ANALYSIS OF THE SHORT CONSENSUS REPEATS OF HUMAN-COMPLEMENT FACTOR-B BY SITE-DIRECTED MUTAGENESIS

Human factor B is required for the initiation and propagation of the c omplement alternative pathway, It also participates in the amplificati on of the complement classical pathway, Alone, factor B is a zymogen w ith Little known biochemical activity, but in the context of the alter native pathway convertases, the factor B serine protease is activated in a process that first involves the association with C3b and subseque ntly the cleavage of factor B into two fragments, Ba and Bb, Ba, the N H2-terminal fragment, is composed mainly of three tandem short consens us repeats, globular domains found in other complement proteins, It di ssociates from the convertase during assembly, leaving the active C3 c onvertase, C3bBb, Previous reports suggest that the Ba region may be i nstrumental in convertase assembly, This hypothesis was tested using s ite-directed mutagenesis of recombinant factor B and monoclonal antibo dy epitope mapping to evaluate the relative importance of specific sho rt consensus repeat amino acid residues, Three sites of interest were identified, Site 1 is a stretch of 19 contiguous amino acids in short consensus repeat 1 that form the epitope of a monoclonal antibody that effectively blocks factor B function. Site 2, composed of 6 contiguou s amino acids in short consensus repeat 2, and site 3, consisting of 7 contiguous amino acids in short consensus repeat 3, were defined by m utations that reduce factor B hemolytic activity to 3% or less, Furthe r analyses indicated that sites 2 and 3 contribute to factor B-C3b int eractions.