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PubMed1988

Expression of functional human C1 inhibitor in COS cells.

Full length human C1 inhibitor cDNA was cloned into a vector suitable for transient expression in COS-1 cells. Transfected COS cells secreted an immunoreactive protein of Mr approximately 110,000 that appeared to be functionally equivalent to the plasma-derived protein as established by the following criteria: 1) ability to form sodium dodecyl sulfate-stable complexes with C1s, factor XIIa, and kallikrein; 2) inhibition of C1s-mediated C4 consumption; and 3) susceptibility to inactivation by the nontarget proteinase elastase. Quantitation of secreted recombinant C1 inhibitor by radioimmunoassay indicated that 72 h after transfection the level was approximately 2.2 micrograms/ml. Treatment of transfected cells with tunicamycin resulted in secretion of a protein of Mr approximately 90,000 that was also capable of complex formation with C1s.

Animals↗
PubMed1987

Concentrations of C1q, factor B, factor D and properdin in healthy children, and the age-related presence of circulating C1r-C1s complexes.

The concentrations of C1q, factor B, factor D and properdin were determined in healthy children belonging to various age groups of one through five years of age. All concentrations were found to be age-dependent, though they varied from one component to another with regard to ontogenetic pattern. Thus, the concentrations of factor B were high, and those of factor D low throughout the age range studied. C1q and properdin levels were lowest in the younger children, who also showed a fairly high incidence of C1r-C1s complexes in excess of C1q. Since the concentrations of C1q are influenced by those of IgG, the presence of C1r-C1s complexes might partly have reflected maturation of immunoglobulin synthesis during ontogeny.

Age Factors↗
PubMed1984

Comparative study of the fluid-phase proteolytic cleavage of human complement subcomponents C4 and C2 by C1s and C1r2-C1s2.

The C3 convertase of the classical pathway of complement is composed of fragments C4b and C2a resulting from cleavage of C4 and C2 by activated C1. The limited proteolysis of these two different substrates by the same protease, C1s, has been studied in the fluid phase using purified proteins. The turnover numbers of C2 and C4 cleavage by C1s were affected to different extents, depending on whether C1s was alone or associated with C1r or with monoclonal antibodies to C1s. The binding of C2 to C4 favours the proteolysis of C2 by C1s, as revealed by the use of I2-treated C2.

Antibodies, Monoclonal↗
PubMed1994

A 100-kDa protein in the C4-activating component of Ra-reactive factor is a new serine protease having module organization similar to C1r and C1s.

Ra-reactive factor (RaRF), a C-dependent bactericidal factor in mice, is composed of one polysaccharide-binding component and one C4/C2-activating component. The former is an oligomer of 28-kDa protein corresponding to the mannose-binding protein of mice. The 100-kDa protein, P100, has been shown to be present in the C4/C2-activating component. This protein generates 29- and 70-kDa polypeptide chains when reduced. In this study, we determined the nucleotide sequence of cDNA coding for P100. cDNAs were prepared by reverse transcription PCR and cassette-ligation-mediated PCR on mRNA from BALB/c mouse liver, using primers synthesized by reference to the sequence determined in a previous study. The results of cDNA sequencing indicate that the precursor protein of P100 containing a 24-residue signal peptide consists of 704 amino acid residues. Taking the results of the previous electrophoretic study into consideration, it is thought that the cleavage of mature P100 protein generates a 29-kDa chain of 251 residues and a 70-kDa chain of 429 residues. Although homology in the amino acid sequence of P100 with that of human C1r and C1s subcomponents of C was less than 40%, a striking similarity in domain organization was found among these proteins, indicating that P100 is a new C4-activating serine protease structurally similar to C1r and C1s. Northern hybridization showed that the liver was the primary site of the expression of the P100 gene.

Amino Acid Sequence↗
PubMed1994

Substrate specificities of murine C1s.

An early step in the initiation of the classical C pathway is the proteolytic activation of component C4 by subcomponent C1-s. We have examined the substrate specificity of murine C1-s (mC1-s) by measuring its proteolytic activity on human and murine C4, and on the murine C4 isotype designated sex-limited protein (Slp). The latter substrate was examined because previous studies have demonstrated that Slp is not cleaved by C1-s, and hence Slp has been assumed to be nonfunctional in the C pathways. Those earlier studies used human, not murine, C1-s, however; a recent report has suggested that Slp is an essential component of a novel complement activation pathway and that the previous failure to observe cleavage of Slp is probably the result of a species incompatibility between Slp and the heterologous human C1-s (hC1-s). The present studies do not support this idea, as we found no evidence of cleavage of Slp by homologous murine C1-s even at enzyme concentrations 10-fold higher than that necessary for 50% cleavage of murine C4 (mC4). We did find a species-specific affect in the cleavage of mC4, where mC1-s is about 10-fold more effective than heterologous hC1-s in cleaving mC4, but mC1-s itself does not distinguish between human and murine C4, cleaving both equally well. Hence mC1-s does not exhibit the species specificity previously found for hC1-s, which shows a several hundred-fold preference for homologous human C4 over murine C4.

Animals↗
PubMed1995

Substrate specificities of the protease of mouse serum Ra-reactive factor.

Ra-reactive factor (RaRF) is a serum bactericidal factor whose function seems to be to activate C in a manner similar to that of C1, but with activation triggered by binding to bacterial polysaccharides instead of to immune complexes. It is composed of multiple polysaccharide-binding subunits associated with a novel serine protease, and its overall structural organization is similar to that of C1. This similarity extends to the serine protease component, which shares a similar modular construction and about 40% sequence identity with the C1r and C1s subcomponents of C1. In this study, we examined the substrate specificity of mouse RaRF by assaying its ability to cleave C components C3, C4, and C5, and its activity against the murine C4 isotype, sex-limited protein. Our results revealed that RaRF preferentially cleaves the C4 alpha-chain with specific activities 20- to 100-fold greater than either human or murine C1s, and that RaRF also cleaves the C3 alpha-chain, but with a lower efficiency than C4 alpha. We also found that RaRF is much less sensitive than C1s to mutations near the proteolytic site and that the two proteases show different reactivities against synthetic substrates. Hence, although the RaRF protease and C1s have similar structures and play similar roles in C activation, they also display clear differences in substrate range and in the details of their substrate recognition mechanisms. Finally, we found that RaRF does not cleave sex-limited protein even at a level 100-fold higher than necessary for C4 cleavage.

Amino Acid Sequence↗
PubMed1989

Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.

Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control. In the present studies, we used site-directed mutagenesis of C4 and expression of the mutant proteins in cultured cells to identify the amino acid substitutions in Slp that are responsible for resistance to C1s cleavage. We focused on sequence changes immediately downstream of the cleavage site in C4 because the arginine at that site is conserved in Slp, but the downstream sequences diverge substantially, with six differences in the first 7 residues followed by a 3-residue deletion in Slp. We found that a C4 mutant carrying only the 3-residue deletion is not cleaved by C1s and has essentially no hemolytic activity, whereas a mutant carrying only the six replacement changes is cleaved by C1s and has normal hemolytic activity. Both mutants have intact thioesters. A third mutant in which two acidic residues in the segment deleted in Slp were replaced by aliphatic residues is also cleaved by C1s, has an intact thioester group, and has normal hemolytic activity. These results indicate that the downstream mutations are responsible for the resistance of Slp to C1s cleavage and suggest that the length rather than the specific sequence of this segment is critical in determining susceptibility to the protease.

Amino Acid Sequence↗
PubMed1990

Neutron scattering study of the (gamma-B) catalytic domains of complement proteases activated C1r and C1s.

The catalytic domains of activated C1r and C1s, comprising the C-terminal region of the A chain (gamma), disulphide-linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For activated C1s (gamma-B), a molar mass of 45,000 +/- 5000 g/mol, and a relatively large radius of gyration (Rg) of 28 +/- 1 A were determined, excluding a single globular domain. The corresponding values for activated C1r (gamma-B)2 (90,000 g/mol, Rg = 34 +/- 1 A) are consistent with a dimer involving the loose packing of two (gamma-B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data.

Chymotrypsin↗
PubMed1987

Quantitation of (C1INH)2 C1r-C1s complexes in glomerulonephritis as an indicator of C1 activation.

C1 activation was assessed in several forms of glomerulonephritis by radioimmunoassay quantitation of circulating (C1INH)2 C1r-C1s complexes (INC). Eight patients with active systemic lupus erythematosus (SLE) and nephritis had elevated serum INC (mean = 15.3 vs control = 5.8, P less than 0.01). Their INC levels were normal during remission. Serum INC had a weak inverse correlation with serum C1q greater than 3 mg/dl (r = 0.42, P = 0.02). In longitudinal studies, serum INC also had a weak inverse correlation with serum C3 and C4. Only 1 of 10 patients with type I and 1 of 15 with type III membrano-proliferative glomerulonephritis (MPGN) had elevated serum INC. No patient with type II MPGN had elevated levels. Two of 10 patients with poststreptococcal glomerulonephritis (P-SGN) had elevated serum INC, but all normalized with convalescence. Patients with IgA nephropathy had normal serum INC. The data demonstrate the importance of C1 activation in SLE and P-SGN. The mechanism of complement activation in types I and III MPGN remains unclear; the data suggest, but do not prove, that C1-independent complement activation may occur in these patients.

Antigen-Antibody Complex↗
PubMed1989

Models for C1. Tools or toys? The real biological challenge.

C1 modelling, based on structural and functional data, does not yet bring the different laboratories to a consensus on C1 activation, activity and associated controls. The heart of C1 beats in its subcomponent C1r2, which, from its domain structure and its twinning with subcomponent C1s, represents the challenge for the knowledge of C1. The 8-shaped model proposed for the C1r2-C1s2 association, with a head-to-tail interaction between the C1r catalytic domains, appears as the hub of an active world in the bosom of C1q. More detail is now required on protein-protein interactions inside C1 to refine the available models or to propose alternatives. Precise data on the interactions of C1 proteins with activators, substrates or control proteins are also likely to bring pertinent help in proposing future models for C1.

Binding Sites↗
PubMed1987

Isolation and analysis of immune complexes from sera of patients with polymyalgia rheumatica and giant cell arteritis.

Serum samples were obtained from patients with polymyalgia rheumatica (PMR: n = 10) or giant cell arteritis (GCA; n = 7), or both. Samples were taken either before treatment or within one week of starting prednisolone. Immune complexes (IC) were concentrated by polyethylene glycol (PEG) precipitation then purified with either IgG anti-C1q-Sepharose or IgG anti-C3c-Sepharose. Complex components were separated by sodium dodecyl sulphate (SDS) gradient polyacrylamide gel electrophoresis then transferred to nitrocellulose by Western blotting. Identification of proteins was carried out using specific antisera. All the IC contained IgM (mu chain), some contained IgA (alpha chain), and IgG (gamma chain). C1r, C1s, C1q, C3, C4, and C reactive protein (CRP), where tested, were found in most but not all IC. The occurrence of properdin, factor B, alpha 2 macroglobulin (alpha 2M), factor H (beta 1H), C1 esterase inhibitor, and C4 binding protein was also investigated. Immune complexes in PMR and GCA differed from those previously characterized in rheumatoid arthritis (RA)1 purified by anti-C1q-Sepharose which contained immunoglobulins and C1q only. No properdin or factor B were detected in RA IC purified with either anti-C1q-Sepharose or anti-C3c-Sepharose.

Aged↗
PubMed1985

Primary structure of the reactive site of human C1-inhibitor.

Human C1-inhibitor (C1-Inh) forms an equimolar complex with complement proteinase C1s that is resistant to dissociation by sodium dodecyl sulfate. The formation of this stable complex results in the cleavage of a peptide bond near the carboxyl terminus of the inhibitor and, whereas the bulk of C1-Inh remains covalently bound to the light chain of C1s, the postcomplex inhibitor peptide can be isolated under denaturing conditions. We have sequenced the amino-terminal region of this peptide and deduced that it represents the carboxyl-terminal side of the reactive site of C1-Inh. Limited proteolysis of C1-Inh by Crotalus atrox protease results in an active derivative lacking an amino-terminal peptide of 36 residues. Further proteolysis of this derivative with Pseudomonas aeruginosa elastase inactivates the inhibitor and a peptide is released. The amino-terminal sequence of this peptide overlaps with that of the postcomplex peptide and indicates that the residue imparting primary specificity to the inhibitor is arginine.

Amino Acid Sequence↗
PubMed1992

Dynamic equilibria between subcomponents of C1, the first component of human complement.

C1r and C1s, the serine protease components of activated C1, form a tetramer in the presence of Ca2+. The stability of this tetramer is sufficient that its association with the third component, C1q, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53-66 (1983)]. We have used the fluorescence anisotropy (A) of fluorescein-labeled C1s (s*) to monitor assembly and subcomponent exchange in 0.15 mol/l NaCl, 0.001 mol/l Ca2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r2s*2 causes a small but measurable delta A of 0.01-0.02. The response is too fast to measure at 37 degrees but can be readily followed at 4 degrees where t 1/2 = 0.6 min when [q] = [r2s*2] = 0.5 mumol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled C1s. Slow incremental addition of q to a solution of r2s*2 produces a dose-dependent delta A from which stoichiometry and dissociation constants can be derived. Measurements of Kd as a function of temperature establish an inverse temperature dependence with delta H = -15 kcal/mol and a value of Kd = 0.031 mumol/l at 37 degrees (delta G = + 11, T delta S = -26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex.

Complement C1r↗