To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neo r gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F 1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P ‫؍‬ 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly ref lecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B−/−), and heterozygous (B+/−) MRL/lpr mice. Compared with B+/− or B+/+ mice, MRL/lpr B−/− mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B−/− mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.

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Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3−/−), heterozygous (C3+/−), and C3 wild-type (C3+/+) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3−/− mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3−/− mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.

The availability of the human genome sequence allowed us to identify a human complement-related, C1r-like protease gene (c1r-LP) located 2 kb centromeric of the C1r gene (c1r). Compared with c1r, c1r-LP carries a large deletion corresponding to exons 4-8 of c1r. The open reading frame of the C1r-LP cDNA predicts a 50 kDa modular protein displaying 52% amino acid residue identity with the corresponding regions of C1r and 75% identity with a previously described murine C1r-LP. The serine protease domain of C1r-LP, despite an overall similarity with the AGY group of complement serine proteases, has certain structural features characteristic of C2 and factor B, thus raising interesting evolutionary questions. Northern blotting demonstrated the expression of C1r-LP mRNA mainly in the liver and ELISA demonstrated the presence of the protein in human serum at a concentration of 5.5+/-0.9 microg/ml. Immunoprecipitation experiments failed to demonstrate an association of C1r-LP with the C1 complex in serum. Recombinant C1r-LP exhibits esterolytic activity against peptide thioesters with arginine at the P1 position, but its catalytic efficiency (kcat/K(m)) is lower than that of C1r and C1s. The enzymic activity of C1r-LP is inhibited by di-isopropyl fluorophosphate and also by C1 inhibitor, which forms stable complexes with the protease. Most importantly, C1r-LP also expresses proteolytic activity, cleaving pro-C1s into two fragments of sizes identical with those of the two chains of active C1s. Thus C1r-LP may provide a novel means for the formation of the classical pathway C3/C5 convertase.

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S 1 pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P 1 -Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp 226 is within the S 1 pocket, whereas Asp 187 is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp 187 caused moderate (50 -60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn 189 resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp 226 completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P 1 -Arg containing thioester by selected mutants confirmed that residue Asp 226 is a primary structural determinant for P 1 -Arg binding and catalysis.Complement is a major effector system of host defense. Activation of complement leads to the generation of protein fragments and protein-protein complexes that mediate acute inflammatory responses, phagocytosis and killing of pathogens, and regulation of adaptive immune responses. Activation-associated production of biologically active protein fragments is catalyzed by a group of eight atypical complement serine proteases (SPs) 1 of the chymotrypsin superfamily (1). Understanding the structural basis for the highly restricted proteolytic activity of these SPs is an important first step toward pharmacologic control of complement activation (2). Members of the chymotrypsin family have very similar three-dimensional structures but distinct substrate specificities. To a great extent specificity is determined by the side chains of the amino acid residues that line up the primary substrate specificity pocket (S 1 site). The pocket has three walls formed by residues 189 -195, 214 -220, and 225-228 (chymotrypsinogen numbering has been used for all SPs or SP domains throughout this paper) (3). The presence at the bottom of the pocket of Asp 189 endows trypsin with preference for positively charged Arg and Lys residues (4, 5), whereas in chymotrypsin the specificity for bulky aromatics is largely determined by Ser 189 (6). Residues at position 216 and 226 also contribute to substrate specificity (7). All complement SPs exhibit trypsinlike specificity for positively charged Arg residues and all have an Asp at position 189, except for factor B and C2 (Fig. 1).Factor B and C2 are structurally similar modular proteins that play a central role in complement activation by providing the catalytic subunits of...

C1r and C1s are the serine proteases that form the catalytic unit of the C1 complex, the first component of complement. In the present study, we found that the genes encoding murine C1r and C1s are duplicated. One set of these genes, referred to as c1rA and c1sA, are primarily expressed in the liver and are therefore the homologues of the human C1r and C1s genes. The other two genes, termed c1rB and c1sB, are expressed exclusively in male reproductive tissues, specifically the coagulating gland and the prostate. The predicted C1rB and C1sB proteins share 96 and 93% amino acid identity with C1rA and C1sA respectively. Most of the substitutions are clustered in the serine protease domains, suggesting differences in catalytic efficiencies and/or substrate specificities or alternatively adaptation to different physiological environments. The high homology of C1rB and C1sB with C1rA and C1sA in the non-catalytic regions indicates that they are probably capable of assembling the C1 complex. The expression of alternative genes encoding isomorphs of activating components of complement in male reproductive tissues raises the possibility of new mechanisms of complement activation in the male genital tract or of novel functions for complement proteases in reproduction.

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