Covalently cross-linked monovalent, divalent, and tetravalent derivatives of concanavalin A

Dimeric succinyl-concanavalin A was cross-linked with ethylenediamine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide a condensing agent. Thus, a divalent dimer and a tetravalent tetramer composed mostly of covalently cross-linked subunits bearing altered net charges were obtained. Photoaffinity labeling of the cross-linked dimer with p-azidophenyl alpha-D-mannopyranoside resulted in a specific label for its saccharide-binding site and yielded a nonvalent dimer and a monovalent dimer (showing no subunit exchange). However, hemagglutination and glycogen precipitation data suggested that the labeled binding site is shielded but not destroyed by the label and can still bind weakly an external saccharide ligand possibly due to unsteadiness of the shielding label. Although nonvalent and monovalent derivatives were mitogenic as well as divalent and tetravalent derivatives for mouse splenic lymphocytes, binding and stimulation experiments indicated that their stimulating efficiencies after binding to the cells were far lower than those of the multivalent counterparts. Their activities were inhibited by methyl alpha-D-mannopyranoside, suggesting that the weak activities of nonvalent and monovalent derivatives were due to the labeled sites entirely and partly, respectively. We suggest that the triggering of lymphocyte mitogenesis by concanavalin A may depend on cross-linkage of cell surface receptors.