Purified proenzyme C1r. Some characteristics of its activation and subsequent proteolytic cleavage

Arlaud, Gérard J.; Villiers, Christian; Chesne, Serge; Colomb, Maurice G.

doi:10.1016/0005-2744(80)90269-7

Cited by 88 publications

(76 citation statements)

References 26 publications

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“…Several authors reported that the autoactivation of Clr was catalyzed not by activated Cli but by an active site possibly generated in a zymogen Clr [4,5]. The intermediary Clr having an autocatalytic site was termed Clr*, which was proposed to be reversibly inhibited by NPGB [4].…”

Section: Discussionmentioning

confidence: 99%

“…An alternative possibility is that a proteolytic site is rapidly generated in parallel to the conformational change of Clr but catalyzes only slowly the activation of altered Clr into active Cl;, which once formed, catalyzes rapidly proteolytic activation of altered Clr. Although this mechanism may well explain the kinetics of autoactivation of Clr, there are several reports suggesting that Clf by itself has no ability to activate Clr [4,5]. However, it was reported in an abstract [ 1 l] that autoactivation of Clr involveda double mechanism: the first intramolecular catalysis due to Clr itself; the second intermolecular reaction due to activated ClI itself.…”

Section: Discussionmentioning

confidence: 99%

“…Although there remain inconsistencies about details of the activation mechanism, the activation of Cl r has been accepted to be an autocatalytic reaction [3-61 and the proteolytic site for autoactivation of Clr has been proposed to be generated in a zymogen Clr through a conformational change [4,5]. However, no report directly indicates that a conformational change occurs in a zymogen Clr prior to the proteolytic activation of Clr.…”

Section: Introductionmentioning

confidence: 99%

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Formation of a conformationally changed C1r, a subcomponent of the first component of human complement, as an intermediate of its autoactivation reaction

et al. 1982

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Formation of a conformationally changed C1r, a subcomponent of the first component of human complement, as an intermediate of its autoactivation reaction

et al. 1982

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“…Proteins-Proenzyme C1s and activated C1r and C1s were purified from human plasma as described previously (15,16). The C1 s ␥-B fragment was obtained by limited proteolysis with plasmin (17) and purified by ion-exchange chromatography on a Mono Q HR 5/5 column (Pharmacia Biotech Inc.) as described previously (12).…”

Section: Methodsmentioning

confidence: 99%

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Rossi

Bally

Thielens

et al. 1998

Journal of Biological Chemistry

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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 (␥-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 (CCP 2 -ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP 2 -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 ␥-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1 r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1 s. Likewise, the activated fragments ␥-B, CCP 2 -ap-SP, and ap-SP retained C1 s ability to cleave C2 in the fluid phase. In contrast, whereas fragment ␥-B cleaved C4 as efficiently as C1 s, the C4-cleaving activity of CCP 2 -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 C1 s, 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 C1 s has no significant effect on catalytic activity.The first component of complement comprises two homologous, yet distinct modular serine proteases C1r 1 and C1s assembled in the form of a Ca 2ϩ -dependent tetramer C1s-C1r-C1r-C1s. C1r is responsible for intrinsic C1 activation, a twostep process first involving autolytic C1r activation and then C1 r-mediated cleavage of proenzyme C1s. The active enzyme C1 s is a highly specific protease with trypsin-like specificity that mediates limited proteolysis of C4 and C2, the two protein substrates of C1 , thereby triggering activation of the classical pathway of complement in response to infection by various microorganisms (2-5). C4 cleavage occurs in the fluid phase and generates fragment C4b, which has the transient ability to bind covalently to the surface of the target. Cleavage of the second substrate C2, in contrast, takes place after prior formation of a C4b-C2 complex and yields C4b-C2a, the protease responsible for cleavage of complement protein C3 (5). This double proteolytic activity of C1 is controlled by C1 inhibitor, a member of the serine protease inhibitor (serpin) family, which reacts stoichiometrically with both C1 r and C1 s to form covalent protease-inhibitor complexes, resulting in disruption of the C1 s-C1 r-C1 r-C1 s tetramer (2).Human C1s is synthesized as a 673-residue single chain zymogen which, upon activation by C1 r, is cleaved between Arg 422 and Ile 423 to yield two disulfide-linked polypeptides, the N-terminal A chain ...

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“…25,26 Isolation of the C1s-C1r-C1r-C1s tetramer was performed as described previously. 27,28 The concentrations of purified C1q and C1s-C1r-C1r-C1s were determined spectrophotometrically using values of absorbance (1%, 1 cm) at 280 nm (A 280 ) of 6Á8 and 13Á5, and MW values of 459 300 and 330 000, respectively.…”

Section: Complement Proteinsmentioning

confidence: 99%

Studies on the interactions between C‐reactive protein and complement proteins

et al. 2007

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Studies on the interactions between C-reactive protein and complement proteins IntroductionThe classical acute-phase reactant C-reactive protein (CRP) is a pentameric, disc-shaped serum protein.1 Its basic features are the control of inflammation, the stimulation of clearance of damaged cell and tissue components, and the initiation of repair functions.2 CRP shows calcium-dependent affinity for phosphate monoesters, such as phosphatidylcholine, but several other ligands of CRP have been characterized, including damaged cell membranes, small ribonucleoprotein particles, apoptotic cells and fibronectin. Native CRP can undergo subunit dissociation into individual monomeric units, for example upon association with negatively charged lipid monolayers. 4 This modified form of CRP can be produced in vitro by urea chelation, acid treatment, heating or direct immobilization of native CRP onto polystyrene.5 This modified form of CRP (mCRP) has reduced solubility and exhibits different electrophoretic characteristics as the result of a decrease of isoelectric point (pI) from 6Á4 to 5Á4. 6 The structural changes releasing CRP subunits from the pentamer are correlated with expression of a new antigenic reactivity and formation of neo-epitopes. 7 The forms of CRP expressing SummarySeveral studies have investigated the interactions between C-reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b-binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme-linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1-INH. Native, ligand-unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea-treatment or by site-directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1-activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.

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Purified proenzyme C1r. Some characteristics of its activation and subsequent proteolytic cleavage

Cited by 88 publications

References 26 publications

Formation of a conformationally changed C1r, a subcomponent of the first component of human complement, as an intermediate of its autoactivation reaction

Formation of a conformationally changed C1r, a subcomponent of the first component of human complement, as an intermediate of its autoactivation reaction

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Studies on the interactions between C‐reactive protein and complement proteins

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