Antonella Circolo

Modulation of Renal Disease in MRL/lpr Mice Genetically Deficient in the Alternative Complement Pathway Factor B

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.

Complement C1r and C1s genes are duplicated in the mouse: differential expression generates alternative isomorphs in the liver and in the male reproductive system

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.

Constitutive Expression of Murine Complement Factor B Gene Is Regulated by the Interaction of Its Upstream Promoter with Hepatocyte Nuclear Factor 4

Factor B (Bf) is a constituent of the alternative pathway of complement activation encoded within the major histocompatibility complex. Transcription of the murine gene from two initiation sites generates two Bf mRNA species differing in size and tissue distribution. Striking genetic, tissue-specific differences in Bf mRNA levels at extrahepatic sites (kidney and intestine) among mouse strains correlate with a DNA sequence polymorphism in the 5′-flanking region of the gene and differential nuclear protein binding at the Bf upstream transcriptional initiation site (UIS). To ascertain the functional consequences of this polymorphism in the Bf promoter, we analyzed the effects of strain-specific sequences in the Bf 5′ region on the expression of a chloramphenicol acetyltransferase (CAT) reporter gene transfected in human and mouse hepatoma cells. The CAT activity and mRNA level produced when transcription was driven by the sequence of strains with high extrahepatic expression were reduced to background levels when the sequence specific to the low expressor strains was used. Eighty percent of this difference was accounted for by a point substitution that affects DNA-protein interaction at the UIS, the sequence of higher affinity conferring higher expression. Hepatocyte nuclear factor 4 (HNF-4), derived from HepG2, mouse liver and kidney or cell-free translation of HNF-4 RNA, is the nuclear protein that preferentially binds to the high expressor UIS. Bf-CAT is not expressed in cells that lack HNF-4 (CV-1). However, co-transfection of HNF-4 into CV-1 cells drives Bf-CAT expression and reproduces the differences derived from the substitution that affect HNF-4 binding in vitro. These data show that interaction of HNF-4 with polymorphic variants of the upstream Bf promoter is the major determinant of strain-specific extrahepatic factor B expression.

Molecular heterogeneity in deficiency of complement protein C2 type I

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child’s fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child’s C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT‐PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base‐pair deletion) on one allele and a previously unrecognized two base‐pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up‐regulated appropriately by interferon‐γ and the flanking sequence containing the Bf promoter was normal in this C2‐deficient patient. Moreover, the concentration of Bf protein was normal in the patient’s plasma.

Mutational Analysis of the Primary Substrate Specificity Pocket of Complement Factor B ASP226 IS A MAJOR STRUCTURAL DETERMINANT FOR P1-ARG BINDING

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S1 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 P1-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp226 is within the S1 pocket, whereas Asp187 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 Asp187caused moderate (50–60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn189resulted 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 Asp226 completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P1-Arg containing thioester by selected mutants confirmed that residue Asp226 is a primary structural determinant for P1-Arg binding and catalysis.

Structure, function and molecular genetics of human and murine C1r.

C1r, the enzyme responsible for intrinsic activation of the C1 complex of complement, is a modular serine protease featuring an overall structural organization homologous to those of C1s and the mannan-binding lectin-associated serine proteases (MASPs). This review will initially summarize current information on the structure and function of C1r, with particular emphasis on the three-dimensional structure of its catalytic domain, which provides new insights into the activation mechanism of C1. The second part of this review will focus on recent discoveries dealing with a truncated, C1r-related protein, and the occurrence in the mouse of two isoforms, C1rA and C1rB, exhibiting tissue-specific expression patterns.

A novel human complement-related protein, C1r-like protease (C1r-LP), specifically cleaves pro-C1s.

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.

Calnexin is associated with and induced by overexpressed human complement protein C2

C2 is a serum glycoprotein that is essential for activation of the classical and lectin pathways of the complement system. We reported previously that in transiently transfected COS cells, C2 accumulates in the endoplasmic reticulum‐Golgi intermediate compartment (ERGIC). Transfection with a cDNA corresponding to a variant C2 mRNA in which exon 17 is spliced out, C2Δ(17), resulted in retention of the mutant polypeptide in the ER. We now show that calnexin, a lectin‐like chaperone, colocalizes with wild‐type (wt) C2 and C2Δ(17). Biosynthetic labeling and sequential immunoprecipitation experiments indicated that colocalization is due to a physical association between calnexin and C2. Immunofluorescence analysis indicated that calnexin was upregulated in cells transfected with either C2 species. Upregulation of calnexin was not affected by castanospermine, which inhibits glucosidases I and II. However, castanospermine inhibited translocation of calnexin to the ERGIC in wt C2 transfected cells. Upregulation of calnexin was also observed in cells transfected with the complement protein factor B, a glycoprotein with extensive structural and functional similarities to C2, but not in cells transfected with complement proteins C3 or factor D, which have no structural similarity to C2, and low or no glycan content, respectively. Calnexin upregulation by transfection with C2 or factor B, but not factor D, was also demonstrated by quantitative analysis of calnexin immunoprecipitates from biosynthetically labeled cells. Increased calnexin expression by overexpressed C2 and factor B appears to be triggered either by the high glycan content of these proteins or, since it also occurs in the presence of castanospermine, by shared features of the structure of these two proteins. Anat Rec 267:7–16, 2002.