A. Sahu et Mk. Pangburn, COVALENT ATTACHMENT OF HUMAN-COMPLEMENT C3 TO IGG - IDENTIFICATION OFTHE AMINO-ACID RESIDUE INVOLVED IN ESTER LINKAGE FORMATION, The Journal of biological chemistry, 269(46), 1994, pp. 28997-29002
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.