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.

A novel murine complement-related gene encoding a C1r-like serum protein.

C1r and C1s are highly specific serine proteases that initiate the classical pathway of complement activation. We recently demonstrated that, in the mouse, the genes encoding these proteins are duplicated. Analysis of the 5'-flanking region of the murine C1rA gene, the homologue of human C1r, revealed the presence of a novel gene encoding a C1r-like protein (c1r-LP).

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.

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.

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, C2Delta(17), resulted in retention of the mutant polypeptide in the ER.

Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice.

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g.

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.

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-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 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.

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene.

Deficiency of human complement protein C4 due to identical frameshift mutations in the C4A and C4B genes.

The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected.

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.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B.

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 neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 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.

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).

Impaired production of both normal and mutant C1 inhibitor proteins in type I hereditary angioedema with a duplication in exon 8.

In the autosomal dominant disorder type I hereditary angioedema, reduced levels of C1 inhibitor may be due in part to increased turnover and decreased synthesis of normal C1 inhibitor protein. A type I hereditary angioedema patient was recently described in whom the C1 inhibitor mutation consisted of a 20-bp duplication of nucleotides 1414 to 1433 in exon 8 that introduced a frame shift predicting the loss of a normal stop codon and the translation of a protein 52 amino acids longer than normal. In this study, we analyzed the expression of C1 inhibitor in fibroblasts obtained from a skin biopsy of this patient.

Translational regulation of murine complement factor B alternative transcripts by upstream AUG codons.

Factor B (Bf), a constituent of the alternative pathway of complement activation, is encoded by a single gene that is located within the MHC. In murine kidney and intestine, two Bf transcripts (Bf short and Bf long), generated from distinct transcriptional initiation sites, are expressed in approximately equal amounts. In the liver, > 95% of Bf mRNA is the short transcript.

Regulation of synthesis of complement protein C4 in human fibroblasts: cell- and gene-specific effects of cytokines and lipopolysaccharide.

Synthesis and secretion of the class III major histocompatibility complex (MHC) gene product, C4, were detected in human skin fibroblasts by metabolic labelling, immunoprecipitation and SDS-PAGE analysis. Pro-C4 (approximately 185,000 MW) was present in intracellular lysates, and the mature protein was present in extracellular media, with three bands of approximately 93,000, 75,000 and 33,000 MW, corresponding to the alpha, beta and gamma chains, respectively. C4 expression was increased in a dose-dependent manner by interferon-gamma (IFN-gamma), but was unaffected by interleukin-1 beta (IL-1 beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) alone, each of which augmented the expression of factor B, C3 and other complement proteins synthesized in fibroblasts.

Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor.

We have previously reported that polypeptide growth factors had an anti-inflammatory effect by decreasing the cytokine-enhanced expression of factor B (FB), an activator of the alternative complement pathway, in human fibroblasts. To further characterize the role of cytokines and growth factors in the inflammatory/repair continuum, we have studied the effects of interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) on the expression of metalloproteinases/antiproteinases of the extracellular matrix in cultured human fibroblasts. Co-incubation of IL-1 and PDGF synergistically increased the expression of stromelysin and interstitial collagenase to 23-fold (for both proteins) over background, while PDGF decreased the IL-1-enhanced expression of FB by 82%.

Antiinflammatory effects of polypeptide growth factors. Platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor inhibit the cytokine-induced expression of the alternative complement pathway activator factor B in human fibroblasts.

The synthesis of complement components in human fibroblasts is modulated by mediators of inflammation such as cytokines. In particular, interleukin-1 (IL-1) and tumor necrosis factor (TNF) induce time- and dose-dependent increases in the synthesis of complement proteins factor B (FB), C3, and factor H (FH). Polypeptide growth factors are also soluble mediators released during inflammation and able to modulate many fibroblast functions.

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution.

Anti-IgM antibody decreases dissociability of cell bound IgM anti-hapten antibodies as measured with fluid phase hapten.

We studied the effect of an IgG anti-IgM on the dissociation of cell bound IgM anti-hapten antibody, as measured with fluid phase hapten. Methotrexate (MTX) covalently bound to the red cells surface was used as hapten; rabbit anti-MTX IgM and rabbit anti-allotype IgG reactive with the IgM were used as antibodies. The amount of cell bound antibodies was measured with 125I-labelled protein A.

Anti-hapten IgG antibodies bound to cell surface hapten: anti-IgG antibody prevents dissociation as measured with fluid phase hapten.

We investigated the dissociation by fluid phase hapten of IgG antibodies bound to cell surface hapten in the presence and absence of anti-IgG antibodies. Dissociation was quantitated with fluid phase hapten, preventing reassociation of the anti-hapten antibodies. More than 90% of the anti-hapten IgG alone was prevented from reassociation by low concentrations of fluid phase hapten (nanogram to microgram range).

Efficiency of activation of complement by anti-hapten antibodies at the red cell surface: effect of patchy vs random distribution of hapten.

Binding and activation of complement (C) by anti-hapten IgG and IgM antibodies (Abs) bound to a cell surface are dependent on the density and presumably on the distribution of cell-bound hapten. The purpose of this study was to find out if altering the distribution of the hapten on a red cell surface could modify the ability of anti-hapten IgG or IgM Ab to activate C. To test this we devised methods for comparing the C binding and activating efficiency of Abs bound to a hapten distributed randomly or in patches on cells.

Activation of hemolytic complement by mouse monoclonal IgG1 antibody: comparison with rabbit IgG.

We investigated the ability of a mouse anti-hapten monoclonal IgG1 antibody (Ab) to bind to cell-bound specific hapten and to fix and activate C1 and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind C1 and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity.

Binding and activation of hemolytic complement by IgG antibodies: cooperativity between antibodies of different hapten specificity.

The ability of IgG antibodies with different hapten specificities to fix C1 and activate C as a function of hapten density on a red cell surface was investigated. Rabbit anti-methotrexate and anti-folinic acid IgG antibodies in a mixture were highly efficient in fixing C1 and activating C when cells carried simultaneously high levels of both haptens. We wished to find out whether in a C-activating IgG complex both IgG molecules had to be in a form that could activate C1.

Lack of binding of C3 to IgG antibodies during the activation of the classical complement pathway on the red cell.

We have studied the interaction between C3 and natural human and rabbit anti-MTX IgM and hyperimmune IgG antibody bound to red cells to which MTX was covalently coupled. IgM Ab molecules bound to the cell surface were measured by their interaction with rabbit anti-human mu-chain IgG Ab: the bound anti-mu-chain or anti-MTX IgG was quantitated with 125I-labelled PA. C3 uptake by EMTX AC142 complexes from purified preparation of C3 was detected by the interaction of bound C3 with rabbit anti-C3 IgG Ab; the bound IgG was then measured with radiolabelled PA.