SummaryNormal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCtL) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.

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.

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Rat hepatocellular carcinomas (HCCs) induced by aflatoxin B1 (AFB) treatment were examined for changes in the p53 tumor suppressor gene and in p53 suppressor gene expression. A high proportion of HCCs (nine of 11 tumors in six of eight animals) exhibited new p53 restriction fragments, indicating genomic alterations of one of the p53 alleles. Each tumor with an altered p53 restriction-fragment pattern exhibited a new fragment in one of two size classes (3 kb or 7 kb with EcoRI digestion) that were missing portions of the 3' end of the p53 gene. These findings indicate that apparently similar genomic rearrangements or deletions occurred independently in AFB-induced tumors. When compared with nontumor liver tissue from the same animal, the tumors with p53 gene alterations showed dramatically reduced levels of p53 mRNA and protein and greatly increased levels of histone H2B and retinoblastoma tumor suppressor (Rb) mRNA. In two HCCs showing no evidence of p53 restriction-fragment alterations, mutant p53 protein was detected. Mutant protein was also detected in two liver samples containing an adenoma and altered foci. These data suggest that alterations of the p53 tumor suppressor gene are involved in the induction of rat HCC by AFB.