apparent association constant (/Ca) for lactose are not altered on immobilization. This is the first report which indicates that the carbohydrate moieties of a lectin may not be essential for its sugar binding property. The polysaccharide (guar gum) binding can be measured much more effectively by the immobilized RCA1 as compared to free RCA~. The pH optimum for the binding of guar gum to RCA1 is broader for the immobilized RCA1 as compared to free RCA1. The immobilized RCA1 shows considerable stability to thermal inactivation and urea denaturation as compared to free RCA1. We suggest that the technique of immobilization, where the functional group of a glycoprotein lectin remains available for its biological activity, could prove an important tool for the study of receptor hormone and antibody-antigen interactions.

INTRODUCTION Earlier work from this laboratory has shown that the lectin from castor bean (RCA1) is a glycoprotein and contains concanavalin A-specific sugars namely mannose and glucose [1]. Concanavalin A binds RCA1 forming an insoluble complex and the stability constants suggested that there was no significant dissociation of the complex in the absence of concanavalin A-specific sugar under the experimental conditions [1]. During the past few years methods have been developed in our laboratory for the immobilization of various glycoprotein enzymes as testicular hyaluronate lyase [2] (EC 4.2.2.1), chicken brain arylsulphatase A [3] (EC 3.1.6.1) and glucose oxidase [2] (EC 1.1.3.4) using concanavalin A. These immobilized enzymes retained their catalytic activity and could be reused several times. All these enzymes are immobilized through their carbohydrate moieties binding to concanavalin A [4]. Considering the fact that a number of lectins [5], immunoglobulins [6] and hormone receptors [7, 8] are glycoproteins, it is of interest to study the binding properties of RCA~ with a low molecular weight sugar and a polysaccharide when it is immobilized by using concanavalin A.

492 In this communication we report the immobilization of RCA1 by concanavalin A, its effect on the number of binding sites and association constant for lactose; polysaccharide binding parameters, its reusability, thermal and storage stability and the role of the carbohydrate moieties of this glycoprotein lectin in its biological activity. The results showed that the number of binding sites and the association constant for the simple sugar remains the same. The polysaccharide binding to immobilized RCA1 follows saturation kinetics. This immobilized lectin is more stable to thermal inactivation and urea denaturation compared to free RCA1. The complex could be stored and reused several times without appreciable loss of its carbohydrate binding property. MATERIALS AND METHODS Analytical reagent grade lactose was obtained from BDH, England. Purified guar gum (a galactomannan [9, I0] from Cyamopsist etragonolobus which does not bind to concanavalin A) was a kind gift from Dr H. C. Srivastava, Atira, Ahmadabad. Soluble concanavalin A and RCA~ were prepared according to the methods of Surolia et al. [11] and Nicolson and Blaustein [12], respectively. Neutral sugar was determined by the phenol-H2SO4 method of Dubois et al. [13]. The concentration of protein in solution was determined spectrophotometrically in a Zeiss PMQ II using ~'~% 13.7 and 14 for concanavalin A [14] and ~280 RCA1 [1], respectively.

Formation of concanavalin A-RCAI complex Excess concanavalin A (200 mg) was added to a solution of 100 mg of RCA~ in 0.05 M phosphate buffer, pH 6.8, containing 0.2 M NaCI in a volume of 10 ml. This was incubated for 1 h at 25 °C following which the complex was centrifuged down at 12 000 x g for 30 min. After washing the complex with buffer, it was suspended in 0.05 M phosphate buffer, pH 6.8, containing 0.2 M NaCI. Since RCA~ contains 8 ~ neutral sugar the amount of RCA~ present in the concanavalin A-RCAx complex was determined by estimating the neutral sugar. The stoichiometry of the insoluble complex concanavalin A-RCAx revealed that one molecule of tetrameric concanavalin A binds per molecule of RCA1 [1]. The same amount of RCA~ was used in the free and bound form for the comparative studies. The RCA~ in solution and bound to concanavalin A has been referred to as free RCA~ and immobilized RCA1, respectively.

Equilibrium measurement Binding studies of free RCA1 with lactose were performed by a conventional equilibrium dialysis method as described earlier [1]. For the measurement of lactose binding to the immobilized RCA1 the incubation medium had 2.5 mg of RCA1 (0.5 ffmole to 50 ffmoles of lactose) 50 ffmoles of phosphate buffer, pH 6.8, and 200 ffmoles of NaC1 in a total volume of 1 ml. This was incubated for 1 h at 5 °C with shaking]following which it was centrifuged for 1 h at 20 000 x g. The supernatant was collected and the pellet was washed once with 0.4 ml of 0.05 M phosphate buffer at pH 6.8. This was centrifuged, and the

493 washing and supernatant were pooled. The neutral sugar was estimated in the pooled supernatant. This corresponds to a free sugar (m) in the Scatchard equation as indicated by Stupp et al. [15]. The decrease in amount of lactose in the supernatant was equal to the lactose bound to immobilized RCA~. The number of binding sites (n) and apparent association constant (Ka) were obtained by plotting the data according to the Scatchard method [16].

r/m = Ka (n -- r) Binding of guar gum to free RCA~ and immobilized RCA1 Experiments were performed in a total volume of 1 ml. The incubation mixtures contained 1.0 mg of RCA1, 50/~moles of sodium phosphate buffer, pH 6.8, and 200 pmoles of NaC1 and varying amounts of guar gum (20-400/~g expressed as neutral sugar using galactose as standard). Incubations were conducted with stirring at 25 °C for 2 h. The tubes were centrifuged at 20 000 x g for 1 h and the pellets washed twice with 0.05 M phosphate buffer, pH 6.8, containing 0.2 M NaC1. The washings and the supernatant were pooled together and estimated for neutral sugar. The decrease in the amount of neutral sugar in the supernatant was a measure of the guar gum bound to immobilized RCA~. Since no dissociation of RCA~ takes place under the experimental conditions from concanavalin A-RCA~ it gave a direct measure of guar gum bound to immobilized RCA~. In the case of free RCA1 the protein was estimated (to determine RCA~) in the supernatant by the method of Lowry et al. [17] and on the basis of its neutral sugar content the amount of neutral sugar contributed by RCA~ [1] in the supernatant was deducted from the total neutral sugar giving a measure of guar gum remaining in the supernatant. The decrease in the amount of guar gum in the supernatant gave a measure of the amount of guar gum bound to RCA~.In subsequent experiments for the determination of the guar gum binding capacity of free and immobilized RCA~. I00 and 350 #g of guar gum were used, respectively, in the incubation medium. Effect of the ionic strength of guar gum binding In order to study the effect of the ionic strength on polysaccharide binding to free RCA~ and immobilized RCA~ the conditions of exl:eriment were the same as described under polysaccharide binding except that varying concentrations of NaC1 (0.05-4 M NaC1) were used and the guar gum was added as the last component in the incubation medium. The pH activity profile of free and immobilized RCA~ The study on the pH activity profile of immobilized RCA~ and free RCA1 was performed as mentioned for polysaccharide binding except that NaCI was omitted and buffers ranging from pH 4 to 10 were added to the incubation medium containing RCA~ prior to the addition of guar gum. The buffers used in an incubation volume of 1 ml were 100 #moles of sodium acetate (pH 4-6), sodium phosphate (pH 6.5-7.5), Tris-HC1 (pH 7.5-8.5) and sodium glycinate (pH 9-10). The relative binding was calculated by comparing the binding~at a particular pH with the maximum binding obtained (pH 7-8 and 6.5-8.5 for free and immobilized RCA1, respectively).

494

Thermal inactivation of free and immobilized RCA~ The following procedure was employed for determining the thermal inactivation of free and immobilized RCA~. The samples of either free RCA~ (containing 1 mg RCAa per ml solution) or immobilized RCAt (containing 1 mg RCA~ per ml suspension) in 50 /~moles of phosphate buffer, pH 6.8, containing 200 /~moles ot NaC1 were placed in a constant temperature water-bath maintained at 50 °C. The test tubes were removed at intervals of 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 8 h. The sugar gum binding capacity of these samples were determined, and compared to that for guar gum binding at 25 °C using the time at zero (no heating) as 100K activity for both the free and immobilized RCA~. Determination of activity in urea solution The effect of urea on guar gum binding by free and immobilized RCAa was determined by preincubating them with varying concentrations of urea (0.5-6 M). After the incubation with urea for 30 min at 37 °C the tubes were removed and assayed for guar gum binding at 25 °C. The binding experiment with guar gum was carried out as indicated earlier. The binding activities were expressed relative to the activity obtained in the absence of urea. For the determination of the effect of urea on the stability of concanavalin A-RCAa, the complex was preincubated for 30 min at 37 °C at different concentrations of urea (0.5-6 M). This complex was then centrifuged for 30 min at 12 000 x g. The neutral sugar was estimated in the supernatant in order to assess the amount of RCAI dissociated from the concanavalin A-RCA~ complex. Reusability of the immobilized RCA~ The guar gum binding capacity of the immobilized RCA~ (1 mg RCA0 was determined as described earlier by incubating with 350 #g of guar gum. The bound guar gum was dissociated by incubating the concanavalin A-RCA~ guar gum complex with 0.25 M lactose in 0.05 M phosphate buffer, pH 6.8, containing 0.2 M NaCI for 1 h. This was centrifuged for 1 h at 20 000 × g, the pellet washed once with the phosphate buffer and suspended in 0.05 M phosphate buffer, pH 6.8, containing 0.2 M NaCI. The suspension was dialyzed exhaustively for 24 h to remove the bound lactose. This suspension was again incubated with guar gum in order to determine the binding capacity of immobilized RCA~ to polysaccharide measured. This procedure was repeated five times. RESULTS AND DISCUSSION

Parameters for the binding of lactose The number of binding sites and the apparent association constant (Ka) for the immobilized RCA1 agrees with the equilibrium dialysis results for free RCA1 [1] (Fig. 1). The number of binding sites (n) per molecule of RCA~ with molecular weight of 120 000 [18] was found to be 1.92 and 1.96 with an association constant (Ka) of 3.9 and 4.3 mM -~ for free and immobilized RCA~, respectively. Identical values for the number of binding sites for RCA~ have been reported by Van Wauwe et al. [19] using a different sugar. Thus the binding parameters of RCA~ for lactose are not changed by immobilization. It has previously been shown that glycoprotein enzymes

495 namely arylsulphatase A, testicular hyaluronidase, glucose oxidase and acid phosphatase (EC 3.1.3.2) [4] retained their catalytic activity on immobilization with concanavalin A. Thus in these glycoprotein enzymes the concanavalin A binding site and substrate binding site (or catalytic site) are different. Moreover, Saraswathi and Bachhawat [20] have shown that there was no change in the kinetic properties of alkaline phosphatase from which sialic acid has been cleaved. These observations lead us to postulate that in glycoprotein lectins their carbohydrate prosthetic groups which are bound to concanavalin A do not play a role in their biological activity. This is the first report in which attempts have been made to understand the role of the carbohydrate moieties of a plant lectin in relation to its sugar binding properties (Sharon, N., personal communication). 7. S 7.0

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Fig. 1. Binding of lactose to free and immobilized RCA1. Scatchard plot of the equilibrium dialysis data with free RCAI in 0.05 M sodium phosphate buffer, pH 6.8, containing 0.2 M NaCI for the binding of lactose at 5 °C (O--O). The Scatchard plot of lactose binding data to the immobilized RCA~ ([]--[]). Experiments were carried out at 5 °C as described in the text.

Binding of the guar gum The binding of guar gum to free RCA1 gives an equivalence-type curve showing inhibition of the precipitation at a very high concentration of guar gum due to the formation of soluble complexes [21] (Fig. 2). At the zone of equivalence 93/zg of

496 guar gum is precipitated per mg of RCA1. At higher concentrations of guar gum (130/~g) even though the binding of guar gum to free RCA1 takes place the exact binding cannot be estimated because of the inhibition of precipitation. For the immobilized RCA~ it is observed that the amount of guar gum bound to it increases with an increase in guar gum concentration in the incubation medium. This shows that the guar gum binding to the immobilized RCA1 follows saturation kinetics. Thus the immobilized RCA1 binds a much higher amount of guar gum as compared to free RCA~. Therefore, the binding studies with two macromolecules in solution (antigenantibody reaction type) may not give a true binding capacity of the lectin because of inhibition at a very high antigen concentration. By the use of such an immobilized lectin or antibody this can be determined. The immobilized RCA~ binds less guar gum as compared to free RCA~ at corresponding lower concentrations of guar gum (20-100/~g). The binding of guar gum to the immobilized RCA1 as compared to free RCAI indicates that an immobilized RCA1 would bind less of a macromolecular substrate as a consequence of the decreased effective concentration of the polysaccharide at RCA~ binding sites may be due to the diffusion limitations and steric hindrance [22, 23]. Since no change in the lactose binding parameters of RCA~ were observed the increased binding for a macromolecular substrate at higher concentrations on immobilization cannot be attributed to the changes in the property of the lectin per se.

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Fig. 2. Binding of guar gum to free and immobilized RCA1. Binding of guar gum to free ( O - - G ) and immobilized RCAI ( D - - D ) was carried out at 25 °C as described in the text. Each tube contained i mg of RCAI or immobilized RCA1 with varying amounts of guar gum.

Effect of ionic strength on guar gum binding The ionic strength does not have any effect on the binding of polysaccharide to free as well as bound RCA1 in the range of 0 . 0 5 4 M NaC1 concentration.

497

Effect of p H on binding The pH dependence curve of guar gum binding to free RCA~ as well as immobilized RCA1 is broad as is evident from Fig. 2. The curve rises rather sharply in the acidic region and falls off more gradually in the alkaline region. However, there are marked differences for the optimal pH of interaction of immobilized RCAa with guar gum as compared to free RCA1. The pH optimum for interaction with guar gum for immobilized RCA1 is broader for maximum binding and the optimum is displaced by 1.0 unit towards acidic pH values and by 0.5 unit towards alkaline pH values. The reason for this shift in pH activity profile in the case of immobilized RCA1 may be due to the buffering action of the supporting material, which results in a different microenvironment in the complex [22, 24].

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F i g . 3. The pH-activity profiles of free and immobilized RCA~. The binding of guar gum to free (O--G) and immobilized RCA1 ([]--[]) determined at various pH as indicated. In an incubation volume of 1 m! the buffers used were 100#moles in sodium acetate (pH 4-6), sodium phosphate (pH 6.5-7.5), Tris-HCl (pH 7.5-8.5) and sodium glycinate (pH 9-10).

Effect of urea on stability and activity of the immobilized RCA1 Even at a 6 M concentration of urea only 15 ~ of the total RCA~ was dissociated from the concanavalin A-RCA~ complex suggesting that the concanavalin A RCA~ complex is very stable. This observation is not surprising as hydrophobic interactions have been suggested in lectin-carbohydrate binding [25]. Earlier studies from this laboratory on the glycoprotein-lectin interactions showed that apart from the initial recognition of the carbohydrate moiety of the glycoprotein by the lectin, the complex is stabilized by nonspecific protein-protein interactions which are ionic and hydrophobic in nature [1, 26, 27]. A comparison of the relative activities of guar gum binding to RCAI at different concentration of urea indicated that the immobilized RCA~ has slightly greater stability towards denaturants like urea at neutral pH, although both the lectins lose a considerable amount of their binding activity at a 6-M urea concentration.

498

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Fig. 4. The effect of urea on activity of free and immobilized RCA~. The guar gum binding activities of free ( © - - © ) and immobilized RCAI ( [ ] - - D ) were determinedin solutions of varying urea concentration (0-6.0 M) at pH 7.0. The binding of guar gum at zero urea concentration was taken as

100 ~ activity.

Stability to thermal inactivation Free as well as immobilized RCA1 are quite stable to mild temperature denaturation and were 40 and 15 ~ inactivated, respectively, at 50 °C in 8 h. However, a comparison of the relative activities with time revealed that the immobilized RCAI had a slightly enhanced thermal stability.

Storage stability The sugar gum binding activity of free and immobilized RCAx were unaltered even after storage at 0-5 °C for 2 months in 0.05 M phosphate buffer, p H 6.8.

Reusability of the immobilized RCA1 The reusability studies showed that the immobilized RCA1 had 80 ~ of its guar gum binding capacity even after it had been used five times. A glycoprotein such as RCA1 immobilized through its carbohydrate moiety to another lectin can be used to study its interaction with small molecules and macromolecules. Although free RCA1 binds with lactose the small molecular weight ligand cannot be isolated since they form a soluble complex. It is apparent from the present investigation that the immobilized lectin can be used for the isolation of glycopeptides and glycolipids having an RCAl-specific terminal sugar which may form a soluble complex with free RCA1. This technique of immobilization where the functional group of lectins remain available for biological activity could be an important tool for studying the receptorhormone interactions. Recent reports have shown that some of the steroid [8, 28] and polypeptide hormone receptors [7, 29] are glycoprotein in nature. Several lines

499 of evidence suggest that the wheatgerm agglutinin [7] and concanavalin A [30] binding site and the insulin binding site are different in insulin receptor on the fat cells. In view of these experiments we wish to suggest that the lectin-insolubilized receptor may have an advantage over the isolated free receptor for kinetic measurements with the small molecular weight hormones. Goldstein et al. [31] have demonstrated that immunoglobulin M forms insoluble precipitates with concanavalin A. It has been established that the carbohydrate residues of the immunoglobulins are situated in the constant regions of their H chains which are far away from the hapten binding sites [32] suggesting that concanavalin A-immobilized antibody could provide an alternative method to the Farr [33] technique for the study of the interaction between an antibody and haptens or nonprecipitating antigens. ACKNOWLEDGEMENT

This work was supported by a grant from the Council of Scientific and Industrial Research, India. A.S. and A.A. are the Junior Research Fellows of the Council of Scientific and Industrial Research, India. REFERENCES 1 Podder, S. K., Surolia, A. and Bachhawat, B. K. (1974) Eur. J. Biochem. 44, 151-160 2 Surolia, A., Bishayee, S., Ahmad, A., Balasubramanian, K. A., Thambi Dorai, D., Podder, S. K. and Bachhawat, B. K. (1974) Proceedings of the Symposium on Biological Approach to Problems in Medicine, Industry and Agriculture, Bombay, in the press 3 Ahmad, A., Bishayee, S. and Bachhawat, B. K. (1973) Biochem. Biophys. Res. Commun. 53, 732736 4 Bishayee, S. and Bachhawat, B. K. (1974) Biochim. Biophys. Acta 334, 378-388 5 Lis, H. and Sharon, N. (1973) Annu. Rev. Biochem. 42, 541-574 6 Porter, R. R. (1959) Biochem. J. 73, 119-126 7 Cuatrecasas, P. and Tell, G. P. E. (1973) Proc. Natl. Acad. Sci. U.S. 70, 485-489 8 Wong, K. C., Kornel, L., Bezkorovainy, A. and Murphy, B. E. P. (1973) Bi•chim. Biophys. Acta 328, 133-143 9 Dea, I. C. M., McKinnon, A. A. and Rees, D. A. (1972) J. Mol. Biol. 68, 153-172 10 Smith, F. and Montogomery, R. (1969) ~Ihe chemistry of plant gums and Mucilages, Reinhold Publishing Corporation, New York 11 Surolia, A., Prakash, N., Bishayee, S. and Bachhawat, B. K. (1973) Ind. J. Biochem. Biophys. 10, 145-148 12 Nicolson, G. L. and Blaustein, J. (1972) Biochim. Biophys. Acta 266, 543-457 13 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1965) Anal. Chem. 28, 350-356 14 McKenzie, G. H., Sawyer, W. and Nichol, L. W. (1972) Biochim. Biophys. Acta 263, 283-293 15 Stupp, Y., Yoshida, T. and Paul, W. E. (1969) J. Immunol. 103, 625-627 16 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-665 17 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265275 18 Nicolson, G. L., Blaustein, J. and Etzler, M. E. (1974) Biochemistry 13, 196-204 19 Van Wauwe, J. P., Loontiens, F. G. and De Bruyne, C. K. (1973) Biochim. Biophys. Acta 313, 99-105 20 Saraswathi, S. and Bachhawat, B. K. (1968) Biochem. J. 107, 185-190 21 Maurer, P. H. (1971) in Methods in Immunology and Immunochemistry (Williams, C. A. and Chase, M. W., eds) Vol. 3, Chap. 13, pp. 1-3, 22 Katchalski, E., Silman, I. and Goldman, R. (1971) in Advances in Enzymology (Nord, F. F . ed.) Vol. 34, pp. 445-536, lnterscience

500 23 Sundaram, P. V., Tweedale, A. and Laidler, K. J. (1970) Can. J. Chem. 48, 1498-1504 24 Saini, R., Vieth, W. R. and Wang, S. S. (1972) Trans. New York Acad. Sci. 34, 664-676 25 Surolia, A., Bishayee, S., Ahmad, A., Balasubramanian, K. A., Thambi Dorai, D., Poddler, S. K and Bachhawat, B. K. (1974) International Symposium on Concanavalin A, Norman Oklahoma, in the press 26 Plow, E. F. and Resnick, H. (1970) Biochim. Biophys. Acta 221, 657-661 27 Bishayee, S. (1974) Ph.D. Thesis submitted to the University of Calcutta 28 Westphal, U. (1971) Steroid-Protein interactions, p. 315, Springer-Verlag, New York 29 Cuatrecasas, P. (1973)Biochemistry 12, 1312-1323 30 Czech, M. P. and Lynn, W. S. (1973) Biochim. Biophys. Acta 297, 368-377 31 Goldstein, I. J., So, L. L., Yang, Y. and Callies, O. C. (1969) J. Immunol. 103, 695-698 32 Edelman, G. M. and Gall, W. E. (1969) Annu. Rev. Biochem. 38, 415-466 33 Farr, R. S. (1958) J. Infect. Dis. 103, 239-262