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Torrey Pines Institute for Molecular Studies, San Diego, CA 92121
We recently suggested that sites of length polymorphisms in protein
families (indels) might serve as useful guides for locating
protein:protein interaction sites. This report describes additional
site-specific mutagenesis and synthetic peptide inhibition studies
aimed at testing this idea for the paralogous complement C3, C4, and C5
proteins. A series of C5 mutants was constructed by altering the C5
sequence at each of the 27 indels in this protein family. Mutants were
expressed in COS cells and were assayed for hemolytic activity and
protease sensitivity. Mutants at five indels showed relatively normal
expression but substantially reduced sp. act., indicating that the
mutations damaged sites important for C5 function. Twenty-three
synthetic 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 one of the C3 peptides showed 50% inhibition of both C
hemolytic and bactericidal activities at a concentration of 100 µM.
In several cases both the mutational and peptide methods implicated the
same indel site. Overall, the results suggest that regions important
for function of both C3 and C5 lie proximal to residues 150–200 and
1600–1620 in the precursor sequences. Additional sites potentially
important for C5 function are near residue 500 in the ß-chain and at
two or three sites between the N-terminus of the 
'-chain and the C5d
fragment. One of the latter sites, near residue 865, appears to be
important for proteolytic activation of C5.
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