Active sites in complement components C5 and C3 identified by proximity toindels in the C3/4/5 protein family

We recently suggested that sites of length polymorphisms in protein familie s (indels) might serve as useful guides for locating protein:protein intera ction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the pa ralogous complement C3, C4, and C5 proteins.;A series of C5 mutants was con structed by altering the C5 sequence at each of the 27 indels in this prote in family. Mutants were expressed in COS cells and were assayed for hemolyt ic activity and protease sensitivity. Mutants at five indels showed relativ ely normal expression but substantially reduced sp, act., indicating that t he mutations damaged sites important for C5 function.;Twenty-three syntheti c peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity,Three of the C5 peptides and on e of the C3 peptides showed 50% inhibition of both C hemolytic-and bacteric idal activities at a concentration of 100 mu M, Tn several cases both the m utational and peptide methods implicated the same indel site, Overall, the results suggest that regions important for function of both C3 and C5 lie p roximal to residues 150-200 and 1600-1620 in the precursor sequences. Addit ional sites potentially important for C5 function are near residue 500 in t he beta-chain and at two or three sites between the N-terminus of the alpha '-chain and the C5d fragment, One of the latter sites, near residue 865, ap pears to be important for proteolytic activation of C5.