Covalent attachment of human complement C3 to IgG. Identification of the amino acid residue involved in ester linkage formation.
C3 (native complement component 3) plays a central role in the activation of complement and in the transport and processing of immune complexes. 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 microorganisms. Recently, a C3 attachment site has been localized on the CH1 domain of IgG1 between Val134 and Lys156. We have synthesized a series of peptide analogs of this region to identify the preferred residue for C3b (the proteolytically activated form of C3) attachment. The parent peptide included all 6 hydroxyl-containing amino acids present in the proposed binding site and was highly reactive with activated C3. The C3b-peptide complex was sensitive to hydroxylamine as was 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-IgG1, and C3b-glycerol complexes. Site-specific amino acid substitution of threonine and serine residues in the peptide indicated that, in contrast to the attachment site in C4b, little or no attachment occurred at serine residues. The threonine corresponding to Thr144 in the CH1 domain of IgG was found to be the major acceptor site for C3b. Thr148 was the second most reactive site on the peptide, but this residue is buried in native IgG.
Department of Biochemistry, University of Texas Health Science Center, Tyler 75710.
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Pangburn
D000595:Amino Acid Sequence; D000818:Animals; D003176:Complement C3 / Q000378:metabolism*; D004952:Esters; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D013912:Threonine / Q000378:metabolism
D003176:Complement C3; D004952:Esters; D007074:Immunoglobulin G; D013912:Threonine