Identification of residues within the 727-767 segment of human complement component C3 important for its interaction with factor H and with complement receptor 1 (CR1, CD35)

Mapping approaches employing blocking antibodies and synthetic peptides hav e implicated the 727-767 segment at the NH2 terminus of C3b alpha'-chain as contributing to the interactions with factor B, factor H, and CR1. Our pre vious mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C 3b to factor B and CR1 but not factor H, We have now extended the charged r esidue mutagenic scan to cover the remainder of the segment (738-767) and h ave assessed the ability of the C3b-like C3(H2O) form of the mutant molecul es to interact with factor H, CR1, and membrane cofactor protein (MCP) usin g a cofactor-dependent factor I cleavage assay as a surrogate binding assay . We have found that the negatively charged side chains of Glu-744 and Glu- 747 are important for the interaction between C3(H2O) and factor H, a resul t in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545-552) which implicated residues within the 7 44-754 segment in H binding. The interactions of the mutants with soluble C R1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of alpha-chain, T hese are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing, The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-7 44 and the Lys-757/Glu-758 pairing, The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribut e basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wide r portion of the 727-767 segment than is the case for factor H, Finally, no ne of the mutations had any effect on the interaction between soluble MCP a nd C3(H2O), indicating that despite its functional homology to H and CR1, M CP differs in its mode of binding to C3b/C3(H2O).