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At least 19 recordsLinked to original sources
PubMed1991

Biochemical characterization and tissue distribution of hamster complement C1s.

Several mAb (PG11, NG7, and ED4) against hamster complement C1s were obtained. PG11 and NG7 were shown to cross-react with human and rat C1s. By using an immunohistochemical method, we examined localization of C1s in tissues of hamsters and rats. Present results revealed a widespread yet specific staining of hamster C1s which is associated with endoderm-, mesoderm-, and neuroectoderm-derived cells. For example, chondrocyte of hyaline cartilage and surface epithelium of the stomach were strongly positive. Intestinal epithelium, muscle cells, pia mater and epithelium of the choroid plexus of the ventricle, and hepatocytes were also stained. The synthesis of hamster C1s in these organs was confirmed by RNA blot hybridization. Secretion of C1s into the culture medium was revealed by immunoblot analysis in cell lines of hepatocytes, kidney cells, and myoblasts of rat or hamster.

Animals↗
PubMed1986

Kinetics of interaction of C1 inhibitor with complement C1s.

The kinetics of inhibition of the complement serine protease, C1s, by its only known inhibitor, C1 inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activity with a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtained by the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appears to be pure second order with a bimolecular rate constant of 6.0 X 10(4) M-1 s-1 at 30 degrees C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all three components (enzyme, inhibitor, and complex) with similar affinity (Kd = 2.0-3.3 microM). The fluorescent probe 1,1'-bis(anilino)-4-,4'-bi(naphthalene)-8,8'-disulfonate [bis(ANS)] bound to the complex with Kd = 0.26 microM under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.

Complement Activating Enzymes↗
PubMed1992

Calcium-linked self-association of human complement C1s.

The weight-average molecular weight of C1s, an activated serine protease subcomponent of human complement C1, has been measured by means of sedimentation equilibrium over a wide range of both protein and calcium ion concentrations. The combined data may be accounted for quantitatively by a simple model for Ca(2+)-dependent self-association of C1s to a dimer. According to this model, the monomer contains a single Ca2+ binding site with K approximately equal to 3 x 10(5) M-1, and the dimer contains three independent Ca binding sites, two having a Ca2+ affinity lower than that of the monomer (K approximately equal to 3 x 10(4) M-1). The third binding site in the dimer, which presumably lies at the interface between the two amino-terminal alpha domains, has a higher Ca2+ affinity (K approximately equal to 1 x 10(8) M-1) and provides the driving force for C1s dimerization in the presence of calcium.

Binding Sites↗
PubMed1990

NH2-terminal calcium-binding domain of human complement C1s- mediates the interaction of C1r- with C1q.

The assembly of C1, the first component of human complement, involves interactions between various domains of each of its three subcomponents, C1q, C1r, and C1s. The isolation, assignment of function, and structural characterization of the individual domains of C1r and C1s are critical for a thorough understanding of this complex assembly. The present study describes a 27-kDa plasmin-generated fragment derived from the NH2-terminal half of the heavy A chain of C1s-, the activated form of C1s. This fragment, C1s-alpha, was shown in the presence of Ca2+ to mimic the ability of whole C1s- to self-associate, bind to C1r-, and facilitate the binding of C1r to C1q. These results directly prove that the Ca2(+)-binding sites of C1s as well as all of the determinants necessary for binding of C1s- to C1r- and C1q are located in the NH2-terminal 27-kDa alpha region of the A chain.

Amino Acid Sequence↗
PubMed1980

Proteolytic cleavage of an activated subcomponent of the first component of rabbit complement, C1s.

When rabbit C1 purified by affinity chromatography on IgG-Sepharose 6B was chromatographed on DEAE-cellulose in the presence of ethylenediaminetetraacetate, C1s was isolated as two forms, C1s(I) and C1s(II), having different molecular weights. On the other hand, incubation of the C1 with soybean trypsin inhibitor before the chromatography resulted in the isolation of C1s(I) alone, indicating that, during the purification, C1s(II) was derived from C1s(I) by proteolytic cleavage of C1s(I) by a contaminating protease, probably plasmin [EC 3.4.21.7]. In fact, C1s(I) was completely converted to C1s(II) or a C1s(II)-like fragment by highly purified plasmin. Analysis of the polypeptide chain structures revealed that C1s(I), which consisted of H and L chains with molecular weights of 70,000 and 36,000, respectively, was converted to C1s(II) by cleavage of the H chain, since C1s(II) consisted of two chains each with a molecular weight of 37,000. This conversion proceeded without any alteration in C1 esterase activity, but was accompanied by loss of the ability to form C1r-C1s complex.

Animals↗
PubMed1991

Identification of the disulfide bonds of human complement C1s.

C1s, one of the three subcomponents of C1, the first component of the complement system, is a complex serine protease. To determine the disulfide-bonding pattern, fragments of C1s were generated by cleavage with pepsin, thermolysin, or subtilisin. Disulfide bonds have been identified by several methods, for example, direct observation of the phenylthiohydantoin derivative of cystine during Edman degradation of isolated peptides and placement in the known cDNA sequence. All of the 26 half-cystines are linked in disulfide bonds occurring at positions 50-68, 120-132, 128-141, 143-156, 160-187, 219-236, 279-326, 306-339, 344-388, 371-406, 410-534, 580-603, and 613-644. All of the disulfide bonds of the earlier described substructures of C1s, the EGF-homologous part, the two SCR units, and the two domains typical for C1s and C1r are localized within these domains.

Amino Acid Sequence↗
PubMed1988

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain.

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.

Amino Acid Sequence↗
PubMed1990

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.

Animals↗
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↗
PubMed1983

The serine proteinase chain of human complement component C1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments.

Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%).

Amino Acid Sequence↗
PubMed1987

Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence.

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.

Amino Acid Sequence↗
PubMed2026

Proteomic profiling of cephalic vein reveals potential biomarkers for arteriovenous fistula neointimal hyperplasia in ESRD patients.

Arteriovenous fistula (AVF) is the preferred vascular access for patients with end-stage renal disease; however, its failure is primarily due to neointimal hyperplasia. Five patients who underwent initial AVF surgery served as the control group, and another five patients with failed AVF surgery served as the experimental group. Herein, we employed mass spectrometry (MS)-based quantitative proteomics coupled with tandem mass tag labeling to screen differentially expressed proteins (DEPs) in the anastomotic cephalic vein, followed by bioinformatics analyses and verification experiments. A total of 121 DEPs were identified in the failed AVF group. GO analysis was primarily enriched in protein binding, nucleic acid binding, enzyme binding, mRNA binding, cadherin binding, catalytic activity, and cell adhesion molecule binding. KEGG pathways were mainly enriched in cell aggregation and adhesion, actin cytoskeleton, extracellular matrix-receptor interaction, PI3K-Akt signaling pathway, complement and coagulation cascades, and cholesterol metabolism. Protein-protein interaction network consisted of 86 (71.07%) DEPs, including complement VII (C7), factor IX (F9), SERPINC1, microfibril-associated glycoprotein 4 (MFAP4), complement C1s subcomponent, complement C1q subcomponent subunit A, complement C1q subcomponent subunit B, tissue factor, and von Willebrand factor, which interacting with numerous other proteins. In the expanded validation for different patients, C7, F9, SERPINC1, and MFAP4, were verified by immunohistochemical staining and Western blotting, which were consistent with the proteomics results. Collectively, this study identifies a series of potential diagnostic biomarkers, and explores the underlying mechanisms associated with AVF dysfunction.

Humans↗
PubMed1987

The complement component C1s catalysed hydrolysis of peptide 4-nitroanilide substrates.

The kinetic parameter kcat/Km has been determined for the hydrolysis of peptide 4-nitroanilides, catalysed by complement component C1s. Substrates based on the C-terminal sequence of human C4a (Leu-Gln-Arg) were synthesised. Replacement of the glutamine residue by glycine or serine increased kcat/Km. Substitution of valine for the leucine residue increased kcat/Km, while substitution of glycine or lysine for the leucine residue decreased kcat/Km slightly. D-Val-Ser-Arg 4-nitroanilide is the most reactive 4-nitroanilide substrate towards C1s, so far. These results are discussed in relation to the amino acid sequences near the bonds cleaved by C1s in C4, C2 and C1 inhibitor.

Amino Acid Sequence↗
PubMed1986

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation.

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.

Amino Acid Sequence↗
PubMed1992

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1979

Staphylococcus aureus opsonization mediated via the classical and alternative complement pathways. A kinetic study using MgEGTA chelated serum and human sera deficient in IgG and complement factors C1s and C2.

Staphylococcus aureus opsonization was studied kinetically by: (1) determination of the uptake of [3H]-thymidine labelled bacteria by human PMN's; (2) fluorescent anti-C3 and anti-IgG staining of opsonized bacteria; and (3) measuring bacterial complement consumption. Maximum opsonization in normal serum occurred within 5 min of incubation. About 80% of staphylococci were then taken up by PMN's, and IgG and C3b could be detected on the bacterial surface. In the absence of a functional classical complement pathway, as in sera deficient in C1s and C2 and in MgEGTA chelated serum, maximal opsonization was only achieved after 30--60 min incubation. Opsonization in IgG deficient serum occurred at a rate similar to that found in C2 deficient or MgEGTA chelated serum. Opsonization was greatly enhanced when sera were reconstituted. It was concluded that in IgG deficient serum Staphylococcus aureus opsonization is mediated via the alternative complement pathway. Dilution of normal serum primarily affected the classical complement pathway, resulting in a decreased rate of opsonization. In normal serum IgG did not appear to be a rate-limiting factor. S. Aureus opsonization was best studied by the phagocytosis assay and the fluorescent-antibody technique. Measuring haemolytic complement consumption was found to be an insensitive indicator of bacterial complement activation and opsonization.

Complement Activation↗