SEGMENT SPANNING RESIDUES-727-768 OF THE COMPLEMENT-C3 SEQUENCE CONTAINS A NEOANTIGENIC SITE AND ACCOMMODATES THE BINDING OF CR-1, FACTOR-H, AND FACTOR-B

CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site. Furthermore, the inhibition of H, CR1, and B binding to C3b by a single MoAb (anti-C3-9) while other MoAbs differentially inhibit the binding of these proteins to C3b suggests that (1) multiple sites exist in the C3 molecule for binding these C3b-binding proteins, (2) at least some of the sites are proximal or share structural elements, and (3) these features may explain observed affinity and specificity differences exhibited by these C3b-binding proteins.