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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↗
PubMed2000

The complement component C1s is the protease that accounts for cleavage of insulin-like growth factor-binding protein-5 in fibroblast medium.

Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions, understanding the chemical identity and regulation of this protease is important for understanding how IGF-I stimulates anabolic functions. The protease was purified from human fibroblast-conditioned medium by hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS-polyacrylamide gel electrophoresis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Sequencing of the high pressure liquid chromatography-purified peptides yielded the complement components C1r and C1s. To confirm that C1r/C1s accounted for the proteolytic activity in the medium, immunoaffinity chromatography was performed. Most of the protease activity adhered to the column, and the eluant was fully active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa band contained only C1r and C1s. C1r in the fibroblast medium underwent autoactivation, and the activated form cleaved C1s. C1s purified from the conditioned medium cleaved C(4), a naturally occurring substrate. The purified protease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit activated C1s, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete C1r and C1s that actively cleave IGFBP-5. The findings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.

Amino Acid Sequence↗
PubMed1995

Structure of the catalytic region of human complement protease C1s: study by chemical cross-linking and three-dimensional homology modeling.

C1s is a multidomain serine protease that is responsible for the enzymatic activity of C1, the first component of the classical pathway of complement. Its catalytic region (gamma-B) comprises two contiguous complement control protein (CCP) modules, IV and V (about 60 residues each), a 15-residue intermediary segment, and the B chain (251 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the gamma-B fragment of C1s, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; then cross-linked peptides were isolated after CNBr cleavage and thermolytic digestion. N-Terminal sequence and mass spectrometry analyses allowed us to identify two cross-links between Lys 405 of module V and Glu 672 of the B chain and between Glu 418 of the intermediary segment and Lys 608 of the B chain. Three-dimensional modeling of the CCP modules IV and V and of the catalytic B chain was also carried out on the basis of their respective homology with the 16th and 5th CCP modules of complement factor H and type I serine proteases. The information provided by both the chemical cross-linking studies and the homology modeling enabled us to construct a three-dimensional model for the assembly of the C-terminal part of the gamma-B region, comprising module V, the intermediary segment, and the B chain. This model shows that module V interacts with the serine protease B chain on the side opposite to both the activation site and the catalytic site. Functional implications of this interaction are discussed in terms of the possible role of module V in the specific recognition and positioning of C4, one of the two substrates of C1s.

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↗
PubMed1998

Baculovirus-mediated expression of truncated modular fragments from the catalytic region of human complement serine protease C1s. Evidence for the involvement of both complement control protein modules in the recognition of the C4 protein substrate.

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (gamma-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP2-ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP2-ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6-3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic gamma-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1s. Likewise, the activated fragments gamma-B, CCP2-ap-SP, and ap-SP retained C1s ability to cleave C2 in the fluid phase. In contrast, whereas fragment gamma-B cleaved C4 as efficiently as C1s, the C4-cleaving activity of CCP2-ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1s has no significant effect on catalytic activity.

Animals↗
PubMed1987

Variations in the enzymatic properties of human complement subcomponent C1s by treatment with human plasma kallikrein.

We have investigated the effect of plasma kallikrein digestion upon hydrolytic activities of human C1s. Incubation of C1s (85 kDa) with plasma kallikrein led to progressive cleavages on the heavy chain to yield C1s-K1 (70 kDa) then C1s-K2 (53 kDa). Although these cleavages caused little change in the C2 hydrolytic and esterase activities of C1s, a marked loss in the C4 hydrolytic activity was observed. C1s-K1 and C1s-K2 were purified by DE-52 chromatography and it was found that the proteolysis of C1s into C1s-K1 was accompanied with a decrease in the C4 hydrolytic activity. Although the turnover numbers for the hydrolysis of C4 by C1s-K1 and C1s-K2 were almost the same as that of intact C1s, the Kms for C4 of C1s-K1 and C1s-K2 were found to be increased to 10 times that of intact C1s. This result suggests that the apparent decrease in the C4 hydrolytic activity upon plasma kallikrein digestion of C1s is not due to disruption in the active site but is due to decrease in the affinity between C4 and the C1s derivatives. In support of this assumption, C1s-K1 was found to be devoid of the ability to bind C4b-Sepharose. C1s is capable of forming a dimer through the C1s-binding domain in the N-terminal side of the heavy chain. Although C1s-K1 is still capable of forming a dimer, C1s-K2 fails to form a dimer, suggesting that the N-terminal C1s-binding site is released during cleavage of C1s-K1 into C1s-K2.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Affinity↗
PubMed1983

Heparin-stimulated modification of C1-inhibitor by subcomponent C1s of human complement.

C1-inhibitor and C1s form a very stable complex which migrates with an apparent molecular mass of 180 kDa in dodecyl sulfate gel electrophoresis. A small fraction of the inhibitor (100 kDa) was found to be converted to a large (95 kDa) and a small (2 to 5 kDa) fragment during this reaction. It is concluded that C1-inhibitor is modified by C1s in a similar way as are the related plasma inhibitors alpha 1-proteinase inhibitor, antithrombin III and antiplasmin by their specific proteinases. The fraction of modified C1-inhibitor increased when heparin was present during complex formation. This reaction was complete after 15 s and is comparable with the fast heparin induced formation of modified antithrombin III. Treatment with hydroxylamine led to a complete dissociation of the inhibitor-enzyme complex by dodecyl sulfate. The large inhibitor fragment (and not unmodified inhibitor as reported by other authors) was released.

Complement Activating Enzymes↗
PubMed1981

Conformational changes of the subunits C1q, C1r and C1s of human complement component C1 demonstrated by 125I labeling.

C1s and C1r proenzymes and enzymes (C1s, C1r) and C1q were labeled with 125I. The distribution of the 125I label between H- and L-chain of C1s was only slightly dependent on the state of activation of C1s, and approx. 90% of the label was found in the H-chain. In the C1r proenzyme molecules 50% of the label was incorporated into the H-chain. The C1r H-chain label was reduced to 10% on activation of C1r to C1r, while the L-chain label increased to 90% of the total label. The presence of either C1s, C1q or C1qs during labeling reduced the C1r H-chain level, although C1r remained in the proenzyme form. The presence of C1s or C1rs enhanced the 125I uptake of C1q in Ca2+ or EDTA medium. This was unexpected because one would have anticipated a diminution of the C1q label due to the apposition of C1r and C1s, similarly as it occurs during C1rs complex and C1s dimer formation for the H-chain label of C1s. The results show that C1r and C1q alter their conformation during activation and C1 complex formation.

Calcium↗
PubMed1986

Functional model of subcomponent C1 of human complement.

The domain organization of the zymogen subunits of the first component of human complement C1s, C1r2 and the complex C1s-C1r2-C1s was studied by electron microscopy. In the absence of Ca2+, monomeric C1s was visualized as a dumb-bell-shaped molecule consisting of two globular domains (center-to-center distance 11 nm) connected by a rod. One of the globular domains is assigned to the light chain (B-chain) of the activated molecule, which is homologous to trypsin and other serine proteases. The second globular domain and the rod are assigned to the heavy chain (A-chain) of CIs. The subunit C1r is a stable dimer in the presence or absence of Ca2+. This dimer C1r2 was visualized as composed of two dumb-bells of dimensions similar to those observed for C1s. These are connected near the junctions between the rod and one of the globular domains. This leads to the structure of an asymmetrical X with two inner closely spaced globules (center-to-center distance 7 nm) and two outer globules at a larger distance (14 nm). By comparison with fragment C1rII2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of C1r2 is readily recognized in the central portion of the thread-like 54 nm long C1s-C1r2-C1s complex formed in the presence of Ca2+. By affinity-labeling of C1s with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that C1s forms the outer portion of the complex. A detailed model of C1s-C1r2-C1s is proposed, according to which two C1s monomers bind to the outer globes of C1r2 by contacts between their heavy chains and those of C1r. According to this model the catalytic domains of C1r are located in the center and those of C1s at the very tips of the C1s-C1r2-C1s complex. On the basis of the structure of C1s-C1r2-C1s, we derived a detailed model of the C1 complex (composed of C1q and the tetrameric complex) and we discuss this model with a view to finding a possible activation mechanism of C1.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activating Enzymes↗
PubMed2004

Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein.

Many secretory proteins are synthesized as proforms that become biologically active through a proteolytic cleavage in the trans-Golgi complex or at a later stage in the secretory pathway. Haptoglobin (Hp) is unusual in that it is cleaved in the endoplasmic reticulum before it enters the Golgi. Here, we present evidence that the recently discovered complement C1r-like protein (C1r-LP) mediates this cleavage. C1r-LP has not previously been shown to possess proteolytic activity, despite its homology to trypsin-like Ser proteinases. We demonstrate that coexpression of the proform of Hp (proHp) and C1r-LP in COS-1 cells effected cleavage of proHp in the endoplasmic reticulum. This cleavage depended on proteolytic activity of C1r-LP because mutation of the putative active-site Ser residue abolished the reaction. Furthermore, incubation of affinity-purified C1r-LP and proHp led to the cleavage of the latter protein. ProHp appeared to be cleaved at the expected site because substitution of Gly for Arg-161 blocked the reaction. C1r-LP showed specificity for proHp, in that it did not cleave the proform of complement C1s, a protein similar to Hp particularly around the cleavage site. C1r-LP accounts for at least part of the endogenous proHp-cleavage activity because suppression of the C1r-LP expression by RNA interference reduced the cleavage of proHp by up to 45% in the cells of a human hepatoma cell line (HepG2).

Amino Acid Sequence↗
PubMed1989

Effect of human mast cell tryptase on human plasma proenzymes.

The effect of human skin mast cell tryptase on human plasma proenzymes (prothrombin, coagulation factor XII, complement C1s, protein C and plasminogen) was investigated. Tryptase had no effect on these proenzymes, when incubated with them at 37 degrees C for up to 90 min, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the ability to hydrolyze specific peptide p-nitroanilide substrates. After prolonged treatment with tryptase, proenzymes could be fully activated with their specific activators. The results indicate that tryptase neither activates these plasma proenzymes nor inactivates the corresponding active enzymes. As a positive control, the tryptase preparation was also incubated with human fibrinogen and rat thymus histones. Prolonged treatment with tryptase increased the thrombin-induced clotting time of fibrinogen. Tryptase also efficiently hydrolyzed histone H1 from rat thymus. Histones H3/H2B and H2A were hydrolyzed less efficiently than H1, and no hydrolysis of histone H4 by tryptase was detected under the experimental conditions.

Animals↗
PubMed1979

The purification and characterization of subcomponent C1s of the first component of bovine complement.

Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.

Amino Acids↗
PubMed1991

C1 inhibitor: different mechanisms of reaction with complement component C1 and C1s.

Inactivation of human complement subcomponent C1-s by its regulator C1 inhibitor at physiological ionic strength proceeded at a 3-fold higher rate when C1-s was in the physiological C1- complex with subcomponents C1q and C1-r rather than as purified subunit. When the C1- complex was disassembled by chelation of calcium, the C1-s subcomponent was inactivated by C1 inhibitor at rates similar to those for the purified proteinase. Increasing ionic strength had little effect on the reaction of purified C1-s with C1 inhibitor but greatly diminished the rate of reaction of intact C1-. Addition of heparin accelerated the inactivation of purified C1-s by C1 inhibitor up to 25-fold but increased the inactivation of intact C1- only about 5-fold. These differences in the inactivation of C1-s by C1 inhibitor, depending on whether the proteinase is free or complexed with other subcomponents of C1-, suggest different mechanisms of reaction. Occurrence of subcomponent C1-s in a macromolecular complex with C1q and C1-r, thus, appears to be critical not only for directing its physiological activation but also its inactivation.

Complement C1↗
PubMed1998

NMR structures of the C-terminal end of human complement serine protease C1s.

Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular alpha-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue alpha-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672.

Amino Acid Sequence↗