C1 Binding to Complexes Consisting of Cross-linked Fc Molecules from Human Immunoglobulin G

Abstract The activation of the classical pathway of complement may occur when C1 is bound to the Fc portion of immunoglobulin G (IgG) in immune complexes. In order to study the properties of C1 binding and activation we have prepared covalently cross-linked complexes of isolated Fc molecules. The Fc of human IgG was isolated from either limited papain digests or trypsin digests by gel filtration and ion exchange chromatography. Using a C1 binding assay we found that monomeric Fc (papain) did not bind C1 even at concentrations as high as 1 mg/ml. Monomeric Fc (trypsin) did bind C1 weakly. The binding properties of polymeric Fc complexes produced by covalent cross-linking with dimethyl suberimidate in polyethylene glycol were remarkably different from those observed with monomeric Fc. Less than 0.2 µg/ml of Fc complexes found more than 50% of the C1 in the assay. The capability of the Fc complexes to bind C1 was comparable to heat aggregated IgG and was more than 500 times more effective than monomeric Fc. Polymeric Fc complexes in the range of monomer to pentamer were prepared and separated by gel filtration. The avidity of C1 for these separated complexes increased with the molecular size and significant inhibition of C1 binding to sensitized erythrocytes was observed with Fc trimers. The C1 binding to Fc complexes was not just a function of increasing size, since reduction and alkylation of the Fc complexes abolished the ability of the complexes to bind C1 without destroying the Fc polymers. We were surprised to find that C1 binding to these Fc complexes did not cause activation of C1. We are presently studying which properties lead to binding as well as activation.