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 complement alternative pathway. It also participates in the amplification of the complement classical pathway. Alone, factor B is a zymogen with little known biochemical activity, but in the context of the alternative pathway convertases, the factor B serine protease is activated in a process that first involves the association with C3b and subsequently the cleavage of factor B into two fragments, Ba and Bb. Ba, the NH2-terminal fragment, is composed mainly of three tandem short consensus repeats, globular domains found in other complement proteins. It dissociates from the convertase during assembly, leaving the active C3 convertase, C3bBb. Previous reports suggest that the Ba region may be instrumental in convertase assembly. This hypothesis was tested using site-directed mutagenesis of recombinant factor B and monoclonal antibody epitope mapping to evaluate the relative importance of specific short 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 contiguous amino acids in short consensus repeat 2, and site 3, consisting of 7 contiguous amino acids in short consensus repeat 3, were defined by mutations that reduce factor B hemolytic activity to 3% or less. Further analyses indicated that sites 2 and 3 contribute to factor B-C3b interactions.
Hourcade
D E
DE
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Wagner
L M
LM
Oglesby
T J
TJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D011415:Complement Factor B / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018604:Epitope Mapping; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D011415:Complement Factor B