Human C1r Research Articles

The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-β family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-β superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.

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.

Complement components C1r/C1s, bone morphogenic protein 1 and Xenopus laevis developmentally regulated protein UVS.2 share common repeats

Property patterns were constructed, based on an alignment of related domains in human complement subcomponents C1r and C1s as well as in the sea urchin protein uEGF. This kind of consensus pattern was able to identify similar domains in a human bone morphogenic protein, in a Xenopus laevis embryonal protein involved in dorsoanterior development and in a calcium-dependent serine protease secreted from malignant hamster embryo fibroblast cells. Because of the high level of overall sequence homology this protease may be the hamsters' equivalent of the human complement subcomponent C1s. The resulting multiple alignment of all studied domains suggests functionally and structurally important regions.

Ca2+ binding properties and Ca2(+)-dependent interactions of the isolated NH2-terminal alpha fragments of human complement proteases C1-r and C1-s.

The NH2-terminal alpha fragments of human complement proteases C1-r and C1-s were obtained by limited proteolysis of the native proteins with trypsin, and isolated. C1-r alpha extended from residues 1 to 208 of C1-r A chain, with at least two cleavage sites within disulfide loops, after lysine 134 and arginine 202. C1-s alpha comprised residues 1-192 of the C1-s A chain, with one cleavage site within a disulfide loop, after arginine 186. C1-r alpha was monomeric either in the presence or absence of Ca2+ but formed Ca2(+)-dependent dimers with native C1-s. C1-s alpha dimerized in the presence of Ca2+ and formed Ca2(+)-dependent tetramers (C1-s alpha-C1-r-C1-r-C1-s alpha) with native C1-r. C1-r alpha and C1-s alpha associated in the presence of Ca2+ to form C1-r alpha-C1-s alpha heterodimers. Equilibrium dialysis studies indicated that each alpha region binds Ca2+ with a dissociation constant ranging from 19 microM (native proteins) to 38 microM (fragments). C1-r alpha, C1-r alpha-C1-s alpha, and the native C1-s-C1-r-C1-r-C1-s tetramer bound 0.9, 1.9, and 4.0 Ca2+ atoms/mol, respectively, whereas dimers C1-s alpha-C1-s alpha and C1-s-C1-s incorporated 2.9 and 3.0 Ca2+ atoms/mol. It is concluded that each alpha region contains one high affinity Ca2+ binding site. This 1:1 stoichiometry is maintained upon heterologous (C1-r-C1-s) interaction, whereas the homologous (C1-s-C1-s) interaction provides one additional binding site.

Isolation and functional characterization of the proenzyme form of the catalytic domains of human C1r

The proenzyme form of C1r catalytic domains was generated by limited proteolysis of native C1r with thermolysin in the presence of 4-nitrophenyl-4'-guanidinobenzoate. The final preparation, isolated by high-pressure gel permeation in the presence of 2 M-NaCl, was 70-75% proenzyme and consisted of a dimeric association of two gamma B domains, each resulting from cleavage of peptide bonds at positions 285 and 286 of C1r. Like native C1r, the isolated domains autoactivated upon incubation at 37 degrees C. Activation was inhibited by 4-nitrophenyl-4'-guanidinobenzoate but was nearly insensitive to di-isopropyl phosphorofluoridate; likewise, compared to pH 7.4, the rate of activation was decreased at pH 5.0, but was not modified at pH 10.0. In contrast, activation of the (gamma B)2 domains was totally insensitive to Ca2+. Activation of the catalytic domains, which was correlated with an irreversible increase of intrinsic fluorescence, comparable with that previously observed with native C1r [Villiers, Arlaud & Colomb (1983) Biochem. J. 215, 369-375], was reversibly inhibited at high ionic strength (2 M-NaCl), presumably through stabilization of a non-activatable conformational state. Detailed comparison of the properties of native C1r and its catalytic domains indicates that the latter contain all the structural elements that are necessary for intramolecular activation, but probably lack a regulatory mechanism associated with the N-terminal alpha beta region of C1r.

Expression of Hemolytically Active Human Complement Component Clr 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.

Genetic polymorphism of human C1R subcomponent of the first complement component in the Japanese population

Genetic polymorphism of the C1R subcomponent of human complement component C1 has been investigated in neuraminidase treated EDTA plasma samples of 440 healthy Japanese individuals living in Tokyo by means of thin-layer polyacrylamide gel isoelectric focusing (PAGIEF) at pH 3.5–9.5 in the presence of 8.0 M urea followed by an electroblotting with enzyme immunoassay. Three common and three rare alleles were detected in the Japanese population. Of these, two common alleles were identical to C1R ∗1 and C1R ∗2 and other new alleles were tentatively designated C1R ∗3, C1R ∗4, C1R ∗5 and C1R ∗6, respectively. The results of the family studies suggested that the genetic model for C1R polymorphism assumed autosomal codominant Mendelian inheritance. The allele frequencies were estimated as C1R ∗1 = 0.4216, C1R ∗2 = 0.3602, C1R ∗3 = 0.2068, C1R ∗4 = 0.0091 and C1R ∗R(C1R ∗5 and C1R ∗6) = 0.0023, respectively. The distribution of allotypes fitted the Hardy-Weinberg equilibrium. The C1R system provides a useful genetic marker for human genetics, anthropologic studies and forensic science.

Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.

Assignment of the complement serine protease genes C1r and C1s to chromosome 12 region 12p13.

C1r and C1s are distinct, but structurally and functionally similar, serine protease zymogens responsible for the enzymatic activity of the first component of complement (C1). Recent comparisons indicate a significant degree of sequence similarity between C1r and C1s and support the hypothesis that they are related by gene duplication. Complementary DNA probes for human C1r and C1s do not cross-hybridize even at mild stringency conditions and are therefore gene-specific. Using a panel of 25 human-rodent cell hybrids, we have independently assigned the C1r and the C1s genes to chromosome 12. In situ hybridization analyses were consistent with these assignments, showing in addition that both C1r and C1s are located on the short arm of the chromosome in the region p13. These data suggest that the homologous C1r and C1s genes have remained closely linked after duplication of a common ancestor. The C1r and C1s loci also provide useful polymorphic DNA markers for the short arm of chromosome 12.

Identification of erythro-β-hydroxyasparagine in the EGF-like domain of human C1r

Previous studies [(1987) Biochem. J. 241, 711–720] have shown that position 150 of human C1r is occupied by a modified amino acid that, after acid hydrolysis, yields erythro-β-hydroxyaspartic acid. In view of further investigations on the nature of this residue, peptide CN1a T8/T9 TL8 (positions 147–155) was isolated from C 1 r A chain by CNBr cleavage followed by enzymatic cleavages by trypsin and thermolysin. Amino acid analysis, sequential Edman degradation and FAB-MS of this peptide indicate that the residue at position 150 is an erythro-β-hydroxyasparagine resulting from post-translational hydroxylation of asparagine.

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.

Cloning and sequencing of full-length cDNA encoding the precursor of human complement component C1r.

The sequencing of human liver cDNA clones encoding the entire C1r precursor protein has confirmed the previously determined peptide sequence and has shown that there is a leader peptide which is 17 amino acids long. A residue tentatively identified as beta-hydroxyaspartic acid [Arlaud, Willis & Gagnon (1986) Biochem. J., in the press] located in the C1r A-chain, within an epidermal-growth-factor consensus sequence, was found to be encoded as asparagine. Two sequence elements, tandemly located in the A-chain, are related to a sequence widespread among proteins which interact with C3b or C4b. Structural comparisons between different clones indicate that multiple polyadenylation sites are responsible for the length heterogeneity observed for C1r mRNA from liver and Hep G2 cells.

Molecular characterization of the catalytic domains of human complement serine protease C1r.

Limited cleavages of human C1r by extrinsic proteases of various specificity (plasmin, elastase, chymotrypsin, thermolysin) yield dimeric associations of two globular domains, each comprised of the intact B chain disulfide linked to gamma, the C-terminal fragment of the A chain. These (gamma-B)2 domains, which are homologous to those obtained from C1r by autolytic cleavage [Villiers, C. L., Arlaud, G. J., & Colomb, M. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4477-4481], represent the core of the C1r molecule and are associated with the catalytic properties of the serine active site. V8 protease also yields (gamma-B)2 associations, although additional cleavages occur in the B chain. Sequence analysis shows that all cleavages generating the gamma fragments occur within a 13-residue sequence extending from positions 274 to 286 of the C1r A chain. Chemical cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide of the (gamma-B)2 catalytic domains obtained from C1r autolytic cleavage indicates that each gamma-B domain interacts with its neighbor in a "head to tail" configuration, the gamma region of one domain interacting with the B chain of the other domain, and conversely. No evidence is found of gamma-gamma or B-B interactions. Such a head to tail configuration, placed in the context of the model proposed for the C1s-C1r-C1r-C1s catalytic subunit of C1 [Colomb, M. G., Arlaud, G. J., & Villiers, C. L. (1984) Philos. Trans. R. Soc. London, B 306, 283-292], is compatible with autolytic activation of C1r through an intramolecular cross-mechanism and with subsequent activation of C1s by activated C1r.

Purification, identification and characterization of chicken C1q, a subcomponent of the first component of complement

A component, having the equivalent haemolytic activity to that of human complement subcomponent C1q, was purified by a combination of precipitation with EGTA, gel filtration, ion exchange and adsorption chromatography from chicken serum. Yields ranged from 8 to 15 mg/litre of serum. The finally purified preparation generates full Cl haemolytic activity when assayed with human complement subcomponents C1r and C1s, and have been identified as chicken C1q. The molecular weight of undissociated C1q, as estimated on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS), is 504 000. Under dissociating but non-reducing conditions, the C1q was shown to consist of 2 subunits having molecular weights of 52 700 and 51 200 in a molar ratio of 2:1. On reduction, the 52 700 molecular weight subunit gave chains with molecular weights of 25 900 and 24 800 in equimolar ratio, and the 51 200 molecular weight subunit decreased to 24 800. The C1q contains hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 7% carbohydrate. Collagenase digestion of C1q caused a rapid loss of haemolytic activity and produced much smaller peptide fragments.

Domain structure and associated functions of subcomponents C1r and C1s of the first component of human complement.

The serine protease subcomponents of the activated form of the first component of human complement (C1), C1r and C1s, were observed by electron microscopy after the native proteins and their limited proteolysis products, obtained from autolytic cleavage (C1r) or from incubation with plasmin (C1s) were rotary shadowed. At the monomeric level, both C1r and C1s comprised two globular domains, a smaller interaction domain (corresponding to the NH2-terminal half of the A chain, alpha, and responsible for calcium binding and C1r-C1s interaction) and a larger catalytic domain (corresponding to the COOH-terminal part of the A chain, gamma, disulfide-linked to the B chain and bearing the serine protease active site). The two globular domains are linked by a connecting strand, beta. The (C1r)2 dimer appeared as a "croissant"-like association, where the two monomers interact through their catalytic domains. On the basis of the domain structure of C1r and C1s, a model of the calcium-dependent C1s dimer is proposed, in which the two monomers interact through their NH2-terminal interaction domains; in the same way, a model of the C1s-(C1r)2-C1s catalytic subunit of C1 is presented, in which (C1r)2 forms a core, its distal interaction domains interacting with the corresponding domains of C1s.

C1r serine proteinase of human complement: a case of intramolecular autolytic activation.

This paper presents a short review of our contribution to the knowledge of the structure and function of human C1r, the activation unit of C1, the first component of the classical pathway of complement. On the basis of the domain structure of C1r, a model accounting for its autolytic activation mechanism is proposed. We suggest that this represents the basic mechanism of C1 function.

Autoactivation of human complement subcomponent C1r involves structural changes reflected in modifications of intrinsic fluorescence, circular dichroism and reactivity with monoclonal antibodies.

Autoactivation of C1r is closely correlated with an irreversible increase of its intrinsic fluorescence. The activation and the fluorescence increase of C1r are accelerated on addition of activated C1r. Ca2+, di-isopropyl phosphorofluoridate and C1 inhibitor, which all inhibit, although to different extents, C1r activation, inhibit in parallel the fluorescence increase. C1r activation is blocked at pH 4.0-5.0, whereas it is accelerated at pH 10.5; under the same conditions the fluorescence increase shows parallel effects. No such fluorescence increase is observed during C1s activation by trace amounts of C1r. Far-u.v. circular-dichroism spectra of C1r indicate 73 and 78% of unordered form in both the proenzyme and the activated species respectively. The slight changes observed on activation are not restricted to C1r, as comparable results are obtained for proenzyme and activated C1s. C1r activation appears thus to involve structural changes leading to an 'activated state' distinct from the 'proenzyme state'. Monoclonal antibody to activated C1r is poorly reactive with proenzyme C1r, a finding that also supports this hypothesis.

Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r.

The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118.

Kinetics of the reaction between human C1-esterase inhibitor and C1r or C1s.

The kinetics of the reactions of C‐esterase inhibitor with Cs or Cr has been studied in purified systems. Both reactions were found to proceed in two steps: a fast reversible second‐order reaction followed by a slower irreversible first‐order transition according to the following scheme: . The rate constant k1 for the reaction between Cs and C‐esterase inhibitor was found to be 1.4 × 105 M−1 s−1 at 25°C and 4.7 × 105 M−1 s−1 at 37°C, while the corresponding values for the reaction between Cr and C‐esterase inhibitor were 2.5 × 103 M−1 s−1 and 4.4 × 104 M−1 s−1, respectively. The dissociation constant K of the initial complex was estimated as 1–2 nM for the complex with Cs and 20–50 nM for the complex with Cr. Heparin was found to accelerate the rate of the Cs/C‐esterase inhibitor reaction by a 16‐fold increase in k1 at heparin concentrations above 100 mg/l, whereas no effect was obtained on the thermodynamic equilibrium. The inhibition of purified Cs added to dilutions of plasma or serum was found to proceed as expected from their known concentrations of C‐esterase inhibitor and the rate constants determined in the purified system. Thus, the half‐life of Cs in plasma under pseudo‐first‐order kinetics would be about 1 s

Characterization of the activation of the human C1r complement molecule

The proenzyme form of Clr 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 Clr had the tendency to spontaneously activate within 60–90 min of incubation at 37 C in presence of EDTA and more slowly in the presence of Ca 2+. The spontaneous activation of Clr 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 Ca 2+ without affecting the hemolytic Clr activity. [ 14C]DFP bound to trace proteins in the 60–90 kD range, but not to Clr proenzyme. The spontaneous activation of Clr was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca 2+ from Clr, thereby changing the conformation of the protein and causing an increased digestibility of the Clr H-chain. At temperatures above 0–4°C this influence destroyed the ability of Clr proenzyme and enzyme to form macromolecular Cl and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous Clr activation in the pH range 6–9 and in the presence of Ca 2+ is due to contaminant proteases. Clr 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 Clr activation in the Cl molecule, because the hemolytic activity of Clr was substantially diminished under the high pH conditions.