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At least 19 recordsLinked to original sources
PubMed1982

Characterization of the activation of the human C1r complement molecule.

The proenzyme form of C1r was isolated by sequential chromatography from the euglobulin fraction of human serum on DEAE-Sepharose 6B-CL, CM-Sepharose 6B-CL and Sepharose S-300-CL. This C1r had the tendency to spontaneously activate within 60-90 min of incubation at 37 degrees C in presence of EDTA and more slowly in the presence of Ca2+. The spontaneous activation of C1r was found to be a bimolecular process and could be completely inhibited by DFP in the pH range 6-9 and in the presence of Ca2+ without affecting the hemolytic C1r activity. [14C]DFP bound to trace proteins in the 60-90 kD range, but not to C1r proenzyme. The spontaneous activation of C1r was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca2+ from C1r, thereby changing the conformation of the protein and causing an increased digestibility of the C1r H-chain. At temperatures above 0-4 degrees C this influence destroyed the ability of C1r proenzyme and enzyme to form macromolecular C1 and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous C1r activation in the pH range 6-9 and in the presence of Ca2+ is due to contaminant proteases. C1r activated also spontaneously at higher pH values between pH 9 and 13.2, but the spontaneous activation ceased abruptly at pH 13.4. An intramolecular process of activation cannot be excluded at these high pH values. It is, however, not clear, whether this activation is a suitable model for the C1r activation in the C1 molecule, because the hemolytic activity of C1r was substantially diminished under the high pH conditions.

Complement Activating Enzymes↗
PubMed2001

Identification of cDNA encoding a serine protease homologous to human complement C1r precursor from grafted mouse skin.

We isolated a cDNA clone from grafted mouse skin that encodes a serine protease homologous to human C1r. The C1r protease is involved in the activation of the first component of the classical pathway in the complement system. In order to identify novel transcripts whose expression is regulated in grafted mouse skin, we first performed differential display reverse transcription polymerase chain reaction analysis and obtained 18 partial cDNA clones whose protein products are likely to play an important role in allograft rejection. One of these showed significant sequence homology with human complement C1r precursor. The other clones displayed no homology to any known sequences, however. Northern blot analysis demonstrated that the level of this transcript was upregulated in day 8 postgrafted skin. The full-length cDNA 2121 nucleotides in length obtained from screening a mouse skin cDNA library contained a single open reading frame encoding 707 amino acid residues with a calculated molecular weight of 80,732 Da. Its deduced amino acid sequence revealed an 81% identity and 89% similarity to the human C1r counterpart. In particular, mouse C1r contained His501, Asp559, and Ser656, which were conserved among this group of serine proteases. This protein was thus designated as mouse C1r. We have expressed a truncated fragment of C1r protein without the N-terminal hydrophobic sequence in Escherichia coli and generated a polyclonal antibody against it. Subsequent immunohistochemical analysis confirmed that mouse C1r was significantly expressed 8 d after the skin graft in both allografted and autografted skins, compared with normal skins. These collective data suggest that a component of the complement system, C1r, might contribute to the graft versus host immune responses in mice.

Animals↗
PubMed1996

Exon structure of the gene encoding the human mannose-binding protein-associated serine protease light chain: comparison with complement C1r and C1s genes.

Mannan or mannose-binding protein (MBP) requires a novel serine protease termed MBP-associated serine protease (MASP) for activation of the complement cascade. In this study, we analyzed MASP genomic clones and found that the light chain (catalytic domain) is encoded by six exons, whereas those of the complement C1 subunits, C1r and C1s, and the haptoglobin segment have been reported to be encoded by a single exon. We confirmed the intron-lacking sequence of C1r by analysis of its genome. These results, in conjunction with those obtained by constructing a phylogenetic tree for these proteins, suggest that the MASP gene is prototype and that the intron-lacking sequences of the other serine proteases have a more recent history.

Binding Sites↗
PubMed1989

Genetic studies of low-abundance human plasma proteins. XIII. Population genetics of C1R complement subcomponent and description of new variants.

Isoelectric focusing and immunoblotting reveals considerable biochemical and genetic variation in the C1R subcomponent of the first complement component. The nature of the intraindividual biochemical variation can be explained by differences in sialic acid content because after digestion with neuraminidase the terminal sialic acids are removed to yield a single major band corresponding to the C1R polypeptide. Plasma samples from a large number of different ethnic groups, consisting of U.S. whites, U.S. blacks, Nigerian blacks, and Inuit, Aleut, and Amerindian populations from the Western Hemisphere have revealed genetically determined charge variation with heterozygous phenotypes consisting of two major asialo bands, indicating that the underlying variation is not due to variation in sialic acid content. Two previously reported common alleles, C1R*1 and CIR*2, have been observed in all studied populations, the notable exception being the Dogrib Indian population, which is devoid of the C1R*2 allele. Several new alleles--designated C1R*3, C1R*4, C1R*5, C1R*6, and C1R*7-have been observed, with variable frequencies ranging from the occurrence in a single individual and related family members to the polymorphic occurrence of certain alleles in several populations. Of these new alleles, the C1R*5 is of considerable interest in population and anthropological genetics studies. The C1R*5 allele is widely distributed, at a frequency of .03 to .17, in all of the North American aboriginal populations screened. This allele is not present in U.S. whites but is present at a polymorphic frequency in U.S. and Nigerian blacks.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed1989

Human complement C1r and C1s proteins and genes: studies with molecular probes.

The isolation of complementary DNA clones for both enzymic subcomponents of C1 has made it possible to derive their complete amino acid sequences and to verify and extend previous protein data. We review here recent advances in studies of the C1r and C1s proteins and of the corresponding genes, using molecular probes. The mosaic structure of these proteins has been compared to the exon-intron organization of the C1s gene. Surprisingly, the C1r and the C1s genes feature an intronless serine protease domain, at variance with all vertebrate serine proteases. Moreover, C1r and C1s are related in evolution to haptoglobin, a serine protease analog lacking enzymic activity. The C1r and C1s genes are closely linked in an unusual tail to tail orientation. These findings are discussed with regard to the apparently coordinate expression of these complement components and to the combined nature of most C1r and C1s deficiencies. We also discuss the implications of the successful production of C1r protein using recombinant DNA technology.

Chromosomes, Human, Pair 12↗
PubMed1986

Nucleotide sequence of the cDNA coding for human complement C1r.

C1r is a zymogen of a serine protease that is involved in the activation of the first component of the classical pathway of the complement system. cDNAs coding for human C1r have been isolated from libraries prepared from poly(A) RNA from human liver and Hep G2 cells. From DNA sequence analysis, the overlapping cDNA inserts were shown to span 2493 nucleotides of the C1r mRNA, not including the poly(A) tail. The cDNA sequence coding for C1r contained a 5' noncoding region, 2115 nucleotides coding for a polypeptide precursor of 705 amino acids, and a 3' noncoding region. Some variability in the length of the 3' noncoding sequence was observed with the cDNA inserts, although most contained a polyadenylation signal followed by a poly(A) tail. The A or noncatalytic chain of C-1r, which originates from the amino-terminal end of the precursor molecule, contains a potential growth factor domain and two different pairs of internal repeats. One pair of these internal repeats is closely related to the amino-terminal sequence of C1s, while the other pair of repeats is homologous to the tandem repeats present in beta 2-glycoprotein I, complement factor B, the b subunit of factor XIII, and a single region present in the alpha 1 chain of haptoglobin. The B chain of C-1r contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.

Amino Acid Sequence↗
PubMed1992

A calcium-binding monoclonal antibody that recognizes a non-calcium-binding epitope in the short consensus repeat units (SCRs) of complement C1r.

C1r is a Ca(2+)-binding serine protease that interacts with two other plasma proteins, C1q and C1s, to form C1, the first component of the complement cascade. A monoclonal antibody, BG6, has been produced which binds to C1r only in the presence of Ca2+, requiring 3-5 microM Ca2+ for half-maximal binding. The antibody reacts with native and heat-denatured C1r, and with zymogen C1r, but does not cross-react with C1s or C1q. BG6 did not significantly affect the esterolytic activity of C1r toward a synthetic thioester substrate nor the hemolytic activity of C1 reconstituted from subcomponents in the presence of the antibody. A tryptic fragment of C1r which consists of the C-terminal gamma region of the A chain disulfide-linked to the B chain (gamma B) binds in a Ca(2+)-dependent manner to BG6-Sepharose. Western blotting experiments have further localized the epitope to the gamma region of the A chain, which is composed of two short consensus repeat (SCR) units. The N-terminal alpha region contains the only previously determined Ca(2+)-binding site in the C1r molecule. Equilibrium dialysis experiments confirmed that C1r-gamma B does not bind Ca2+, and showed that antibody BG6 and the gamma B/BG6 complex do bind Ca2+. Thus, the Ca(2+)-dependent nature of this interaction is due exclusively to binding of the metal ion to the antibody. Equilibrium dialysis and immunoblotting have further localized the Ca(2+)-binding site to the Fab fragment of BG6, indicating that the metal-induced conformational change residues in or near the variable region of the IgG. BG6 may set a precedent for the preparation of Ca(2+)-dependent antibodies to non-Ca(2+)-binding epitopes in other proteins.

Antibodies, Monoclonal↗
PubMedDate not supplied

Familial deficiency of two subunits of the first component of complement. C1r and C1s associated with a lupus erythematosus-like disease.

Complete absence of C1r and almost complete absence of C1s were found in 4 of 8 living siblings. Two of the 4 suffer from a syndrome that combines discoid lupus erythematosus and nondeforming rheumatoid-like arthritis; one of the siblings has mild nephritis. The other 2 C1 deficient family members are clinically well. Evidence from this and other families suggests that deficiency of C1 components or C4 is associated with higher risk of developing a lupus-like disease than is deficiency of C2.

Adolescent↗
PubMed2003

Complete sequencing and expression of three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway of the complement system in rainbow trout Oncorhynchus mykiss.

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M(r) of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M(r) of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a alpha-chain of 773 aa, a beta-chain of 635 aa and a lambda-chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M(r) of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.

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↗
PubMed1986

Characteristics of complement subcomponents C1r and C1s synthesized by Hep G2 cells.

The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.

Animals↗
PubMed1989

Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector.

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.

Animals↗
PubMed1992

Complement component C1r mediated cleavage of the heavy chain of the major histocompatibility class I antigens.

Apart from cleaving C1s, we demonstrate for the first time that: 1) at concentrations found in serum, the activated forms of the complement components C1r in addition to C1s can cleave the heavy chain of MHC class I antigens, 2) the cleavage by C1r and C1s is seemingly dependent upon a native configuration of the MHC class I antigen, since heat denaturation of the HLA antigens reduce the cleavage. The proteolytic fragments following C1 cleavage were characterized by precipitation with Con A-Sepharose, anti-MHC class I and anti-beta 2-microglobulin antibodies. The proteolysis of the alpha-chain of MHC class I was shown to take place between the alpha 2- and alpha 3- domains as estimated by the Con A-Sepharose precipitation pattern on SDS-PAGE. The alpha 1/alpha 2 fragment was still shown to interact with beta 2-microglobulin as shown by immunoprecipitation.

Antibodies, Monoclonal↗
PubMed1999

The N-terminal CUB-epidermal growth factor module pair of human complement protease C1r binds Ca2+ with high affinity and mediates Ca2+-dependent interaction with C1s.

The Ca2+-dependent interaction between complement serine proteases C1r and C1s is mediated by their alpha regions, encompassing the major part of their N-terminal CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In order to define the boundaries of the C1r domain(s) responsible for Ca2+ binding and Ca2+-dependent interaction with C1s and to assess the contribution of individual modules to these functions, the CUB, EGF, and CUB-EGF fragments were expressed in eucaryotic systems or synthesized chemically. Gel filtration studies, as well as measurements of intrinsic Tyr fluorescence, provided evidence that the CUB-EGF pair adopts a more compact conformation in the presence of Ca2+. Ca2+-dependent interaction of intact C1r with C1s was studied using surface plasmon resonance spectroscopy, yielding KD values of 10.9-29.7 nM. The C1r CUB-EGF pair bound immobilized C1s with a higher KD (1.5-1.8 microM), which decreased to 31.4 nM when CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+ concentrations ranging from 5 microM with intact C1r to 10-16 microM for C1ralpha and CUB-EGF. The isolated CUB and EGF fragments or a CUB + EGF mixture did not bind C1s. These data demonstrate that the C1r CUB-EGF module pair (residues 1-175) is the minimal segment required for high affinity Ca2+ binding and Ca2+-dependent interaction with C1s and indicate that Ca2+ binding induces a more compact folding of the CUB-EGF pair.

Animals↗
PubMed1991

Isolation of human complement subcomponents C1r and C1s in their unactivated, proenzyme forms.

We have modified a standard isolation procedure for C1r and C1s, which employs IgG-Sepharose affinity chromatography followed by DEAE chromatography. As usual, all steps were performed at low temperature and two proteolytic inhibitors, PMSF and NPGB, were added during affinity chromatography on IgG-Sepharose. The novel condition was to keep the pH at pH 6.1 during the entire procedure, where activation was markedly depressed. In addition, purification was improved by washing the IgG-Sepharose column with a buffer free of added divalent cations immediately prior to elution of the C1r and C1s with EDTA. The final yields of highly purified C1r and C1s were about 20%; little or no activated material was detected in these highly purified fractions.

Chromatography, Affinity↗
PubMed1987

Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r.

Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗