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PubMed1985

The C1q subunit of the first component of complement binds to laminin: a mechanism for the deposition and retention of immune complexes in basement membrane.

The C1q subunit of complement component C1 is known to bind to immune complexes, which often are deposited in basement membrane. We investigated the possibility that this deposition is a result of binding to laminin, a large basement membrane glycoprotein. C1q showed saturable binding to immobilized laminin; this binding was increased at reduced ionic strength. Intact C1 did not bind laminin. A ternary complex was formed by laminin, C1q, and aggregated IgG. This complex formation was dependent on and proportional to the amount of C1q bound to the aggregated IgG. Binding of laminin to C1q occurred with a Kd of 2 nM and was stronger than the binding of C1q to fibronectin. Preliminary data, including electron micrographs of rotary-shadowed preparations, suggest that laminin binds to the collagen-like tail of C1q. Electron microscopy localized the site of interaction with C1q to a short arm of laminin. Since laminin is found only in basement membranes, the interaction between laminin and C1q could be involved in the deposition and retention of immune complexes in these structures.

Antigen-Antibody Complex↗
PubMed1990

Limited proteolysis of beta 2-microglobulin at Lys-58 by complement component C1s.

We have now demonstrated that activated complement component C1s cleaves beta 2-microglobulin at the position identical to that at which beta 2-microglobulin is cleaved in serum of patients suffering from lung cancer. The main cleavage is in the disulphide loop C-terminal to Lys-58, generating a modified form of beta 2-microglobulin with a two-chain structure. The C-terminal Lys-58 in the A chain is highly susceptible to removal by a carboxypeptidase-B-like activity causing the formation of des-Lys58-beta 2-microglobulin. This is the first demonstration of a noncomplement protein substrate for the proteolytic activity of C1s. The C1s-induced cleavage of beta 2-microglobulin can be inhibited in the presence of C1 esterase inhibitor, demonstrating a regulatory function of C1 esterase inhibitor in the C1s-induced cleavage of beta 2-microglobulin.

Amino Acid Sequence↗
PubMed1988

Role of EAC1q4 in C1a transfer reaction (C1aTR) and further information on the nature of the EAC1q4 site.

The complement intermediates EAC1, EAC4, and EAC1q4 were prepared with guinea pig, porcine, as well as human complement. EAC4 and EAC1q4 were made from EC4 and EAC14 respectively. The C1a transfer reaction (C1aTR), the second step of Borsos' C1a fixation and transfer test, was carried out with various combinations of these intermediates. It was found that the EAC1q4, instead of the EAC4, was the C1a acceptor, and the C1rs subcomponents rather than the whole C1 molecule should have to transfer in the C1aTR. The EAC41 derived from EC4 generated no EAC1q4 in EDTA medium as the EAC14 did. This presented evidence for the joining of C4 to A in the EAC1q4 site.

Animals↗
PubMed1989

Autoantibody facilitated cleavage of C1-inhibitor in autoimmune angioedema.

C1-inhibitor (C1-Inh) is an important inhibitor of the inflammatory response and deficiency of this inhibitor, which may be hereditary or acquired, is associated with recurrent episodes of edema. Recently, an autoimmune form of angioedema has been described that is associated with functional deficiency of C1-Inh and an autoantibody that impedes C1-Inh function. In this report we describe the isolation of C1-Inh from the monocytes and plasma of a patient with autoimmune angioedema and demonstrate that the patient's monocytes secrete structurally and functionally normal C1-Inh, but show that this protein circulates in the patient's plasma in an inactive, structurally altered form. Furthermore, using analytic gel electrophoresis techniques it is demonstrated that the patient's autoantibody facilitates cleavage of normal C1-Inh, by its target proteases, to the same species of C1-Inh that is found circulating in the patient's plasma. This autoantibody facilitated cleavage of normal C1-Inh is apparently a consequence of destabilization of protease/inhibitor complexes. These findings contribute to our understanding of protease/C1-Inh interactions and document important observations on pathogenic mechanisms in autoimmune disease.

Angioedema↗
PubMed1990

Receptor expression and functional status of cultured human eosinophils derived from umbilical cord blood mononuclear cells.

Selective use of recombinant human cytokines has enabled the culture of large numbers of eosinophils from human cord blood mononuclear cells, raising the possibility of their use as a model of eosinophil function. Cultured eosinophils (CE) were compared with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and function. Fc gamma R and CR1 expression of CE and PBE was similar. In contrast, the specific mean fluorescence for LFA-1 alpha, p150,95 alpha, ICAM-1, and HLA-DR was significantly elevated for CE compared with PBE. CE responded in PAF-induced chemotaxis in a similar fashion to PBE. CE gave higher numbers of both resting and platelet activating factor (PAF)-stimulated immunoglobulin G (IgG)- and C3b-dependent rosettes than PBE. CE and PBE had comparable capacity to kill IgG- and C-opsonized schistosomula in terms of both baseline values and PAF-induced enhancement of cytotoxicity. Baseline adherence by CE and PBE to plasma-coated glass was essentially the same, but stimulated adhesion (PAF) of CE was lower. Compared with PBE, CE generated less than half the amounts of extracellular and cell-associated PAF induced by calcium ionophore A23187 stimulation. Unlike PBE, CE did not generate PAF after exposure to IgG-coated Sepharose particles. CE stimulated with IgG-coated beads generated small quantities of LTC4, while A23187 stimulation resulted in approximately half the LTC4 levels observed with PBE. The total cell content of eosinophil peroxidase (EPO) was similar for CE and PBE. These data suggest that although CE and PBE have many phenotypic and functional properties in common there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

Animals↗
PubMed1990

Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q.

The serum lectin, mannan binding protein (MBP), was isolated in a yield of 40 micrograms/liter from pooled normal human serum by affinity chromatography on mannan-Sepharose, followed by gel-filtration and ion-exchange chromatography and finally by passage down an anti-IgM Sepharose column. A rabbit antiserum was prepared against the purified MBP and an enzyme-linked immunoassay developed that used both the specificity of the polyclonal antibody and the Ca+(+)-dependent carbohydrate binding property of MBP. Assay of the sera from 103 blood-donors showed a wide range of MBP levels, ranging from 0 to 870 micrograms/liter. MBP, after interaction with zymosan, caused efficient activation of a C1r2 125I-C1s2 complex that was prepared by incubation of 125I-C1s2 with serum, from a patient with a complete genetic deficiency of C1q, followed by gel-filtration on Sepharose 6B. The purified MBP is composed of a mixture of trimers, tetramers, pentamers, and hexamers of an approximate 90-kDa structural unit as judged by chromatography, SDS-PAGE and electron microscopy studies. Only the molecules in the pentamer/hexamer fraction, which have a similar overall structure to that of C1q, appeared to cause efficient, zymosan-dependent, activation of C1s within the C1r2C1s2 complex. The pentamer/hexamer form of MBP may therefore play an important role in antibody-independent activation of the C system during the early stages of certain infections.

Calcium↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1985

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.

Complement Activating Enzymes↗
PubMed1986

Biosynthesis of the subcomponents C1q, C1r and C1s of the first component of complement (C1) by guinea pig hepatocyte primary cultures.

Thus far, the synthesis of C1q by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10(6) cells/ml) were cultured under serum-free conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4-8) antibody directed against a nonpolymorphic Ia determinant and preabsorbed rabbit serum. The hemolytic activity of C1 and its subcomponents C1q and C1r/C1s was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 X 10(3) effective C1q molecules/cell/24 h and 34 X 10(3) effective C1r/C1s molecules/cell/24 h. The synthesis of C1q and C1r/C1s could be reversibly inhibited by cycloheximide (50 micrograms/ml). Furthermore, to demonstrate de novo synthesis of the C1q subcomponent, endogeneous labeling with 3H-proline (or 14C-proline) was performed. The immunoprecipitated C1q from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig C1q from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize C1 subcomponents, whereby the synthesis of C1q and C1r/C1s occurs independently.

Animals↗
PubMed1989

FN-C1q and C1 INH C1r-C1s complexes as indicators of complement activation in patients with chronic lymphocytic leukaemia.

We have previously found low levels of C1 and C4 INH in the sera of chronic lymphocytic leukaemia (CLL) patients. Hypocomplementaemia was supposed to be the consequence of a permanent activation of the classical pathway. We have compared the levels of C1 INH-C1rC1s and C1q-FN complexes in the sera of 95 CLL patients and 100 healthy controls, because these complexes are known to be formed in the early stage of classical pathway activation. A significant increase in the level of both types of complexes was found in sera of CLL patients as compared to the controls. These findings support the assumption that the classical complement pathway is activated in the patients with CLL.

Antigen-Antibody Complex↗
PubMed1985

Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides.

Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue.

Amino Acids↗
PubMed1986

Genetic studies of low abundance human plasma proteins. III. Polymorphism of the C1R subcomponent of the first complement component.

Genetic polymorphism of the C1R subcomponent of human complement component C1 has been detected in normal plasma samples using the high resolving power of isoelectric focusing in 6 M urea followed by immunoblotting. There are two common alleles at the C1R structural locus that show autosomal codominant inheritance. The C1R*1 and C1R*2 allele frequencies in U.S. white and U.S. black blood donors are: .934, .066, and .899, .101, respectively.

Alleles↗
PubMed1988

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.

Animals↗
PubMed1986

The functional inhibition of activated C1 inhibitor in normal human serum causes spontaneous consumption of the complement components C2, C3, C4, and factor B.

The human complement components C1r, C1s, C4, C3, factor B, and/or activated C1INH were functionally blocked in normal human serum (NHS) and EGTA- or EDTA-treated NHS by polyclonal monospecific Fab'-fragments to the individual components. The results of inhibition experiments are compatible with the formation of a classical pathway fluid-phase C3 convertase (C4b2a) spontaneously generated by the inhibition of activated C1INH. This process in both NHS and EGTA-NHS was accompanied by the consumption of C2, C4, C3, and factor B but only by poor enhancement of C5 conversion. Blocking subcomponent C1r, completely inhibited spontaneous activation of the complement components, indicating that the control of C1r hydrolysis is the essential role of activated C1INH as a regulator of C1 activation in NHS. Non-complement serum proteases were inactive during the initiation of the activation process. The presence of blood cells during functional inhibition of activated C1INH in NHS slightly decreased the consumption of C3 but not of C2 and C4.

Angioedema↗
PubMed1987

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.

Amino Acid Sequence↗