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PubMed1998

2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease.

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Complement C1r↗
PubMed1989

Genetic studies of low-abundance human plasma proteins. XI. Linkage analysis and population genetics of the C1S subcomponent of the first complement component.

Although subcomponents C1r and C1s of the first complement component, C1, have been established to be in the same linkage group as the proline-rich protein gene cluster on chromosome 12p13.2, no direct analysis of linkage between the C1R and C1S structural gene loci has been available. We have detected through a population screening study 5 families which are heterozygous at the structural loci for both C1R and C1S. Three of the 5 families, 21 individuals, were informative for linkage. A maximum lod score of 1.505 at theta = 0.00 was found in a two-point analysis between C1R and C1S. Ten populations were screened for structural variation at the C1S locus. Only US Whites and a Kodiak Island Eskimo group expressed heterogeneity. The frequencies of the designated alleles, C1S*1 and C1S*2, were 0.992 and 0.007, respectively, in the US White population and 0.998 and 0.002, respectively, in the Kodiak Island Eskimo population. In addition, the product of a putative new allele, designated C1S*4, was observed in a single US White individual but segregation of this variant was not observed in the limited family data available.

Blood Proteins↗
PubMed1994

A 100-kDa protein in the C4-activating component of Ra-reactive factor is a new serine protease having module organization similar to C1r and C1s.

Ra-reactive factor (RaRF), a C-dependent bactericidal factor in mice, is composed of one polysaccharide-binding component and one C4/C2-activating component. The former is an oligomer of 28-kDa protein corresponding to the mannose-binding protein of mice. The 100-kDa protein, P100, has been shown to be present in the C4/C2-activating component. This protein generates 29- and 70-kDa polypeptide chains when reduced. In this study, we determined the nucleotide sequence of cDNA coding for P100. cDNAs were prepared by reverse transcription PCR and cassette-ligation-mediated PCR on mRNA from BALB/c mouse liver, using primers synthesized by reference to the sequence determined in a previous study. The results of cDNA sequencing indicate that the precursor protein of P100 containing a 24-residue signal peptide consists of 704 amino acid residues. Taking the results of the previous electrophoretic study into consideration, it is thought that the cleavage of mature P100 protein generates a 29-kDa chain of 251 residues and a 70-kDa chain of 429 residues. Although homology in the amino acid sequence of P100 with that of human C1r and C1s subcomponents of C was less than 40%, a striking similarity in domain organization was found among these proteins, indicating that P100 is a new C4-activating serine protease structurally similar to C1r and C1s. Northern hybridization showed that the liver was the primary site of the expression of the P100 gene.

Amino Acid Sequence↗
PubMed1998

Structure and function of the serine-protease subcomponents of C1: protein engineering studies.

Our protein engineering studies on human C1r and C1s revealed important characteristics of the individual domains of these multidomain serine-proteases, and supplied evidence about the cooperation of the domains to create binding sites, and to control the activation process. We expressed the recombinant subcomponents in the baculovirus-insect cell system and checked the biological activity. Deletions and point mutants of C1r were constructed and C1r-C1s chimeras were also produced. Our deletion mutants demonstrated that the N-terminal CUB domain and the EGF-like domain of C1r together are responsible for the calcium dependent C1r-C1s interaction. It seems very likely that these two modules form the calcium-binding site of the C1r alpha-fragment and participate in the tetramer formation. The deletion mutants also demonstrated that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine-protease module. The substrate specificity of the serine-protease is also determined by the five N-terminal noncatalytic domain of C1r/C1s chimera, which contains the catalytic domain of C1s preceded by the N-terminal region of C1r, could replace the C1r in the hemolytically active C1 complex. The C1s/C1r chimera, in which the alpha-fragment of the C1r was replaced for that of the C1s exibits both C1r- and C1s-like characteristics. We stabilized the zymogen form of human C1r by mutating the Arg(463)-Ile(464) bond. Using our stable zymogen C1r we showed that one active C1r in the C1 complex is sufficient for the full activity of the entire complex. Further experiment with this mutant could provide us with important information about the structure of the C1 complex.

Amino Acid Substitution↗
PubMed1994

Functional analysis of the serpin domain of C1 inhibitor.

To analyze the role of the heavily glycosylated amino-terminal domain of C1 inhibitor in protease inhibitory activity, two truncated C1 inhibitor molecules were constructed. The abilities of the recombinant truncated inhibitors to complex with target proteases were compared with that of the wild-type recombinant protein. One recombinant truncated molecule consisted of amino acid residues 76 to 478 (C-serp(76)) and the other of residues 98 to 478 (C-serp(98)). The recombinant proteins were each expressed in similar quantities. The thermal denaturation profiles of the two truncated proteins were similar to that of the wild-type protein. Identical binding of C1s, C1r, kallikrein, and beta factor XIIa was observed with the three molecules. Furthermore, the truncated molecules also effectively inhibited C1 activity in hemolytic assays. These studies therefore clearly demonstrate that the amino-terminal domain of C1 inhibitor does not influence complex formation with target proteases.

Complement C1 Inactivator Proteins↗
PubMed1988

A monoclonal antibody to C1q which appears to interact with C1r2C1s2-binding site.

A monoclonal antibody (SB-4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 10(10) M-1. It has been shown that the antibody binds to the A-B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of performed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2-C1s2 complex.

Antibodies, Monoclonal↗
PubMed1993

Protein engineering studies on C1r and C1s.

1. C1r and C1s cDNAs were placed downstream the strong polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus and the recombinant proteins were expressed in insect cells, in biologically active form. The yield of expression is high enough to get recombinant components for chemical and functional studies (5 micrograms/ml cell culture supernatant). 2. The biological activity and the post-translational modifications of the recombinant subcomponents were checked. The rC1r and rC1s proved to be biologically active in the hemolytic assay, although their glycosylations were different compared to that of the serum proteins. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. 3. Two deletion mutants of C1r cDNA were constructed in order to clarify the role of domain I and II. The results show that both, domain I, and II are absolutely necessary for the tetramer formation and both have a regulatory role in the autoactivation. The autoactivation of the mutants is accelerated significantly. 4. Hybrid cDNA constructions were also made, and one of them was expressed. In the C1s alpha R hybrid the C1s alpha part cannot dimerize in presence of Ca2+, but it can form a tetramer with C1r2, that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. 5. In order to control the autoactivation process point mutant cDNAs were constructed through altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation. These results do justify our approach of using domain-domain interchange, domain deletion and point mutations in combination, to reveal the structural background of C1 function at intramolecular level.

Animals↗
PubMed1987

Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles.

C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.

Antigen-Antibody Complex↗
PubMed1989

Biosynthesis of C1r and C1s subcomponents.

Biosynthesis of C1r and C1s subcomponents has been studied using monocytes and macrophages, hepatocytes and hepatoma cell lines or fibroblasts. Both proteins have been detected in supernatants and cell lysates as proenzymic monocatenar molecules. C1r and C1s were secreted by stimulated monocytes and by Hep G2 cells, according to a 1:1 stoichiometry. Monocyte C1s secretion was enhanced by lymphokines, such as alpha- or gamma-interferon or by placental soluble factors. Expression of both proteins was coordinately modulated by a newly purified 14 kDa lymphokine at a pretranslational level. Data from in vitro RNA translation are discussed.

Animals↗
PubMed1981

Binding of purified C1 subcomponents, C1 inactivator and their complexes to immobilized heparin.

Under specified conditions purified C1q, activated C1r and C1s and C1r-C1s complexes were bound independently of Ca2+, to heparin-Sepharose, and could be eluted by an increasing salt gradient. Zymogen C1r and C1s, C1r-C1s complexes, C1 inactivator, and C1r-C1s-C1 activator complexes were not bound. However, at lower conductance Ca2+ independent binding of C14 occurred, which was utilized in the purification of C14 and C1s. In the presence of C1t (serum amyloid P component), C1s was firmly retained on heparin-Sepharose, which was probably due to formation of a C1s-C1t complex.

Amyloid↗
PubMed1989

Models for C1. Tools or toys? The real biological challenge.

C1 modelling, based on structural and functional data, does not yet bring the different laboratories to a consensus on C1 activation, activity and associated controls. The heart of C1 beats in its subcomponent C1r2, which, from its domain structure and its twinning with subcomponent C1s, represents the challenge for the knowledge of C1. The 8-shaped model proposed for the C1r2-C1s2 association, with a head-to-tail interaction between the C1r catalytic domains, appears as the hub of an active world in the bosom of C1q. More detail is now required on protein-protein interactions inside C1 to refine the available models or to propose alternatives. Precise data on the interactions of C1 proteins with activators, substrates or control proteins are also likely to bring pertinent help in proposing future models for C1.

Binding Sites↗
PubMed1987

Quantitation of (C1INH)2 C1r-C1s complexes in glomerulonephritis as an indicator of C1 activation.

C1 activation was assessed in several forms of glomerulonephritis by radioimmunoassay quantitation of circulating (C1INH)2 C1r-C1s complexes (INC). Eight patients with active systemic lupus erythematosus (SLE) and nephritis had elevated serum INC (mean = 15.3 vs control = 5.8, P less than 0.01). Their INC levels were normal during remission. Serum INC had a weak inverse correlation with serum C1q greater than 3 mg/dl (r = 0.42, P = 0.02). In longitudinal studies, serum INC also had a weak inverse correlation with serum C3 and C4. Only 1 of 10 patients with type I and 1 of 15 with type III membrano-proliferative glomerulonephritis (MPGN) had elevated serum INC. No patient with type II MPGN had elevated levels. Two of 10 patients with poststreptococcal glomerulonephritis (P-SGN) had elevated serum INC, but all normalized with convalescence. Patients with IgA nephropathy had normal serum INC. The data demonstrate the importance of C1 activation in SLE and P-SGN. The mechanism of complement activation in types I and III MPGN remains unclear; the data suggest, but do not prove, that C1-independent complement activation may occur in these patients.

Antigen-Antibody Complex↗
PubMed1987

Isolation and analysis of immune complexes from sera of patients with polymyalgia rheumatica and giant cell arteritis.

Serum samples were obtained from patients with polymyalgia rheumatica (PMR: n = 10) or giant cell arteritis (GCA; n = 7), or both. Samples were taken either before treatment or within one week of starting prednisolone. Immune complexes (IC) were concentrated by polyethylene glycol (PEG) precipitation then purified with either IgG anti-C1q-Sepharose or IgG anti-C3c-Sepharose. Complex components were separated by sodium dodecyl sulphate (SDS) gradient polyacrylamide gel electrophoresis then transferred to nitrocellulose by Western blotting. Identification of proteins was carried out using specific antisera. All the IC contained IgM (mu chain), some contained IgA (alpha chain), and IgG (gamma chain). C1r, C1s, C1q, C3, C4, and C reactive protein (CRP), where tested, were found in most but not all IC. The occurrence of properdin, factor B, alpha 2 macroglobulin (alpha 2M), factor H (beta 1H), C1 esterase inhibitor, and C4 binding protein was also investigated. Immune complexes in PMR and GCA differed from those previously characterized in rheumatoid arthritis (RA)1 purified by anti-C1q-Sepharose which contained immunoglobulins and C1q only. No properdin or factor B were detected in RA IC purified with either anti-C1q-Sepharose or anti-C3c-Sepharose.

Aged↗
PubMed1999

Grafting an RGD motif onto an epidermal growth factor-like module: chemical synthesis and functional characterization of the chimeric molecule.

A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.

Amino Acid Sequence↗
PubMed2001

C1 inhibitor: analysis of the role of amino acid residues within the reactive center loop in target protease recognition.

Previous analysis of a naturally occurring C1 inhibitor P2 mutant (Ala(443)-->Val) indicated a role for P2 in specificity determination. To define this role and that of other reactive center loop residues, a number of different amino acids were introduced at P2, as well as at P6 (Ala(439)) and P8'/9' (Gln(452)Gln(453)). Ala(439)-->Val is a naturally occurring mutant observed in a patient with hereditary angioedema. Previous data suggested that Gln(452)Gln(453) might be a contact site for C1s. Reactivity of the inhibitors toward target (C1s, C1r, kallikrein, beta factor XIIa, and plasmin) and nontarget proteases (alpha-thrombin and trypsin) were studied. Substitution of P2 with bulky or charged residues resulted in decreased reactivity with all target proteases. Substitution with residues with hydrophobic or polar side chains resulted in decreased reactivity with some proteases, but in unaltered or increased reactivity with others. Second order rate constants for the reaction with C1s were determined for the mutants with activities most similar to the wild-type protein. The three P2 mutants showed reductions in rate from 3.35 x 10(5) M(-1)s(-1) for the wild type to 1.61, 1.29, and 0.63 x 10(5) for the Ser, Thr, and Val mutants, respectively. In contrast, the Ala(439)-->Val and the Gln(452)Gln(453)-->Ala mutants showed little difference in association rates with C1s, in comparison with the wild-type inhibitor. The data confirm the importance of P2 in specificity determination. However, the P6 position appears to be of little, if any, importance. Furthermore, it appears unlikely that Gln(452)Gln(453) comprise a portion of a protease contact site within the inhibitor.

Amino Acid Substitution↗
PubMed1981

New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin.

p-Guanidinobenzoate derivates were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r and C1 esterase were examined. Among the various inhibitors tested, 6'-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(beta-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein. plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r and C1 esterase, and then strongly inhibited complement-mediated hemolysis.

Benzamidines↗
PubMed1998

Aspects of leukocyte function and the complement system following aerobic exercise in young female gymnasts.

Recent studies have reported reduced immunity in trained athletes. Scant information exists on changes in the immune function among trained children. The purpose of this study was to assess the effect of aerobic exercise on the phagocytic process of neutrophils and the complement system in young athletes. Subjects included prepubertal elite female gymnasts (n = 7) and untrained girls (n = 6) aged 10-12 years. Venous blood was withdrawn before, immediately post and 24 h following a 20-min run at a heart rate of 170-180 beats.min-1. Neutrophil random migration, chemotactic activity, bactericidal function and PMA/FMLP-stimulated superoxide anion release as well as various complement components were assessed. Net chemotaxis was found reduced (P < 0.05) 24 h following exercise (58 +/- 11 vs. 36 +/- 11 cells/field in gymnasts and 47 +/- 7 vs. 42 +/- 8 cells/field in untrained girls pre- and 24 h post-exercise, respectively). The basal values, as well as post-exercise values of bactericidal activity were lower (P < 0.05) in gymnasts as compared with the control group (0.8 +/- 0.3, 0.8 +/- 0.2 and 0.8 +/- 0.1 log decrease of colonies in gymnasts at pre-, immediately post-, and 24 h post-exercise, respectively and 1.1 +/- 0.1, 1.1 +/- 0.1 and 1.0 +/- 0.2 log decrease of colonies in controls, respectively). No significant effect on the bactericidal activity was observed in either group following exercise. The addition of homologous sera did not correct the bactericidal activity. PMA-stimulated superoxide anion release decreased (P < 0.05) among gymnasts immediately following exercise (5.7 +/- 0.4 vs. 4.4 +/- 1.0 mmol O2/10(6) PMN.min) and remained low 24 h later. The same trend was observed in FMLP-stimulated neutrophils but the data were not significant. Significantly decreased levels (P < 0.05) of the early complement components (C1Q, C1R) were also found following exercise (1.34 +/- 0.64 vs. 1.27 +/- 0.28 and 1.09 +/- 0.07 vs. 1.02 +/- 0.06 pre- and post-exercise in gymnasts and untrained, respectively). Furthermore, consistently lower C2 and C3 were observed in gymnasts compared with controls. Neutrophil dysfunction as well as impairment of the complement system seem to occur following exercise.

Analysis of Variance↗
PubMed1990

Trimer and tetramer complexes containing C1 esterase inhibitor, C1r and C1s, in serum and synovial fluid of patients with rheumatic disease.

During activation, the first component of complement C1q (C1r-C1s)2 is dissociated in conjunction with the formation of complexes containing C1 esterase inhibitor (C1-INH). Trimer complexes, with zymogen C1s associated with a firm C1-INH-C1r complex (C1-INH-C1r-C1s) can be distinguished from tetramer complexes C1-INH-C1r-C1s-C1-INH) in which C1-INH is firmly bound to both proteases. In the present study a two-stage electroimmunoassay was developed for the specific measurement of C1-INH-C1r-C1s. In the first step, C1-INH and its complexes were immunoprecipitated with anti-C1-INH during electrophoresis in the presence of Ca2+. In the second step, C1s contained in C1-INH-C1r-C1s was dissociated in the presence of EDTA and was measured by immunoprecipitation with anti-C1s. C1-INH-C1r-C1s were consistently found in normal sera. Normal sera did not contain C1-INH-C1r-C1s-C1-INH as assessed with a previously described ELISA procedure. Sera and synovial fluids from two groups of patients with inflammatory arthritis were investigated. In rheumatoid arthritis patients (n = 15) C1-INH-C1r-C1s complexes were usually found at high concentration both in serum and synovial fluid. C1-INH-C1r-C1s-C1-INH complexes were also present with values that were higher in synovial fluid than in serum, in accord with previous findings of classical pathway activation in the inflamed joints of the patients. Patients with spondylarthritic syndromes (n = 7) had serum and synovial fluid C1-INH-C1r-C1s concentrations that were comparable to those of the rheumatoid arthritis patients. If at all present, C1-INH-C1r-C1s-C1-INH were detected in trace amounts. Thus, C1 activation in patients with spondylarthritic syndromes appeared to be efficiently controlled at the C1r level. Distinguishing between C1-INH-C1r-C1s and C1-INH-C1r-C1s-C1-INH may prove of value in further studies of the activation and control of C1 in disease.

Animals↗