Substrate specificities of the protease of mouse serum Ra-reactive factor.
Ra-reactive factor (RaRF) is a serum bactericidal factor whose function seems to be to activate C in a manner similar to that of C1, but with activation triggered by binding to bacterial polysaccharides instead of to immune complexes. It is composed of multiple polysaccharide-binding subunits associated with a novel serine protease, and its overall structural organization is similar to that of C1. This similarity extends to the serine protease component, which shares a similar modular construction and about 40% sequence identity with the C1r and C1s subcomponents of C1. In this study, we examined the substrate specificity of mouse RaRF by assaying its ability to cleave C components C3, C4, and C5, and its activity against the murine C4 isotype, sex-limited protein. Our results revealed that RaRF preferentially cleaves the C4 alpha-chain with specific activities 20- to 100-fold greater than either human or murine C1s, and that RaRF also cleaves the C3 alpha-chain, but with a lower efficiency than C4 alpha. We also found that RaRF is much less sensitive than C1s to mutations near the proteolytic site and that the two proteases show different reactivities against synthetic substrates. Hence, although the RaRF protease and C1s have similar structures and play similar roles in C activation, they also display clear differences in substrate range and in the details of their substrate recognition mechanisms. Finally, we found that RaRF does not cleave sex-limited protein even at a level 100-fold higher than necessary for C4 cleavage.
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Medical Biology Institute, La Jolla, CA 92037.
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Kawakami
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D000595:Amino Acid Sequence; D000818:Animals; D001770:Blood Bactericidal Activity; D003167:Complement Activation; D003173:Complement C1s / Q000235:genetics / Q000378:metabolism; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism; D003181:Complement C4 / Q000235:genetics / Q000378:metabolism; D003182:Complement C5 / Q000235:genetics / Q000378:metabolism; D003165:Complement System Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics; D012697:Serine Endopeptidases / Q000097:blood* / Q000235:genetics; D013379:Substrate Specificity
D003176:Complement C3; D003181:Complement C4; D003182:Complement C5; D010455:Peptides; D003165:Complement System Proteins; D012697:Serine Endopeptidases; D003173:Complement C1s