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PubMed1989

Sequence of the gene for murine complement component C4.

The gene for murine complement component C4 lies in the S region of the murine major histocompatibility (H-2) complex; in this paper, we report the nucleotide sequence of this gene. The present sequence extends from a SmaI restriction enzyme cleavage site near the 5' end of the gene to a KpnI restriction enzyme cleavage site 569 nucleotides 3' of the polyadenylation site. The sequence spans 15,956 base pairs and together with previously reported data provides a complete sequence extending from the site of transcriptional initiation to the polyadenylation site. The sequence reveals that the C4 gene has 40 introns which range from 75 to 1089 base pairs in length and which include three murine B1 middle repetitive elements, a MT repeat element, and an apparently novel repeat sequence that is also found in noncoding regions of the murine beta-glucuronidase, lymphotoxin (TNF-beta), and rat alpha-crystallin genes. An intron splits the protein coding sequence precisely at the site of proteolytic activation of C4 by complement protease C1s; however, except for this one case, the intron positions show no striking relationship to the structural features of the C4 protein. The length of the murine C4 gene relative to the isotypic C4A and C4B genes in man suggests the independent loss of a 6-kilobase intron from both murine and human C4 genes.

Animals↗

PubMed1998

Structure and function of the serine-protease subcomponents of C1: protein engineering studies.

Our protein engineering studies on human C1r and C1s revealed important characteristics of the individual domains of these multidomain serine-proteases, and supplied evidence about the cooperation of the domains to create binding sites, and to control the activation process. We expressed the recombinant subcomponents in the baculovirus-insect cell system and checked the biological activity. Deletions and point mutants of C1r were constructed and C1r-C1s chimeras were also produced. Our deletion mutants demonstrated that the N-terminal CUB domain and the EGF-like domain of C1r together are responsible for the calcium dependent C1r-C1s interaction. It seems very likely that these two modules form the calcium-binding site of the C1r alpha-fragment and participate in the tetramer formation. The deletion mutants also demonstrated that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine-protease module. The substrate specificity of the serine-protease is also determined by the five N-terminal noncatalytic domain of C1r/C1s chimera, which contains the catalytic domain of C1s preceded by the N-terminal region of C1r, could replace the C1r in the hemolytically active C1 complex. The C1s/C1r chimera, in which the alpha-fragment of the C1r was replaced for that of the C1s exibits both C1r- and C1s-like characteristics. We stabilized the zymogen form of human C1r by mutating the Arg(463)-Ile(464) bond. Using our stable zymogen C1r we showed that one active C1r in the C1 complex is sufficient for the full activity of the entire complex. Further experiment with this mutant could provide us with important information about the structure of the C1 complex.

Amino Acid Substitution↗

PubMed1987

Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles.

C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.

Antigen-Antibody Complex↗

PubMed1983

A new simple method for determination of C1-esterase inhibitor activity in plasma.

A convenient method for the determination of C1-esterase inhibitor activity in plasma samples is described. The method is based on addition of purified C1s to plasma and measuring excess C1s with a new chromogenic tripeptide-p-nitroanilide substrate with a recording spectrophotometer. Addition of C1s to C1-esterase inhibitor-depleted plasma did not result in any appreciable inactivation of the enzyme for three hours. The concentration of C1-esterase inhibitor in 19 healthy individuals was estimated as 1.63 +/- 0.27 (SD) mumol/l. The correlation with C1-esterase inhibitor antigen in these individuals and 19 patients with varying concentrations of C1-esterase inhibitor was excellent. The correlation with an antikallikrein assay was found to be poor.

Complement Activating Enzymes↗

PubMed1997

Regulation of C1-inhibitor function by binding to type IV collagen and heparin.

Serpins inhibit proteinases by a branched pathway, in which an intermediate serpin-proteinase complex can either form a stable covalent serpin-proteinase complex or produce reactive center cleaved serpin in a substrate reaction. It was tested whether these competing reactions could be regulated for the serpin C1-inhibitor by ligand binding. C1-inhibitor bound to type IV collagen, laminin, and entactin. Type IV collagen (10 microg/ml) caused an increase in the stoichiometry of inhibition for C1s inhibition by C1-inhibitor to 1.48 from 1.09 in the absence of ligand. A dose-dependent increase in the stoichiometry up to 1.27 in the presence of 100 microg/ml heparin was also observed. At low ionic strength the stoichiometry increased to 2.55. These data provide the first report that C1-inhibitor can bind to type IV collagen and also show that C 1-inhibitor can be regulated by ligand binding.

Binding, Competitive↗

PubMed1992

C1 inhibitor hinge region mutations produce dysfunction by different mechanisms.

Heterozygosity for a mutant dysfunctional C1 inhibitor protein, a member of the serine proteinase inhibitor (serpin) superfamily, results in type II hereditary angioneurotic oedema. We identified a "hinge" region mutation in C1 inhibitor with a Val to Glu replacement at P14 Val-432. Recombinant C1 inhibitors P10 Ala-->Thr and P14Val-->Glu did not form stable complexes with fluid phase C1s or kallikrein. The P14 Val-->Glu mutant, however, was cleaved to a 96K form by C1s, while the P10 Ala-->Thr mutant was not. The recombinant P10 mutant also did not complex with C1s, kallikrein or beta-factor Xlla-Sepharose. The two mutations, therefore, result in dysfunction by different mechanisms: in one (P14 Val-->Glu), the inhibitor is converted to a substrate, while in the other (P10 Ala-->Thr), interaction with target protease is blocked.

Alanine↗

PubMed1999

Hepatitis C virus NS3 serine protease interacts with the serpin C1 inhibitor.

Both NS3 protein (1007-1657) and its protease moiety (NS3p, 1027-1207) were able to interact in vitro with C1 Inhibitor (C1Inh) to give a 95-kDa Mr C1Inh cleavage product similar to that obtained upon proteolysis by complement protease C1s. High-Mr reaction products were also detected after incubation of C1Inh with NS3 but not with NS3p; they correspond to ester-bonded complexes from their hydroxylamine lability. Similar reactivity of NS3 was observed upon incubation with alpha2-antiplasmin. Serpin cleavage was prevented by treatment of NS3 with synthetic serine protease inhibitors. This interaction between viral NS3 and host serpins suggests that NS3 is likely to be controlled by infected cell protease inhibitors.

Complement C1 Inactivator Proteins↗

PubMed1987

Proteolysis and deglycosylation of human C1 inhibitor. Effect on functional properties.

The effects of proteolysis and deglycosylation on C1 inhibitor (C1Inh) were tested with respect to both its ability to form complexes with C1s and its capacity to block C1 autoactivation. Limited proteolysis of C1Inh by Staphylococcus aureus V8 proteinase, proline-specific endopeptidase or elastase generated a major high-Mr (approximately 86,000) fragment. In contrast with the fragment produced by elastase, which was inactive, the fragments resulting from V8 proteinase and proline-specific endopeptidase treatment retained activity. Deglycosylation with N-glycanase or O-glycanase, or both, had no major effect on the functional activity of C1Inh.

Complement C1 Inactivator Proteins↗

PubMed1991

Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status.

The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor.

Amino Acid Sequence↗

PubMed1975

Cationic proteins of human granulocytes. VI. Effects on the complement system and mediation of chemotactic activity.

The chymotrypsin-like cationic proteins of human granulocytes are shown to possess the ability to produce conversion of the complement components C1s, C4, C3, and C5 as detected by crossed immuno-electrophoresis. This ability seems to be a direct proteolytic effect. Incubation of cationic proteins with serum or functionally pure preparations of C3 and C5 is shown to generate the formation of chemotactic activity which is abolished by prolonged incubation. Also, the chemotactic activity of porcine C5a or spontaneously activated C5 is abolished by incubation with cationic proteins. It is suggested that the chymotrypsin-like cationic proteins of human granulocytes after extrusion from the phagocytic cell play an important role for generation of inflammatory mediators.

Antigen-Antibody Reactions↗

PubMed1995

Improved method for measuring C1-r-C1-s-(C1 inh)2 complexes by an enzyme-linked immunosorbent assay.

Measurement of C1-r-C1-s-(C1 inh)2 complexes in serum or plasma by enzyme-linked immunosorbent assay (ELISA) has been proposed as a relatively convenient and sensitive means for assessing C1 activation. However, interference by unactivated C1q (r-s)2 at low serum or plasma dilutions has resulted in estimates that vary widely with the degree of serum or plasma dilution. Precipitating the interfering C1q (r-s)2 with 6% polyethylene glycol has been proposed to resolve this problem, but here it is shown that this procedure also precipitates or coprecipitates some of the C1-r-C1-s-(C1 inh)2 complexes. Satisfactory results have been achieved without PEG precipitation by testing high plasma dilutions under conditions where there is a sufficient excess of anti-C1s coating the microtitration plate wells that removal of C1q (r-s)2 is not necessary. Optimizing conditions for quantitating these complexes at high dilution have been investigated. The mean normal EDTA plasma C1-r-C1-s-(C1 inh)2 complex measurement was 36.6 +/- 7.0 (S.D.) ELISA units with a 95% confidence interval of 19.5-47.6u. Besides providing a sensitive assay for C1 activation, measuring C1-r-C1-s-(C1 inh)2 complexes may help to clarify the pathophysiologic mechanisms resulting from C1 inh deficiency under various conditions.

Adult↗

PubMed1993

Protein engineering studies on C1r and C1s.

1. C1r and C1s cDNAs were placed downstream the strong polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus and the recombinant proteins were expressed in insect cells, in biologically active form. The yield of expression is high enough to get recombinant components for chemical and functional studies (5 micrograms/ml cell culture supernatant). 2. The biological activity and the post-translational modifications of the recombinant subcomponents were checked. The rC1r and rC1s proved to be biologically active in the hemolytic assay, although their glycosylations were different compared to that of the serum proteins. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. 3. Two deletion mutants of C1r cDNA were constructed in order to clarify the role of domain I and II. The results show that both, domain I, and II are absolutely necessary for the tetramer formation and both have a regulatory role in the autoactivation. The autoactivation of the mutants is accelerated significantly. 4. Hybrid cDNA constructions were also made, and one of them was expressed. In the C1s alpha R hybrid the C1s alpha part cannot dimerize in presence of Ca2+, but it can form a tetramer with C1r2, that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. 5. In order to control the autoactivation process point mutant cDNAs were constructed through altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation. These results do justify our approach of using domain-domain interchange, domain deletion and point mutations in combination, to reveal the structural background of C1 function at intramolecular level.

Animals↗

PubMed1985

Evaluation of euglobulin methods for the study of blood fibrinolytic activity: results for patients with rheumatoid arthritis and in the postoperative period.

Euglobulin fractionation is a frequently employed pretreatment of plasma for the determination of fibrinolytic activity. The fractionation procedure suffers from possible in vitro artifacts, e.g., variable precipitation of C1-inactivator. This is illustrated by the following two situations. It is shown that increased amounts of C1-inactivator not related to an increased plasma concentration are present in euglobulin fractions in cases of classic rheumatoid arthritis. Similarly, postoperatively, a disproportional increase in C1-inactivator in euglobulin fractions occurs. In both cases, an artificially reduced fibrinolytic activity is recorded due to increased inhibition by C1-inactivator. This is circumvented and recognized by adding sodium flufenamate or C1s-esterase to euglobulin fractions to uniformly eliminate C1-inactivator. Two specific assays for tissue-type plasminogen activator activity in euglobulin fractions (as C1-inactivator-resistant activator activity and a parabolic rate assay on a synthetic substrate) correlate excellently (r = 0.8728; p less than 0.001; n = 108). The first mentioned is corrected for variable endogenous C1-inactivator; the latter assay is found to be insensitive to inhibition by C1-inactivator. It is concluded that with euglobulin methods a misinterpretation of blood fibrinolytic activity is possible in rheumatoid arthritis patients. In the postoperative period, the fibrinolytic shutdown concerns tissue-type plasminogen activator activity; the pattern of the shutdown can be misjudged in using traditional euglobulin methods.

Abdomen↗

PubMed1996

Biotinylation of proteins via amino groups can induce binding to U937 cells, HL-60 cells, monocytes and granulocytes.

The use of biotinylated ligands for the flow cytometric detection of cell surface receptors has become a popular alternative to radioreceptor assays. Although the biotinylation of a protein is a relatively mild chemical reaction several reports have mentioned the fact that the number and location of biotin moieties coupled to amino groups of a protein can alter its physicochemical properties and impair biological activity. In the present study we show for a variety of biotinylated functionally unaltered ligands that biotinylation by N-hydroxysuccinimide (NHS) esters of biotin can induce a binding to cell surfaces, which is not specific for the respective unlabelled ligand. C1q, C1 inhibitor (C1-INH), alpha 1-antitrypsin (AT), ovalbumin (OV), transferrin and soybean trypsin inhibitor (STI) were labelled with S-NHS-LC-biotin and activated C1s (C1s) with NHS-biotin. Biotinylation of C1q, C1s and C1-INH exerted negligible effects on biological function, antigenicity or electrophoretic mobility but when labelled and unlabelled proteins were assayed for binding to monocytic U937 cells, promyelocytic HL-60 cells, monocytes and granulocytes, a remarkable binding was observed for biotinylated C1q, C1-INH and C1s. In contrast, no binding was observed when we used unlabelled C1q, C1s and C1-INH and employed specific antibodies, alpha-mouse-FITC or alpha-rabbit-FITC for detection. Increasing molar ratios of biotin-to-protein (B : P) for biotinylated AT, OV and STI evoked increased fluorescence intensities of the cells. Most importantly the unlabelled ligands did not compete for cell binding with their biotinylated derivatives, with the exception of transferrin. Preincubation of the cells with an excess of free d-biotin did not reduce binding of biotinylated proteins, thus excluding a potential involvement of biotin receptors. Hydrophobic interaction chromatography revealed a remarkable increase in hydrophobicity of the biotinylated proteins compared to their unlabelled counterparts, suggesting that the biotinylation-induced binding is due to increased hydrophobicity. Our findings indicate that biotinylation by the common amino acid esterification method may be critical for proteins if they are to be used as ligands for receptor binding studies.

Amino Acids↗

PubMed1981

Immunofluorescence studies on the subcomponents of the first component of complement (C1): detection of C1q and C1s in different cells of biopsy material and on human as well as on guinea pig peritoneal macrophages.

The first component of complement (C1) is a macromolecule consisting of three distinct subcomponents, C1q, C1r, and C1s. In regard to its production site and its role in phagocytic processes it was of interest to find out whether these different subcomponents could be detected in human biopsy material only as a complex in individual cells or whether C1 subcomponents could be found on different cells. To study this question, monospecific fluorescein-labelled anti-human-C1q IgG and monospecific rhodamine-labelled anti-human C1q IgG were used. Biopsy material from human rectum was stained with fluoresceinated antisera, either by use of one antiserum or by double staining. Using this technique, these observations were made: C1q as well as C1s were detectable in individual cells in the subepithelial area of the gut. Furthermore, C1q and C1s could be found together in the same cell or separately in different cells. These findings were supported by experiments with cultured peritoneal macrophages either from human or from guinea pig. The examination of the cultured cells with the two antisera revealed that individual cells were stained either by anti-C1q or by anti-C1s antibodies. The specificity of the detection of the individual subcomponents was also proven by the peroxidase technique and by using fluoresceinated anti-human C1q F(ab')2. The membrane immunofluorescent staining revealed the presence of C1q on the membrane of the macrophage.

Animals↗

PubMed1981

Purification and characterization of two functionally distinct forms of C1 inhibitor from a patient with angioedema.

A minority of patients with hereditary angioedema (HAE) have normal concentrations of a dysfunctional C1 inhibitor protein (C1INH) in their plasmas. We purified C1INH from the plasmas of one such patient before and during treatment with the anabolic steroid stanozolol. Both the pretreatment plasma and plasma obtained during stanozolol treatment contained varying amounts of two extremely similar C1INH proteins that were functionally distinct. The pretreatment plasma contained primarily (94%) dysfunctional C1INH that did not inactivate or complex with either purified C1s, activated Hageman factor, or kallikrein and small amounts (6%) of functionally normal C1INH. Stanozolol treatment increased the plasma concentrations of both of these proteins as well as the proportion (23%) of functional C1INH in the plasma. The purified dysfunctional and functional C1INHs had identical or nearly identical molecular sizes, charges, amino acid compositions, and amino sugar contents, and could not be distinguished physicochemically from each other or from normal C1INH. From these studies of purified C1INH proteins we concluded that HAE associated with dysfunctional C1INH is due to a defect at the structural locus for one C1INH gene and that both the dysfunctional C1INH gene and the normal C1INH gene products are present in the plasma of the affected subject. Treatment with stanozolol comparably increased the synthesis of both C1INH proteins. The disproportionate rise in the level of the normal C1INH protein is consistent with the view that it is more rapidly catabolized as a consequence of its interaction with the proteases it inactivates.

Adult↗

PubMed1997

C1 inhibitor-C1s complexes are internalized and degraded by the low density lipoprotein receptor-related protein.

Like other serpin-enzyme complexes (SECs), proteinase-complexed C1 inhibitor (C1-INH) is rapidly cleared from the circulation and thought to be a neutrophil chemoattractant, suggesting that complex formation causes structural rearrangements exposing a domain which is recognized by specific cell surface receptors. However, the cellular receptor(s) responsible for the catabolism and potential mediation of chemotaxis by C1-INH-protease complexes remained obscure. To determine whether the SEC receptor mediates the binding and potential chemotaxis of C1-INH.Cs, we performed binding assays with HepG2 cells, neutrophils, and monocytes, and the results show that C1-INH.Cs neither bind to these cells nor cause a chemotactic response of neutrophils and monocytes. Furthermore, C1-INH.Cs, the COOH-terminal C1 inhibitor peptide, or the tetrameric C1-INH.Cs.Cr. C1-INH complex were found to be significantly less effective in competing with the SEC receptor ligand 125I-peptide 105Y for the binding to HepG2 cells than unlabeled 105Y, indicating that the SEC receptor does not sufficiently recognize C1-INH-protease complexes. The asialoglycoprotein receptor was also ruled out to be responsible for the removal of the heavily glycosylated C1-INH.Cs complex, since asialoorosomucoid did not compete for the clearance of C1-INH. 125I-Cs and asialoglycoprotein receptor knockout mice showed no alterations in the C1-INH.125I-Cs clearance rate. We found that C1-INH.125I-Cs complexes were efficiently degraded by normal murine fibroblasts expressing the low density lipoprotein receptor-related protein (LRP) and cellular degradation was significantly reduced by chloroquine and the receptor-associated protein, which is a potent inhibitor of the binding of all known ligands to LRP. Moreover, receptor-associated protein inhibited the in vivo clearance of C1-INH.125I-Cs and murine fibroblasts genetically deficient for LRP did not degrade C1-INH.125I-Cs. Our results demonstrate that C1-INH. Cs complexes do not stimulate neutrophil or monocytic chemotaxis but are removed by LRP, further underscoring its role as a serpin-enzyme complex clearance receptor.

Animals↗

PubMed1984

Lack of activation of C1, despite circulating immune complexes detected by two C1q methods, in patients with rheumatoid arthritis.

The activation of C1 by circulating immune complexes in patients with rheumatoid arthritis was investigated. C1rC1s(C1-In)2 complexes in EDTA-plasma, reflecting C1 activation in vivo, were slightly raised in 35 of 57 patients with rheumatoid arthritis, though most patients had elevated levels of circulating immune complexes as measured with either the 125I-C1q binding test or the C1q solid phase assay. The activation of C1 by circulating immune complexes in vitro was investigated by measuring the generation of C1rC1s(C1-In)2 complexes during 60 minutes at 37 degrees C in diluted recalcified EDTA-plasma. In 16 of the 57 patients, a slightly increased C1 activation in vitro was observed. These patients tended to have high levels of circulating immune complexes. However, the majority of the patients with high levels of circulating immune complexes showed a normal C1 activation in vitro. Therefore, it was concluded that measurement of circulating immune complexes by either of the two C1q methods in patients with rheumatoid arthritis does not imply that these circulating immune complexes are able to activate C1.

Aged↗