Concanavalin A Derivatives with Altered Biological Activities ( lectins / lymphocyte stimulation / membrane receptors )

Chemical derivatization of tetrameric concanavalin A (Con A) with succinic anhydride or acetic anhydride converts the protein to a dimeric molecule without altering its carbohydrate-binding specificity. At low concentrations, the dose-response curves for the mitogenic stimulation of mouse spleen cells by native Con A and succinyl-Con A are similar. Above lectin concentrations of 10 ug/ml, however, the response to Con A is diminished, while that for succinyl-Con A does not decrease until much higher doses are reached. We have attributed this difference mainly to the higher rate of cell death induced by the native Con A molecule. Con A also shows a greater capacity than succinyl-Con A to agglutinate sheep erythrocytes and to inhibit cap formation by immunoglobulin receptors on spleen cells. Moreover, at low concentrations, Con A induced its glycoprotein receptors to form caps, but succinyl-Con A did not induce cap formation. Addition of antibodies directed against Con A to succinyl-Con A bound on cells restored the properties of agglutination, inhibition of immunoglobulin receptor cap formation, and induction of cap formation by Con.A receptors. Similar results have been obtained for acetylCon A. These data suggest that the altered biological activities ofsuccinyl-Con A and acetyl-Con A are attributable to their reduced valence. The critical initial event in stimulation of lymphoid cells by specific ligands such as antigens, and nonspecific ligands such as antibodies and lectins, is the binding to appropriate receptor sites on the cell surface. There is evidence that the stimulation of lymphocytes both by multivalent antigens and by divalent antibodies requires cross-linkage or multipoint attachment to cell surface receptors (1-3). We have examined several chemical derivatives of the lectin, concanavalin A (Con A), in order to determine whether some of its biological activities are dependent upon its valence. Con A agglutinates various somatic and germ line cells (4, 5) and is mitogenic for lymphocytes (6, 7). Binding of Con A both restricts the mobility of immunoglobulin receptors on the lymphoid cell surface (8) and inhibits phagocytosis by polymorphonuclear leukocytes (9). Studies on the subunit and three-dimensional structure of Con A (10, 11) indicate that at pH 7 it is a tetramer with four saccharide binding sites, suggesting that receptor crosslinkage by the tetravalent Abbreviations: Con A, concanavalin A; anti-Ig, rabbit immunoglobulin directed against mouse immunoglobulin; fl-anti-Ig, fluorescein-labeled anti-immunoglobulin; fl-Con A, fluoresceinlabeled concanavalin A; anti-Con A, rabbit immunoglobulin directed against concanavalin A; PBS, phosphate-buffered saline (pH 7.4) (8.00 g of NaCl-0.20 g of KC1-0.20 g of KH2PO4-0.15 g of Na2HPO4 per liter); HBSS-FBS, Hank's balanced salt solution containing 5% (v/v) fetal bovine serum. Con A molecule may play an important role in its action on cells. Because it is multivalent, Con A may function to crosslink receptors between two different cells leading to their agglutination. It has also been suggested (8, 9) that inhibition of immunoglobulin receptor mobility and of phagocytosis by Con A may result from interference of membrane movement due to crosslinking of adjacent binding sites. We report here studies of two dimeric Con A derivatives, succinyl-Con A and acetyl-Con A, which differ from the native tetrameric structure in their properties. In addition, we suggest a hypothesis to explain the alteration in biological activities resulting after derivatization. MATERIALS AND METHODS The preparation and purification of Con A have been described (12). Fluorescein-labeled antibodies and fluoresceinlabeled Con A were prepared (13) from rabbit Ig fractions containing antibodies to mouse Ig and from Con A using fluorescein isothiocyanate (BBL, Division of BioQuest, Cockeysville, Md.). Succinyl-Con A was prepared by dissolving 100 mg of Con A in 25 ml of saturated sodium acetate at room temperature. After centrifugation, the supernatant was transferred to a 50-ml flask containing 30 mg of succinic anhydride (Matheson Coleman and Bell, East Rutherford, N.J., Lot no. 2503). The solution was stirred in an ice bath for 1 hr, dialyzed overnight against H20, and lyophilized. The lyophilized protein was subjected to a second derivatization by dissolving it in 20 ml of saturated sodium acetate. After removal of precipitate, the protein solution was added to a flask containing 30 mg of succinic anhydride and stirred at room temperature for 90 min. The solution was then dialyzed exhaustively against H20 and lyophilized. The number of succinyl groups per polypeptide chain of Con A was determined byuse of [I4C]succinic anhydride (ICN Chemical & Radioisotope Division, Irvine, Calif., Lot no. 7300-65) to prepare [I4C ]succinyl-Con A. Acetyl-Con A was prepared by dissolving 25 mg of Con A in 7 ml of saturated sodium acetate. 40 ,l of acetic anhydride (Eastman Organic Chemicals, Rochester, N.Y., Lot no. 691A) was added to the protein solution, which was stirred for 1 hr in an ice bath. The protein was dialyzed against H20 and lyophilized. Molecular weight was determined by equilibrium sedimentation at 36,000 rpm as described by Waxdal et al. (14). Sedimentation velocity experiments were done at 52,500 rpm. Polyacrylamide gel electrophoresis was performed by the method of Ornstein (15). Affinity con-