SUBSTRATE SPECIFICITIES OF THE PROTEASE OF MOUSE SERUM RA-REACTIVE FACTOR

Ra-reactive factor (RaRF) is a serum bactericidal factor whose functio n seems to be to activate C in a manner similar to that of C1, but wit h activation triggered by binding to bacterial polysaccharides instead of to immune complexes. It is composed of multiple polysaccharide-bin ding subunits associated with a novel serine protease, and its overall structural organization is similar to that of C1. This similarity ext ends to the serine protease component, which shares a similar modular construction and about 40% sequence identity with the C1r and C1s subc omponents of C1. in this study, we examined the substrate specificity of mouse RaRF by assaying its ability to cleave C components C3, C4, a nd C5, and its activity against the murine C4 isotype, sex-limited pro tein. Our results revealed that RaRF preferentially cleaves the C4 alp ha-chain with specific activities 20- to 100-fold greater than either human or murine C1s, and that RaRF also cleaves the C3 alpha-chain, bu t with a lower efficiency than C4 alpha. We also found that RaRF is mu ch less sensitive than C1s to mutations near the proteolytic site and that the two proteases show different reactivities against synthetic s ubstrates. Hence, although the RaRF protease and C1s have similar stru ctures 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 se x-limited protein even at a level 100-fold higher than necessary for C 4 cleavage.