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PubMed1983

Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.

Complement C3↗
PubMed1981

Complement levels in cigarette smokers: elevation of serum concentrations of C5, C9, and C1-inhibitor.

Serum levels of 13 individual complement component and control proteins were measured in 25 male cigarette smokers and 25 male non-smokers. A significant elevation of the mean serum levels of C5, C9 and C1-inhibitor was demonstrated for the smokers while levels of C1q, C2, C4, C3, C6, C8, Factor B, properdin, C3b inactivator and beta 1H did not differ significantly between the two groups. Serum level of C9 in individual smokers correlated significantly with both the present amount of consumption and cumulative consumption, while C5 level correlated with present consumption. The complement profile in the cigarette smoker is similar to that observed in certain inflammatory conditions. The elevation of C5, C9 and C1-inhibitor levels may reflect the presence of a low grade inflammatory process in the cigarette smoker.

Adolescent↗
PubMed1980

Cleavage of C4b by C3b inactivator: production of a nicked form of C4b, C4b', as an intermediate cleavage product of C4b by C3b inactivator.

We have investigated the mechanism of cleavage of C4b into C4c and C4d by the C3b inactivator (C3bINA) and revealed the formation of a nicked form of C4b as an intermediate cleavage product. The cleavage of C4b by the C3bINA was a two-step reaction. The first cleavage occurred on the alpha-chain (89,000 daltons) yielding two fragments, 73,000 daltons and 16,000 daltons. These fragments were bound to each other or to the beta or gamma chain through disulfide linkages. Therefore, an altered form of C4b, C4b', consisting of four disulfide-linked polypeptide chains with the same m.w. as C4b, was produced as an intermediate cleavage product. Subsequently, the second cleavage occurred on the alpha-chain fragment of 73,000 daltons to produce the two generally recognized fragments, C4c and C4d. In both cleavage reactions, a high m.w. cofactor protein, C4bC3bINACo, which is the same protein described as the C4 binding protein, was required, suggesting that both of the proteolytic processes are catalyzed by the C3bINA.

Complement C3b Inactivator Proteins↗
PubMedDate not supplied

Failure of the synthetic androgen, danazol, to increase protein S levels and prevent thrombosis in a patient with severe protein S deficiency.

A 34-year-old man with recurrent deep and superficial thromboses was found to have severe protein S deficiency. Treatment with both warfarin and adjusted-dose subcutaneous heparin failed to completely prevent thrombosis. Based on reports of increases in the endogenous anticoagulants (protein C, protein S, antithrombin III and plasminogen) with synthetic androgen therapy, the patient was treated with danazol for 8 weeks. Although the levels of antithrombin III, protein C and plasminogen increased, no change in the levels of total or free protein S or C4b binding protein was observed. Treatment was discontinued at 8 weeks when the patient developed a recurrence of superficial thrombophlebitis. The role of synthetic androgens in the treatment of patients with inherited thrombotic disorders is reviewed and potential reasons for treatment failure in this patient are discussed.

Adult↗
PubMed1989

IL-1 and tumor necrosis factor. Similarities and differences in stimulation of expression of alternative pathway of complement and IFN-beta 2/IL-6 genes in human fibroblasts.

IL-1 and TNF induced concentration-related increases in the synthesis of factor B, C3, and IFN-beta 2/IL-6 in human skin fibroblasts. Effects of both stimuli were apparent with concentrations as low as 0.1 ng/ml and maximal responses were observed between 1 and 10 ng/ml; only for IL-1 induction of IFN-beta 2/IL-6 was there a further increase in response up to 100 ng/ml. For factor B and C3, maximal increases induced by IL-1 and TNF were similar: 119- and 109-fold for factor B and 15-fold and 11-fold for C3, respectively. Although both IL-1 and TNF increase synthesis of factor B and C3 in hepatocytes, the increases observed in fibroblasts were approximately 50- and 8-fold more for factor B and C3, respectively. Neither protein synthesis nor mRNA for IFN-beta 2/IL-6 was present in HepG2 cells either before or after stimulation with IL-1 or TNF. In contrast to the similarities between the effects of IL-1 and TNF on synthesis of factor B, C3, and IFN-beta 2/IL-6, only TNF increased synthesis of factor H. Because TNF induces membrane IL-1 in fibroblasts, it is possible to speculate that the effects of TNF on fibroblasts are due to induction of IL-1. An autocrine action of TNF through IL-1 is possible for TNF-induced synthesis of IFN-beta 2/IL-6, but the effects of TNF on synthesis of factor B, C3, and factor H indicated that TNF has effects on fibroblasts separate from IL-1. The effects of IL-1 and TNF on the synthesis of factor B and C3 in fibroblasts may be a part of an acute phase response occurring at a local level. However, the large responses in synthesis of factor B and C3 to IL-1 and TNF may suggest that factor B and C3 have a role, as yet undescribed, in tissues in addition to the role these proteins are known to play in inflammation.

Adult↗
PubMed1985

Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s.

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.

Calorimetry↗
PubMed2003

Angioedema and oral contraception.

BACKGROUND: Oral contraceptives can precipitate attacks of hereditary angioedema (ANE) or induce acquired forms. OBJECTIVE: We studied 5 patients who had an ANE which had begun under oral contraception and disappeared after stopping the pill. METHODS: We explored the clinical and biological characteristics of these patients. RESULTS: The symptoms developed during the first year or later after starting contraception; the patients reported relapsing swelling of the lips, hands, larynx and abdomen. All women had normal serum C4 and C1 inhibitor (C1Inh) antigen levels, but a lowered C1Inh activity, with a marked protein cleavage on the immunoblot. The suppression of the pill was associated with the regression of the edema and normalization of C1Inh function. CONCLUSION: The mechanism of these ANE is unknown. The could be due to a modulation of C1Inh expression upon androgens or an imbalance between coagulation proteins favoring C1Inh cleavage by its target proteases.

Adult↗
PubMed1981

Hypocomplementemia in Reye syndrome: relationship to disease stage, circulating immune complexes, and C3b amplification loop protein synthesis.

Measurement of C1q, C2, C4, C5, C6, factor B, properdin, beta1H, and C3bINA were made in acute sera from 31 patients with Reye syndrome. Abnormalities were found in 18 patients. The magnitude of the complement component depression correlated with disease severity. Sera from patients with stage IV illness had significantly lower complement levels than did sera from patients with state I (P less than 0.001), II (P less than 0.05), and III (P less than 0.05) disease. Circulating immune complex measurements were performed on all 31 acute sera and were present in six (19%). However, from the results of the present study, it would appear that in the majority of the patients circulating immune complexes are not the cause of the lowered levels of, at least, C3 and factor B. Rather, these low levels could be explained as secondary to reductions in the levels of the C3b amplification loop control proteins beta1H and C3bINA.

Antigen-Antibody Complex↗
PubMed1994

The inhibitory effect of factor J on the alternative complement pathway.

Factor J (FJ) is a cationic glycoprotein with inhibitory activity in C1, the first component of the classical complement pathway. This study demonstrates that FJ is able to regulate the activity of the alternative complement pathway. FJ inhibits the generation of fluid-phase and cell-bound alternative pathway C3 convertase, C3b,Bb (C3-cleaving enzyme). Thus, FJ interferes with the generation of alternative pathway C3 convertase when sheep erythrocytes bearing antibody and activated C3 and C4 (EAC4b,3b) are incubated with the individual complement components, factors B, D, and P. FJ accelerates the decay of C3 convertase with a time course similar to that of factor H, and when both regulators are present together, the decay of enzyme activity is faster than when they are added separately. Furthermore, FJ is able to inhibit the cleavage of C3 by factor B in a fluid-phase assay. FJ prevents the initiation of alternative pathway activation in "more stabilized systems" with well known activators of alternative pathway C3 convertase such as C3 nephritic factor (an autoantibody against alternative pathway C3 convertase), cobra venom factor, and rabbit erythrocytes. In these systems, FJ has no effect on C3 convertase stabilized by rabbit erythrocytes or cobra venom factor. In contrast, FJ promotes the dissociation of C3 convertase stabilized by C3 nephritic factor, but with much lower efficiency than in preventing initiation. Direct interaction of FJ with individual components of C3 convertase was shown by a solid-phase binding assay using plates coated with C3, C3b, B, Bb, or FJ. FJ inhibitory activity in the alternative pathway can be modulated by polyanions like heparin. FJ-mediated inhibition in the alternative complement pathway can be modified by surface interactions, as occurs during alternative pathway C3 convertase activation. Thus, when FJ is adsorbed by and eluted from hydroxylapatite and reverse-phase columns, its inhibitory effect on more stabilized systems is lost. This loss of inhibitory activity is fully reversed when FJ is rechromatographed on heparin-Sepharose or Sepharose columns. Taking into account these data, FJ may be included in the group of highly charged molecules that inhibit the activation of classical and alternative complement pathways (i.e. eosinophil major basic protein, protamine, and heparin).

Animals↗
PubMed1996

Biotinylation of proteins via amino groups can induce binding to U937 cells, HL-60 cells, monocytes and granulocytes.

The use of biotinylated ligands for the flow cytometric detection of cell surface receptors has become a popular alternative to radioreceptor assays. Although the biotinylation of a protein is a relatively mild chemical reaction several reports have mentioned the fact that the number and location of biotin moieties coupled to amino groups of a protein can alter its physicochemical properties and impair biological activity. In the present study we show for a variety of biotinylated functionally unaltered ligands that biotinylation by N-hydroxysuccinimide (NHS) esters of biotin can induce a binding to cell surfaces, which is not specific for the respective unlabelled ligand. C1q, C1 inhibitor (C1-INH), alpha 1-antitrypsin (AT), ovalbumin (OV), transferrin and soybean trypsin inhibitor (STI) were labelled with S-NHS-LC-biotin and activated C1s (C1s) with NHS-biotin. Biotinylation of C1q, C1s and C1-INH exerted negligible effects on biological function, antigenicity or electrophoretic mobility but when labelled and unlabelled proteins were assayed for binding to monocytic U937 cells, promyelocytic HL-60 cells, monocytes and granulocytes, a remarkable binding was observed for biotinylated C1q, C1-INH and C1s. In contrast, no binding was observed when we used unlabelled C1q, C1s and C1-INH and employed specific antibodies, alpha-mouse-FITC or alpha-rabbit-FITC for detection. Increasing molar ratios of biotin-to-protein (B : P) for biotinylated AT, OV and STI evoked increased fluorescence intensities of the cells. Most importantly the unlabelled ligands did not compete for cell binding with their biotinylated derivatives, with the exception of transferrin. Preincubation of the cells with an excess of free d-biotin did not reduce binding of biotinylated proteins, thus excluding a potential involvement of biotin receptors. Hydrophobic interaction chromatography revealed a remarkable increase in hydrophobicity of the biotinylated proteins compared to their unlabelled counterparts, suggesting that the biotinylation-induced binding is due to increased hydrophobicity. Our findings indicate that biotinylation by the common amino acid esterification method may be critical for proteins if they are to be used as ligands for receptor binding studies.

Amino Acids↗
PubMed1998

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

Amino Acid Sequence↗
PubMed2002

Normal transcription of the C1 inhibitor gene is dependent upon a polypurine-polypyrimidine region within the promoter.

Analysis of the transcriptional activity of C1 inhibitor (CIINH) promoter reporter constructs with mutations in the R-Y region indicate that triplex formation by this region is not a predictor of transcriptional activity and that normal promoter function depends on the interaction of trans acting factors with specific elements within this region. Electrophoretic mobility shift assay (EMSA) of Hep3B nuclear extracts using the wild type promoter probe (nucleotides -98 to -9) yielded four major bands. Incubation of the same extracts with probes lacking the HNF-1 site resulted in the disappearance of one band. Supershift assays indicate that HNF-1alpha is the only previously identified protein that is present in the EMSA bands. Southwestern blot analysis detected four bands (M(r)s -130, 75, 65 and 20 kDa). These data suggest that the -98 to -9 region of the C1INH promoter interacts with at least four proteins, one of which is HNF-1alpha.

Blotting, Southwestern↗
PubMed1989

Analysis of complement factor H mRNA expression: dexamethasone and IFN-gamma increase the level of H in L cells.

Murine complement protein H is encoded by a 100-kb gene on chromosome 1. A 3.2-kb fragment of the 5' flanking region of the H gene was sequenced, and two transcription start sites for this gene were identified by RNase protection and S1 nuclease analyses, each of which had upstream TATA and CAAT boxes. This region shares sequence homology with known regulatory elements, including the SV40 enhancer consensus, the Sp1 binding site, and two glucocorticoid-responsive core elements (GRE). Tissue and cell-line specificity has been examined by Northern analysis, and the 4.4-kb full-length H messenger RNA was identified in liver, kidney, spleen, thymus, liver cell line 1469, and L cells. IFN-gamma did not induce H mRNA expression in the macrophage cell line P388D.1 but had a positive effect on both the mRNA and protein levels of H in L cells. PMA, LPS, and vitamin D did not increase H mRNA levels in L cells. Pursuant to the discovery of two GRE in the 5' regulatory region of the H gene, we examined the effects of glucocorticoids on H mRNA expression. Dexamethasone (10(-7) M) was found to increase markedly the levels of H mRNA and protein after 24 h of incubation, and the effect on the mRNA was detectable by 30 min. The fact that H is a down-regulator of complement activation is consistent with the known immunosuppressive role of glucocorticoids. To our knowledge, this is the first time that dexamethasone has been shown to increase the levels of a complement protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CR1) and comparison with H, C4b-binding protein (C4bp), and decay accelerating factor (DAF).

Four CR1 variants have been found in the normal population and are designated CR1-A (190,000 daltons), CR1-B (220,000 daltons), CR1-C (160,000 daltons), and CR1-D (250,000 daltons). In the present study, we first developed an improved chromatographic purification scheme for CR1 that does not employ a C3b affinity step. CR1 variants (A, B, and C) were then isolated, and their individual functional activity was assessed. Each possessed similar co-factor activity for I-mediated cleavage of C3(H2O), as well as for the inhibitory activity for fluid phase C3 convertases. These results indicate that, despite relatively large Mr differences, in the purified state these three CR1 variants have similar functional activities. The functional activity of CR1 was also compared with C4bp, H, and decay accelerating factor (DAF) in fluid phase assays designed to assess the inhibition of the C3 convertases and co-factor activity. On a molar basis, CR1 had approximately the same inhibitory activity as C4bp for the classical pathway convertase, and had the same as H for the alternative pathway convertase. These results indicate that CR1 encompasses the functional capabilities of both proteins. They also raise a number of interesting genetic and structural questions in regard to these complement regulatory proteins, because C4bp is thought to have multiple C4b binding domains, whereas H is reported to bind one C3b. DAF was an approximately fourfold better inhibitor of the alternative pathway convertase than CR1 or H, but was a fourfold less efficient inhibitor of the classical pathway convertase than CR1 or C4bp. The effective inhibitory capacity of DAF in these fluid phase assay systems suggests that the DAF substrate specificity is for the convertases. Fluid phase CR1 was twofold less efficient than H in serving as a co-factor for the first cleavage of fluid phase C3b, and hardly mediated the second cleavage. These data are in contrast to the co-factor activity of CR1 on a cell membrane, and provide additional evidence for the local environment being a critical modulator of the function of proteins that regulate the activation of C3.

Complement Activating Enzymes↗
PubMed1986

Partial characterization of human complement factor H by protein and cDNA sequencing: homology with other complement and non-complement proteins.

Factor H, a control protein of the human complement system, is closely related in functional activity to two other complement control proteins, C4b-binding protein (C4bp) and complement receptor type 1 (CR1). C4bp is known to have an unusual primary structure consisting of eight homologous units each about 60 amino acids long. Such units also occur in the N-terminal regions of the complement proteins C2 and factor B, and in the non-complement serum glycoprotein beta 2I. Amino acid sequencing, and sequencing of a factor H cDNA clone, show that factor H also contains internal repeating units, and is homologous to the proteins listed above.

Amino Acid Sequence↗
PubMed2000

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds.

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.

Binding Sites↗
PubMed1991

Determination of serum levels of complement component C4b-binding protein: influence of age and inflammation.

C4b-binding protein (C4b-BP) is a high molecular weight plasma protein which inhibits the activity of the classical complement pathway C3 convertase. In addition to multiple binding sites for C4b, C4b-BP possesses a single binding site for vitamin K-dependent protein S, an inhibitor of blood coagulation. As protein S bound to C4b-BP has no anticoagulant activity, C4b-BP participates in the regulation of both the complement and the coagulation pathways. We have produced and immunochemically characterized a series of murine monoclonal antibodies to human C4b-BP. A mixture of four monoclonal antibodies precipitating C4b-BP both in agarose gel and in solution was used to develop a highly reproducible radial immunodiffusion method for the measurement of C4b-BP in human serum. C4b-BP levels were measured in sera from 284 patients referred to our central laboratory. Samples from subjects with an increased erythrocyte sedimentation rate (ESR), a1-acid glycoprotein (a1-AGP) or C-reactive protein (CRP) had significantly higher C4b-BP levels (307 mg/l, 292-322 mg/l, geometric mean and 95% confidence limits of the mean) than those from subjects without elevation of the aforementioned established acute phase reactants (231 mg/l, 226-237 mg/l, P less than 0.00001). C4b-BP was significantly (P less than 0.001) correlated with ESR (r = 0.715), a1-AGP (r = 0.692) and CRP (r = 0.567). There was no gender-related difference in C4b-BP levels. In subjects with no increased acute phase reactants there was a significant correlation between C4b-BP levels and age (r = 0.387, P less than 0.001). High C4b-BP might contribute to the increased thrombotic risk associated with inflammation and aging.

Adult↗