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PubMed1981

The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.

The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.

Binding, Competitive↗

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↗

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↗

PubMed1984

Lysine residues, but not carbohydrates, are required for the regulatory function of H on the amplification C3 convertase of complement.

Lysine epsilon-amino groups of human factor H were selectively converted to guanidino groups by treatment with 0.1 M O-methylisourea at pH 10.4. Guanidination resulted in a dose-dependent decrease in the capacity of the regulatory protein to accelerate decay dissociation of P-stabilized amplification C3 convertase sites, to serve as a co-factor for cleavage of cell-bound C3b by I, and to compete for binding of 125I-untreated H to C3b. Modification of approximately 75% lysine epsilon-amino groups suppressed 97% of H functional activity. Biochemical analysis of native H demonstrated a total carbohydrate content of 18.5% (w/w) and the presence in the molecule of 11 biantennary oligosaccharidic chains of the N-acetyl-lactosaminic type. Total desialation of H by using Clostridium perfringens neuraminidase, and total deglycosylation of desialated H by using beta-endo-N-acetylglucosaminidase resulted in a 1.5- to 2-fold increase in H activity on a weight basis. Deglycosylation did not alter the capacity of H to discriminate between activating and nonactivating surfaces of the alternative pathway. Thus, lysine residues are important determinants of the binding capacity of H for cell-bound C3b, whereas the carbohydrate portion of the molecule is not required for the regulatory function of the protein on the amplification C3 convertase.

Acetylglucosamine↗

PubMed1983

Activation of complement via the alternative pathway.

Activation of complement via the alternative pathway represents one means of natural resistance to infection because it is capable of neutralizing a wide variety of potential pathogens in the total absence of antibody. The pathway involves six serum proteins and possesses a unique amplification system capable of depositing large numbers of C3b molecules on the surfaces of activating particles. C3b deposition enhances phagocytosis and results in activation of the membrane attack pathway of complement. C3b attachment is covalent, arising from a reaction between an intramolecular thiolester bond in nascent C3b and nucleophiles such as hydroxyl groups on surface carbohydrates. The reactions that initiate C3b attachment are not specific interactions like those initiating other biological cascade systems, but involve slow, spontaneous hydrolysis of the thiolester bond in C3 and subsequent random deposition of C3b onto all nearby surfaces. Once bound, C3b is capable of discriminating between host-derived cells and activating particles. Recognition is evidenced by a lower affinity between activator-bound C3b and the complement control protein factor H. Measurements of the association constant between unbound, soluble C3b and factor H suggest that activator-bound C3b recognizes structures on activators that inhibit factor H binding.

Complement C3↗

PubMed1982

Intestinal glycoprotein activates the alternative complement pathway by reacting with factor H.

Previous observations indicating complement activation via the alternative pathway of chelated human serum by a rat intestinal glycoprotein fraction were substantiated by using C2-deficient serum. Some inactivation of C6 by the glycoprotein was observed both in normal and C2-deficient serum. As estimated by conglutination, the glycoprotein largely abolished the ability of serum to convert EAC3b to EAC3bi, indicating inhibition of the control factors H (beta 1H globulin) and I (C3bINA), or inhibition of C3b deposition on the cells. The glycoprotein caused no change in the electrophoretic mobility of factors H or I. By immunoelectrophoresis the glycoprotein interfered with the precipitation line of plasminogen and beta-lipoprotein and some other serum proteins. The absence of plasminogen or beta-lipoprotein in serum seemed unimportant for the C3 conversion by the glycoprotein. Factor H was retained when heat-inactivated serum, heat-inactivated serum depleted of 99% albumin or 85% IgG, or heat-inactivated hypogammaglobulinaemic serum was eluted through a column of the intestinal glycoprotein coupled to epoxy-activated Sepharose 6B. Presence of EDTA abolished factor H retention. Factor H from methylamine-treated serum was also adsorbed to the glycoprotein. When serum depleted at 5 degrees C or 31% of the factor H content was heated, the alternative pathway was activated spontaneously. Addition of some of the previously removed factor H reduced the C3 and factor B conversion rate.

Animals↗

PubMed1990

Identification and sequence analysis of four complement factor H-related transcripts in mouse liver.

Mouse liver RNA analyzed by Northern blotting with a full-length complement factor H cDNA probe demonstrates the 4.4-kilobase (kb) H mRNA as well as three additional hybridizing species of 3.5, 2.8, and 1.8 kb, respectively. Further characterization of these alternative transcripts was pursued by isolation of additional cDNAs from a liver library using a full-length H probe. Twelve clones homologous to but distinct from H were isolated, analyzed by restriction mapping, and divided into four classes, A, B, C, and D, based on their sequences. Clones from classes A, B, and C all contained nearly identical 5'-untranslated regions and leader sequences that differed from H at more than 50% of their nucleotide positions. The 5'-untranslated and leader sequences of the class D clone were unrelated to the corresponding regions of H or the class A, B, or C clones. The remaining portions of the H-related cDNAs were made up of short consensus repeats, 7 in class A, 4 in class B, 13 in class C, and 5 in class D. To determine the relationship between the H-related transcripts and the cDNA clones, Northern blots of liver RNA were analyzed by hybridization with two probes, one specific for the class D cDNA and the other reacting specifically with the class A, B, and C cDNAs. The class A/B/C probe detected transcripts of 3.5, 2.8, and 1.8 kb in liver RNA, and the class D probe hybridized to a distinct 1.8-kb message. Additionally, a cosmid genomic library was screened with H cDNA, and nine H-related clones were isolated. They spanned a region of approximately 120 kb, defining at least two discrete H-related gene loci. These results identify new members of the super-family of C3b/C4b binding protein genes.

Amino Acid Sequence↗

PubMed1983

[The alternative complement pathway].

The alternative complement pathway comprises three component proteins C3, B, D and three regulatory proteins P, H and I. These plasma proteins represent the major humoral defense mechanism against infection in a non-immune host. The following topics are reviewed: biochemistry of the alternative pathway proteins; molecular mechanisms of activation and regulation of the pathway; involvement of the alternative pathway in human diseases.

Complement Activation↗

PubMed1990

Expression of complement alternative pathway proteins by endothelial cells. Differential regulation by interleukin 1 and glucocorticoids.

We have studied the secretion of proteins of the alternative pathway of complement C3, factor B and factor H by human umbilical vein endothelial cells (HUVEC). Results showed that factor H and factor B are quantitatively secreted in abundance whereas C3 could only be detected when the cells are maintained in culture during long periods of time. Interferon-gamma stimulated factor H, factor B and, to a lesser extent, C3 secretions. Interleukin (IL) 1 had a differential effect on spontaneous C3, factor B and factor H secretions. In the presence of IL 1, there was a significant secretion of C3 occurring within a short period of culture. IL 1 also stimulated factor B secretion. There was a synergistic stimulating effect between IL 1 and interferon-gamma to bring C3 and factor B productions by HUVEC to very high levels. In contrast, factor H secretion was consistently inhibited by IL 1. Local increase in C3 and factor B secretions by endothelial cells in the presence of IL 1 may have important implications in the inflammatory reaction. In striking contrast, the glucocorticoid dexamethasone (DXM) had modulatory effects which are consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, decreased C3 and factor B secretions and increased factor H secretion. Local modulation of complement protein secretion by DXM appears to be a new mechanism by which this glucocorticoid may control inflammation.

Complement C3b Inactivator Proteins↗

PubMed1985

[The complement system in hepatopathies].

Low serum complement is often observed in cirrhosis of the liver. This is the result of two mechanisms: a failure to synthesise a certain number of components and regulatory proteins of complement, and an increased consumption due to activation of the complement system. This acquired deficit in complement contributes to the increased risk of infection in patients with cirrhosis.

Acute Disease↗

PubMed1984

[Alternative complement pathway].

Antibody Formation↗

PubMed1990

Enhanced synthesis of factor B induced by tumor necrosis factor in human skin fibroblasts is decreased by IL-4.

IL-4 is a T cell-derived lymphokine that has multiple biologic functions, affecting B cells, T cells, mast cells, monocytes, macrophages, and hematopoietic progenitor cells. We report that IL-4 also affects human skin fibroblasts. These effects were primarily in modulating the effects of other mediators of inflammation on these cells, with IL-4 producing little or no effects by itself. Synthesis of C proteins in human skin fibroblasts is modulated by TNF and other mediators of inflammation. TNF increased synthesis of factor B by 18.6-fold; IL-4 reduced the TNF effect on factor B synthesis by 83%. This effect started with concentrations of IL-4 as low as 0.1 ng/ml. The effect of IL-4 on the TNF-induced increase in synthesis of factor B occurred after 2 h incubation, the time when the effects of TNF alone were first observed. IL-4 also abrogated the effects of IL-1 and LPS on factor B synthesis, but did not affect the increase in synthesis of factor B induced by IFN-gamma. In contrast to the pattern for effect of IL-4 on factor B synthesis, the TNF-induced rate of synthesis of C3 was augmented by IL-4. The specificity of the IL-4 effect was also apparent when comparing the effect of IL-4 on the TNF-induced synthesis of factor B to the effects on total protein synthesis (increased 1.5-fold) and the TNF-induced increases in synthesis in C1r, C1s, C1 inhibitor, C2, and factor H (no changes). The IL-4-induced decrease in synthesis of factor B was mediated at a pretranslational level and was dependent on synthesis of a new protein. This interaction between IL-4 and mediators of inflammation can potentially modulate the inflammatory response in the setting of activation of Th cells.

Cells, Cultured↗

PubMed1990

Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene.

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.

Animals↗

PubMed1987

Sequence analysis of a cDNA clone encoding the C-terminal end of human complement factor H.

Peptide sequencing of the complement system regulatory protein, factor H, permitted the synthesis of a mixed sequence oligonucleotide probe. Human liver cDNA libraries were screened and factor H-specific clones selected. No full-length clone was obtained, but the largest available clone, R2a, was found to encode the C-terminal 657 amino acids of factor H. The derived amino acid sequence consists of 10 contiguous internally homologous segments, each about 60 amino acids long. Sequences homologous to these are found in several other complement and non-complement proteins. Such sequences are likely to represent a particular type of tertiary structure subunit.

Amino Acid Sequence↗

PubMed1985

Isolation of rabbit C3, Factor B, and Factor H and comparison of their properties with those of the human analog.

The rabbit complement components C3, Factor B, and Factor H were isolated and characterized and were compared to the corresponding proteins of human serum. Chromatographic behavior, chemical properties, and functional interactions show great similarities between the components in both species. By SDS polyacrylamide gel electrophoresis, the m.w. were estimated to be 195,000 for C3, 86,000 for Factor B, and 155,000 for Factor H. The amino acid compositions of the rabbit proteins resembled those of the human analog. The total carbohydrate content of rabbit C3 and Factor H was approximately one-half that of the human proteins. In addition, a qualitative difference in the carbohydrate moieties of the C3 proteins was observed. The serum concentration of the rabbit proteins was markedly lower than that of the human proteins. The rabbit C3b,Bb enzyme resembled the human analog with respect to half-life, control by Factor H, and stabilization by nickel ions.

Amino Acids↗

PubMed1978

Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway.

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO(4)). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and beta1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by beta1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in beta1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO(4) was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of beta1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and beta1H is essential to prevent the particle from effectively activating the alternative pathway.

Animals↗

PubMed1989

Regulation of the human alternative complement pathway: formation of a ternary complex between factor H, surface-bound C3b and chemical groups on nonactivating surfaces.

Sephadex [alpha(1----6) cross-linked dextran] activates the human alternative pathway of complement. Substitution of hydroxyl groups of Sephadex with carboxymethyl groups (CM) results in a dose-dependent decrease of the activating capacity of the polymer in normal human serum. Sephadex bearing one CM group/glycosyl unit (CM-Seph 0.95) exhibited no activating capacity. CM groups did not interfere with the ability of the polymer to covalently bind C3b in the presence of purified alternative pathway proteins nor with the capacity of bound-C3b to form a C3 convertase in the absence of regulatory proteins. C3b that was bound to CM-Seph 0.95 was more susceptible to inactivation by factors H and I in serum than C3b bound to Sephadex. Binding studies using 125I-labeled H demonstrated that H bound with a similar affinity to the activating particle Sephadex, to Sephadex bearing C3b and to the nonactivating particle CM-Seph 0.95. However, factor H bound with a 5- to 7-fold higher affinity to CM-Seph 0.95 bearing C3b. These results demonstrate a requirement for both CM groups and C3b molecules in order for H to bind with high affinity to C3b on the non-activating surface, and indicate that H formed a ternary complex with surface-bound C3b and CM groups on CM-Seph 0.95. Using a chemically defined model system, the present study provides a molecular basis for the enhanced interaction between surface-bound C3b and factor H on nonactivators of the human alternative pathway.

Complement Activation↗

PubMed1984

Control of immune complex and zymosan-mediated anaphylatoxin generation by proteins B and H of the alternative complement pathway.

The generation of histamine releasing activity (HRA) from human basophils in fresh serum by tetanus toxoid (Te)/anti-Te complexes or by zymosan can be modulated through introduction of incremental amounts of proteins B and H of the alternative complement pathway. Serum treated at 50 degrees in order to abolish alternative pathway-mediated haemolytic activity, lost 90% of its capacity to generate HRA upon addition of Te/aTe; such loss could be reversed through additions of purified B. Amounts of B sufficient to restore normal alternative pathway haemolytic activity also restored HRA induced by Te/aTe; as little as a 33% increase above the normal serum concentration of B increased the capacity to support Te/aTe induced HRA by a factor of 1.4. In contrast, additions of incremental doses of purified H to fresh serum reduced generation of HRA by both Te/aTe and zymosan. Total inhibition was achieved by increasing the serum H concentration by 12.5-30%; further increases of H up to 200% again permitted HRA generation induced by immune complexed aTe. H also inhibited Te/aTe induced HRA in a serum heated at 50 degrees but only 30% inhibition of HRA could be achieved over a range of H inputs up to 187% above normal serum concentration. Additions of H also inhibited HRA generation in fresh serum when induced with plain or C3b-coated zymosan (Z) particles. By increasing the serum concentration of H from 12.5 to 125%, dose-dependent inhibition of HRA generation was observed; the H input necessary to suppress 48% of HRA generation was ten times higher when HRA was generated by Z-C3b than by plain zymosan. Thus, the complement-dependent generation of HRA from fresh serum strongly depends on modest variations in the concentrations of the two regulatory proteins B and H of the alternative complement pathway, suggesting their direct effect on generation of anaphylatoxins C3a and C5a.

Anaphylatoxins↗