COVALENT ATTACHMENT OF HUMAN-COMPLEMENT C3 TO IGG - IDENTIFICATION OFTHE AMINO-ACID RESIDUE INVOLVED IN ESTER LINKAGE FORMATION

C3 (native complement component 3) plays a central role in the activat ion of complement and in the transport and processing of immune comple xes. Proteolytic activation of C3 exposes a highly reactive thioester bond which preferentially reacts with the hydroxyl groups of acceptor molecules on activators such as immune complexes or carbohydrates on m icroorganisms. Recently, a C3 attachment site has been localized on th e CH1 domain of IgG(1) between Val(134) and Lys(156). We have synthesi zed a series of peptide analogs of this region to identify the preferr ed residue for C3b (the proteolytically activated form of C3) attachme nt. The parent peptide included all 6 hydroxyl-containing amino acids present in the proposed binding site and was highly reactive with acti vated C3. The C3b-peptide complex was sensitive to hydroxylamine as wa s C3b-IgG indicating that both were ester linked. The kinetic profile of hydrolysis of the C3b-peptide complex under physiologic conditions was found to be nearly identical to the profiles of C3b-IgG, C3b-IgG(1 ), and C3b-glycerol complexes. Site-specific amino acid substitution o f threonine and serine residues in the peptide indicated that, in cont rast to the attachment site in C4b, little or no attachment occurred a t serine residues. The threonine corresponding to Thr(144) in the CH1 domain of IgG was found to be the major acceptor site for C3b. Thr(148 ) was the second most reactive site on the peptide, but this residue i s buried in native IgG.