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PubMed1988

Structural and functional relationships among receptors and regulators of the complement system.

The classical and alternative pathway of complement activation are regulated by a series of fluid phase and cell-bound factors, some of which at the same time serve as receptors for fragments of C3 and C4. These molecules are factor H, CR1 (C3b/C4b receptor), CR2 (C3d/EBV receptor), C4BP (C4b binding protein), DAF (decay accelerating factor), MCP (membrane cofactor protein; earlier designated p45/70), CR3 (iC3b receptor or Mac-1) and CR4 (protein 150/95). Due to structural, genetic and functional features these factors are members of one or several newly recognized large families of proteins: (1) molecules with 60 amino acids long repeats (H, CR1, CR2, C4BP, DAF); (2) proteins with 1,2-diacylglycerol membrane anchoring (DAF); (3) proteins with a heterodimer structure and preference for ligands containing the tripeptide arginine-glycine-asparagine (CR3, CR4). Recognizing the above mentioned regulators and receptors of the complement system as belonging to these protein families opens new perspectives for further genetic and functional research of mutual interest to complement and noncomplement scientists.

Amino Acid Sequence↗

PubMed1990

Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

Animals↗

PubMed1988

Structure of the murine complement factor H gene.

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.

Animals↗

PubMed1983

Requirement for human red blood cells in inactivation of C3b in immune complexes and enhancement of binding to spleen cells.

We showed previously that soluble immune complexes (IC) added to normal human serum and unseparated autologous blood cells bind predominantly to red blood cells (RBC) bearing C3b receptors. RBC-associated IC are processed both by C3b/C4b inactivator (I) cleavage of C3b and by alternative pathway-dependent C3b generation, and are returned to the serum in a modified form. In the present study, soluble IC were prepared by using 125I-BSA and guinea pig anti-BSA, and were treated with diluted human serum to optimize sustained C3b-dependent binding to human RBC. This binding to RBC could not be abrogated by preincubation of IC with heat-inactivated human serum or purified serum regulatory proteins, I and beta 1H (H), but after binding, pretreated IC dissociated. When IC were prepared with serum containing 125I-C3, treatment with I + H did not release labeled C3 from the complexes. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of 125I-C3b in IC showed similar labeled peaks whether IC were pretreated with buffer or with I + H or were bound to RBC without pretreatment. In contrast, when IC containing 125I-C3b were exposed to I + RBC, nearly 70% of the label was released from the IC. The released material analyzed by SDS-PAGE showed major 125I-C3 peaks corresponding in m.w. to free beta-chain and alpha 1 cleavage products of 30,000 to 33,000 daltons. IC in undiluted serum after incubation with RBC gave less binding to human polymorphonuclear leukocytes than after incubation without RBC, but notably more binding to guinea pig spleen cells and human lymphoblastoid cells. These findings argue that exposure to serum regulatory proteins does not prevent subsequent binding of experimental IC to RBC, that RBC binding promotes degradation of C3b in IC which proceeds beyond iC3b, and that this I-mediated breakdown renders IC reactive with spleen cells.

Animals↗

PubMed1989

Human complement factor H: an additional gene product of 43 kDa isolated from human plasma shows cofactor activity for the cleavage of the third component of complement.

In addition to the 150-kDa factor H protein, we have previously described a 43-kDa factor H molecule in human plasma, which probably represents a translational product of the additional 1.8-kb mRNA for factor H. This factor H was isolated from human plasma by means of immunoaffinity chromatography and high-performance liquid chromatography. When tested for its functional activity, this purified 43-kDa H protein was shown to act as cofactor for factor I- mediated cleavage of fluid-phase C3b to iC3b.

Antibodies, Monoclonal↗

PubMed1989

Biosynthesis of complement factor H by human umbilical vein endothelial cells. Regulation by T cell growth factor and IFN-gamma.

The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity for C3bBb formation, which is present in human umbilical vein endothelial cells (HUVEC). Therefore, human endothelial culture supernatants (HECS) were analyzed for the presence of human complement factor H by ELISA. It was found that H was present in HECS. An immunoblot analysis of affinity purified H from HECS showed that the size of HUVEC H was identical to that of plasma H. The mean production of H by HUVEC of first passage cultures was 40 ng/10(6) cells/day. The synthesis of HUVEC H was fully inhibitable by the addition of cycloheximide to the cultures, suggesting that H is de novo synthesized. Additional evidence for de novo synthesis was obtained by using biosynthetic labeling with [35S] methionine, immunoprecipitation, and SDS-PAGE. It was demonstrated that, indeed, HUVEC produce and secrete factor H. Two forms of the protein were identified, the 150-kDa form and also a 45-kDa form, both forms have been identified in plasma. The functional activity of HUVEC H is identical to that of plasma H. IFN-gamma induced enhanced synthesis of H by HUVEC, whereas it had no effect on C3 synthesis. Supernatant from stimulated PBMC, T cell growth factor, enhanced synthesis of both H and C3. The present studies indicate that H is produced by HU-VEC and that H may function as an inhibitor of complement activation at the endothelial cell level and, thereby, together with molecules like decay accelerating factor and membrane cofactor protein, may influence resistance of endothelial cells to complement mediated damage.

Binding, Competitive↗

PubMed1981

Activation of the alternative pathway of complement: efficient fluid-phase amplification by blockade of the regulatory complement protein beta1H through sulfated polyanions.

Current concepts of activation of the alternative pathway of complement (APC) focus on the central role of an amplification mechanism triggered by C3b which is covalently bound to the surfact of activating substances. Using sulfated polyanions as model substances, an efficient fluid-phase activation of complement is demonstrated in contrast to solid-phase activation. It is shown that particulate high-molecular weight sulfated polyanions are capable of reversible binding the guinea pig and human regulatory protein beta1H. This fixation leads to an extensive activation of C3 and factor B because the regulatory function of beta1H is blocked in the fluid-phase C3b-dependent amplification system of the APC. Addition of beta1H-depleted C4-deficient guinea pig serum reconstitutes the physiological control mechanisms of the APC. Guinea pig beta1H, purified to homogeneity, is described as a 160000 dalton protein of a single-chain structure. In addition, highly specific and sensitive test systems for beta1H are described.

Absorption↗

PubMed1990

Demonstration of an unusual allelic variation of mouse factor H by the complete cDNA sequence of the H.2 allotype.

Three allotypes of murine factor H have been identified serologically in the previous study (denoted H.1, H.2, and H.3). A cDNA clone coding for the entire length of murine factor H was isolated from a library constructed from the livers of STR/N mice which have H.2 allotype and was fully sequenced. The insert of this clone (STR309) contained 4184 nucleotides and consisted of a 47-bp 5' noncoding region, a 54-bp coding for leader peptide, a 3648 bp for the mature factor H protein, and a 435-bp 3' noncoding region. Compared with the previously reported sequence of the cDNA clone (MH8) isolated from B10.WR mice that have H.1 allotype, the size of the protein coding region was exactly the same, but 21 nucleotide substitutions resulting in 15 amino acid replacements were observed. The amino acid replacement/nucleotide substitution ratio (0.71) is far higher than those observed in the allotypic variations of other proteins. Four 15-base oligonucleotide probes specific for either STR309 or MH8 were synthesized and used in Northern blot analysis. The probes specific for STR309 hybridized with mRNA isolated from the livers of STR/N mice but not with mRNA from the livers of BALB/c mice that have H.1 allotype, whereas the reverse pattern was observed with the oligonucleotide probes specific for MH8. These results strongly suggest that the nucleotide sequence of STR309 represents H.2 allotype of factor H protein, providing an example of an unusual allotype with high ratio of amino acid replacements to nucleotide substitutions.

Alleles↗

PubMed1979

[Alternative complement pathway (author's transl)].

The dual role of the alternative complement pathway in recognition of foreign substances by a non-immune host and in the intrinsic regulation of the complement sequence is now well recognized. Activation of this pathway occurs through escape from its regulatory mechanisms induced by the activating principle; its functional expression depends on the respective levels of the component proteins C3, factor B, factor D and properdin, and on the control proteins beta 1H and C3bINA. This article presents recently acquired knowlege on the molecular mechanisms of activation, regulation and behaviour under pathological conditions of the alternative complement pathway.

Complement Activation↗

PubMed1982

Characterization of the lymphocyte membrane receptor for factor H (beta 1H-globulin) with an antibody to anti-factor H idiotype.

Antibody to the binding site (idiotype) of anti-factor H was shown to have specificity for both B lymphocyte membrane H receptors and C3b. Goat F(ab')(2) anti-human H was purified by absorption and elution from H agarose and used for rabbit immunization to produce anti-anti-H (aaH). After absorption with nonimmune goat IgG, (125)I-labeled aaH bound to B lymphocytes and to sheep erythrocytes coated with C3b (EC3b) but did not bind to T lymphocytes or to EC3d. All B cell- and C3b-specific activities of the aaH were removed and subsequently recovered by absorption and elution of the antibody from either C3-agarose or goat-anti-H-agarose. This indicated that the aaH probably recognized a single common antigenic structure that was shared by anti-H, C3b, and the membranes of B cells. Affinity-purified aaH resembled H in that it bound to B cells, blocked the uptake of H onto B cell H receptors, and triggered B cells to release endogenous factor I (C3b inactivator). In addition, aaH functioned with factor I as either a cofactor for cleavage of fluid-phase C3b or a potentiator for cleavage of bound C3b. This same spectrum of C3 binding functions could not be demonstrated with either sheep anti-C3b or rabbit-anti-C3c. Analysis by sodium dodecyl sulfate- polyacrylamide get electrophoresis of the [(3)H]leucine intrinsically labeled B cell proteins reactive with the purified aaH revealed proteins of 100,000 M(r) and 50,000 M(r) without reduction, and after complete reduction of disulfide bonds, a single protein band of 50,000 M(r). This same protein molecular weight profile was also demonstrated with labeled B cell proteins that were absorbed and eluted from H-agarose. Thus, aaH is apparently specific for both B cell H receptors and C3b. However, because parallel analysis of C3b confirmed its known 115,000- and 75,000-M(r) polypeptide chain structure, the H receptor is probably not C3b and shares only the structure of the H binding site with C3b.

Animals↗

PubMed1989

Analysis of the mechanism of recognition in the complement alternative pathway using C3b-bound low molecular weight polysaccharides.

The human complement (C) system recognizes bacterial, fungal and viral activators of the alternative pathway following covalent attachment of the protein C3b to carbohydrates (CHO) on the surface of the organisms. Recognition first manifests itself as a 3- to 10-fold reduction in the affinity of C3b for factor H, a regulatory protein of C. This report describes the use of a fluorimetric assay which is sensitive to the C3b-H interaction to study the characteristics of recognition. Fluid phase C3b covalently bound to CHO (C3b-CHO) was prepared by activating C3 in the presence of the small homopolymers dextran (alpha 1-6 polyglucose) or inulin (beta 1-2 polyfructose). In particulate form both polysaccharides are activators of C. The conjugates exhibited increased resistance to inactivation in the factor H-dependent assays compared to C3b not bound to CHO and to C3b bound to mono- or disaccharides. The dextran-induced restriction of inactivation was partially reversed by treatment of the conjugate with dextranase. C3b-CHO conjugates failed to bind to factor H-Sepharose and when introduced into serum behaved as though C3b was attached to particulate activators of C, suggesting that the fluorimetric assay accurately reports recognition. The results suggest that the recognition site which induces a reduction in the affinity of C3b for factor H is distinct from the thioester site of C3b and can recognize structural features of polysaccharides including size, sialic acid content, and possibly aspects of three-dimensional oligosaccharide structure.

Binding Sites↗

PubMed1988

gp 58/68, a parasite component that contributes to the escape of the trypomastigote form of T. cruzi from damage by the human alternative complement pathway.

A glycoprotein of apparent molecular weight 58,000 (unreduced)/68,000 (in its reduced form) (gp 58/68), which is one of the fibronectin/collagen receptors of Trypanosoma cruzi, was purified to homogeneity from the trypomastigote forms of the Tehuantepec and Y strains of the parasite. Purified gp 58/68 inhibited formation of cell-bound and fluid-phase alternative pathway C3 convertase in a dose-dependent fashion, as assessed using purified human complement components. Gp 58/68 differed from the human regulatory proteins H, DAF, MCP and CR1 and from previously reported regulatory proteins on the parasite membrane in that it was unable to enhance decay-dissociation of preformed alternative pathway C3 convertase sites, did not serve as a co-factor for I-mediated cleavage of C3b and had no inhibitory activity on the classical pathway convertases. The inhibitory effect of gp 58/68 was most likely dependent on an interaction of the protein with factor B rather than with C3b. Gp 58/68 provides trypomastigotes with an additional potential mechanism for escaping complement lysis by the human alternative pathway.

Animals↗

PubMed1991

Two major serum components antigenically related to complement factor H are different glycosylation forms of a single protein with no factor H-like complement regulatory functions.

Factor H is a 150-kDa serum glycoprotein with key regulatory functions in the alternative pathway of complement activation. Two glycoproteins with a molecular mass of approximately 42 and 37 kDa that react with an antiserum against factor H were purified from human plasma. The two glycoproteins have identical N-terminal amino acid sequences but differ in glycosylation. Sequence comparisons indicated that they both correspond to a 1.4-kb mRNA recently cloned from human liver cDNA. The serum concentration of the two glycoproteins together was estimated to be approximately 40 mg/liter. They were found not to exert factor H-like regulatory functions in the alternative pathway. Thus, the 42-kDa glycoprotein described here appears to be distinct from the previously characterized factor H-related protein of similar size, suggesting that human serum contains two factor-H related molecules which both have a molecular mass of 41 to 43 kDa but which differ largely in structure.

Amino Acid Sequence↗

PubMed1984

Release of prostaglandin E and thromboxane from macrophages by stimulation with factor H.

Recently novel actions of factor H of complement other than regulation of alternative pathway activation have been described. We examined the influence of H on the arachidonic acid (AA) metabolism of macrophages. Guinea-pig peritoneal macrophages cultured for up to 18 h under serum free conditions were supplied with homologous factor H. H, tested over a concentration range of 12.5-100 micrograms/ml, promoted an indomethacin sensitive release of prostaglandin E and thromboxane B2 in a dose-dependent manner. Stimulation of AA conversion to prostanoids in response to H was shown to be specific as evidenced by immunoabsorption experiments. This novel effect attests to the potential of H to act not only as regulatory protein of the complement pathway but also as an inducer of cellular release reactions. Moreover, these findings emphasize the close functional links that exist between the three main constituents of the inflammatory process: macrophages, the complement system and the AA cascade.

Animals↗

PubMed1982

Complement behavior in infectious mononucleosis: possible mechanisms for the prevention of immune complex injury.

The acute-phase complement reaction was examined in patients with uncomplicated infectious mononucleosis (IM). Concentrations of both complement and complement inhibitor protein were significantly elevated during the acute phase, and turnover studies with purified 125I-labeled C3 showed this increase to result from increased C3 synthesis. Nine of 16 patients had evidence of in vivo complement activation during this period. Despite high levels of C1q-binding immune complexes in most acute-phase sera from patients with IM, the capacity of these sera to solubilize immune precipitates in vitro was normal. In contrast, four of five sera from patients with active systemic lupus erythematosus showed reduced solubilization. The level of C3b inactivator in sera from patients with IM and the percentage conversion of C3 after in vitro complement activation by aggregated IgG were inversely correlated (P less than 0.01). It is proposed that an acute-phase complement reaction limits immune complex-mediated injury to tissue by maintaining those complement functions that are required for the effective processing of circulating antigen/antibody material.

Adolescent↗

PubMed1989

Analysis of recognition in the alternative pathway of complement. Effect of polysaccharide size.

Covalent attachment of the complement (C) protein C3b to polysaccharides on biologic particles which activate the alternative pathway leads to changes in the affinity of C3b for factor H, a regulatory protein of the C system. In this study the size of the site with which the polysaccharides interact and its special relationship to the thioester site were investigated using a fluorimetric assay and soluble C3b attached to low m.w. polysaccharides. Oligomers of alpha 1-6 and alpha 1-4 polyglucose and beta 1-2 polyfructose were prepared and attached to C3b at the thioester site. C3b bound to monomeric, dimeric, or trimeric sugars exhibited the same interaction with factor H as free C3b, i.e., there was no effect due to attachment alone. Beginning with tetrameric oligosaccharides a linear decrease in factor H binding was observed with increasing oligosaccharide size and the effect reached an apparent maximum with large polysaccharides. Maximum inhibition of factor H function was estimated to occur at a length of 16 saccharide units. The results suggest that this site, which regulates the inactivation rate of surface-bound C3b and thus the activation of the alternative pathway of C, spans a maximum of 13 sugar units (less than 65 A) starting four units (approximately 15 A) from the thioester site in C3b.

Binding Sites↗

PubMed1987

Human complement factor H: expression of an additional truncated gene product of 43 kDa in human liver.

The human complement factor H is an important factor in the control of the alternative pathway and also induces the stimulation of B cells and macrophages in vitro. Using a human factor H cDNA clone as probe, two factor H-specific transcripts of 4.4 and 1.8 kb were detected in four human livers. Both mRNAs were found independently of the expression of an acute phase marker SAA, in these livers, indicating that their presence is not linked to an acute phase state. The shorter transcript was cloned in two cDNA plasmids H-19 and H-20, lacking only seven amino acids of the N-terminus of the mature factor H protein. The deduced protein sequence showed that this protein is identical to the N-terminal portion of the large classical factor H of 150 kDa mol. mass. Parallel to the finding that the N-terminal sequence of factor H is expressed by two distinct mRNA species, evidence is presented that the C-terminal sequence is also contained on two different transcripts, the common 4.4-kb mRNA and an additional 1.0-kb mRNA. A novel, short form of human complement factor H of 43 kDa was detected in human sera which represents the translation product of the 1.8-kb factor H-specific mRNA detected in human liver. Five distinct epitopes detected with 6 monoclonal antibodies are present on both the 150-kDa factor H protein and the truncated 43-kDa molecule. We conclude that additional H-specific mRNAs are found in human liver and that at least one of them is translated yielding a truncated form of factor H.

Amino Acid Sequence↗

PubMed1989

Factor C3f is a spasmogenic fragment released from C3b by factors I and H: the heptadeca-peptide C3f was synthesized and characterized.

C3f, a heptadeca-peptide having the amino acid sequence of NH2-Ser-Ser-Lys-Ile-Thr-His-Arg-Ile-His-Trp-Glu-Ser-Ala-Ser-Leu-Leu-Arg- COOH, is liberated during the catabolic degradation of C3b in serum. The amino acid sequence of C3f is known both from the cDNA-derived structure of C3 and from protein analysis after isolation of the natural factor. C3f was synthesized by solid phase peptide synthesis. Both natural and synthetic C3f had identical retention times by RP-18 high performance liquid chromatography (HPLC) analysis and the respective amino acid compositions agreed with the expected theoretical values. C3f, but not des-Arg-C3f, was weakly spasmogenic inducing contraction of guinea pig ileum at a level of 5-10 x 10(-6) M. Since C3f and C3a were cross-tachyphylactic, it was concluded that these two spasmogens compete for the same receptors. Both C3f and des-Arg-C3f at concns of 1-4 x 10(-4) M enhanced vascular permeability in guinea pig skin. These observations further suggest that C3f functionally resembles C3a anaphylatoxin. Formation of C3f in human serum following CVF activation of C3 could be demonstrated by radioimmunoassay (RIA). Digestion of C3f with purified human serum carboxypeptidase N produced C3f-desArg. These observations suggest that when serum complement protein C3 undergoes conversion to C3b, further degradation by Factors H and I readily generates C3f. C3f is a weak spasmogen that functions like C3a anaphylatoxin and C3f-desArg is a major metabolite in serum.

Amino Acid Sequence↗