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PubMed2005

Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins.

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the alpha' chain of C3b between residues 954-955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.

Complement C3b Inactivator Proteins↗

PubMed1989

Monoclonal anti-human C3d antibodies: stabilization of the alternative pathway C3 convertase.

IgG mouse monoclonal antibody (mAb) was prepared by fusion of spleen cells from mice immunized with human C3d (mAb:C3d) using syngeneic thymocytes as feeder cells. mAb:C3d was assessed for its effect on the stabilization of the cell-bound alternative pathway C3 convertase EAC3bBb. It bound to cell-bound C3b and stabilized C3bBb at 30 degrees in the presence of EDTA-GVB. The plasma protein H reduced the stabilization effect of the stabilized C3 convertase. These results suggest that binding of antibody to C3d may stabilize C3bBb. It seems likely that such antibody induces in C3b conformational change, which increases the C3bBb complex stability.

Antibodies, Monoclonal↗

PubMed1985

[Localization of functionally important areas of the regulator protein factor H using monoclonal antibodies].

Four monoclonal antibodies to the control protein of the complement system, factor H, were used to try to localize functionally important domains on the molecule. Attempts to inhibit the interaction of C3b and H in ELISA and agglutination assays by means of these monoclonal antibodies showed that two of them, namely MAH 1 and MAH 2, recognized an epitope in close proximity to the binding site for C3b on H. The determinants defined by MAH 3 and MAH 4 are localized at a certain distance from this binding site, the MAH 4 epitope being situated closer to it than the MAH 3 epitope. The cofactor function of H with respect to C3b inactivator was inhibited by the same monoclonal antibodies which interfered with the binding of H to C3b. Since MAH 1, MAH 2, MAH 3 and MAH 4 all bind to the same tryptic 38 KD fragment of H, the binding site for C3b on H, as well as the cofactor activity seem to reside on this fragment.

Agglutination Tests↗

PubMed1984

Control of the immune complex-complement interaction by protein H of the alternative complement pathway and the natural inhibitor heparin.

The potential of the negative regulatory protein H of the alternative complement pathway convertase and of heparin in modulating the complement-dependent capacity of fresh serum to inhibit immune complex precipitation (CIICP) between bovine serum albumin (BSA) and rabbit anti-BSA as well as tetanus toxoid (TT) and human anti-TT was assessed. Additions of purified H to serum to increase the intrinsic concentration of this protein by 80% (BSA-anti-BSA system) and 190% (TT-anti-TT system) resulted in an inhibition of CIICP by 50% and 60%, respectively, whereas further increase of the amount of H lead to a decrease of its inhibitory activity. A similar effect was observed with heparin: at a concentration of 400 U/ml a 90% inhibition of CIICP in the TT-anti-TT system was obtained which diminished at higher heparin concentrations. The effect of H on C3 deposition to immune aggregates was assessed through its influence on C3b-mediated immune adherence hemagglutination; factor H dose-dependently suppressed such hemagglutination induced by aggregated human IgG or preformed TT-anti-TT complexes when added to the immune complex-fresh serum mixture at the outset but not after 45 min of the 37 degrees C incubation period which means that H inhibited more likely decoration of immune complexes with C3b than it did inhibit the interaction of C3b-coated immune complexes with erythrocytes. This suppressive effect of H was reversed by the simultaneous addition of the activating protein B. Complement-mediated binding of tritiated C3 to latex-bound human IgG was assessed and H was found to dose-dependently inhibit such binding with a maximal inhibition of 37% at a H concentration of 7 micrograms/ml.

Animals↗

PubMed1982

Circular dichroism studies of human factor H. A regulatory component of the complement system.

Factor H of the human complement system exhibits an unusual circular dichroism spectrum. The CD spectrum of Factor H exhibits a positive extreme at 230 nm and a negative extreme at 190 nm. No apparent alpha-helical or beta-sheet conformations were present in the native protein structure. However, when the disulfide bridges are reduced, followed either by reoxidation or alkylation, the structure of Factor H is modified so that it now exhibits conventional protein secondary structure as determined from its CD spectra in the far ultraviolet region. Factor H also fails to mediate its regulatory function of inhibiting the alternative pathway convertase once the disulfides have been ruptured and conformational rearrangement has occurred. CD studies indicate that minor conformational changes take place when Factor H and C3b associate in free solution.

Alkylation↗

PubMed1978

Capillary transport of immunoglobulins and complement proteins to the interstitial fluid and lymph.

Low concentration of immunoglobulins G, A and M and complement component proteins were found in leg lymph of normal men. The lymph/serum ratios of different proteins ranged from 0.07 to 0.24 and depended on the molecular weight of the measured protein. Major variations in concentration were found dependent on the rate of capillary filtration and lymph formation. Hemolytic activity of total complement and of the 9 components were low, with the L/S ratio from 0.06 to 0.25. The C3H50 in lymph was much lower than of the remainder of components. Remarkably low immune adherence capacity of lymph C was noted.

Capillary Permeability↗

PubMed1988

Interferon gamma induces synthesis of complement alternative pathway proteins by human endothelial cells in culture.

Human umbilical vein endothelial cells grown in vitro under standard conditions contain a high level of mRNA specific for the complement regulatory factors H and I. An additional 1.8-kb mRNA encoding a truncated form of factor H is also present. IFN-gamma stimulation of the cells causes a 6-7 fold increase in both factor H mRNA species, and a greater than 10-fold increase in factor I mRNA. IL-1 and LPS slightly suppressed factor H mRNA, while TNF had no effect. mRNA for factor B is also detectable in IFN-gamma-stimulated cells, but messengers for C1q, C4bp, and CR3 beta chain were not found. Secretion of factor H protein was also stimulated by IFN-gamma. The presence of mRNA for factors H, B, and I, together with C3 secretion, demonstrated by others, suggests that endothelial cells can assemble the complete alternative complement pathway. Endothelial cell complement may be involved in leukocyte-endothelium interactions mediated by leukocyte C3 receptors.

Blotting, Northern↗

PubMed1984

Use of monoclonal antibodies against factor H to investigate the role of a membrane-associated protein antigenically related to H in C3b-receptor function.

Three murine monoclonal IgG1 kappa-antibodies, MAH-1, MAH-2, and MAH-3, were raised against factor H purified from human plasma. In cross-inhibition studies MAH-3 did not compete with MAH-1 and MAH-2, and vice versa, for the binding to H, whereas MAH-1 and MAH-2 inhibited each other. MAH-1 and MAH-2 inhibited the binding of H to C3b attached to an ELISA plate as well as to C3b bound to sheep erythrocytes by means of the classical pathway convertase and of C3b to H attached to an ELISA plate. The determinant defined by MAH-1 and MAH-2 was no longer accessible on H bound to C3b. In contrast, MAH-3 interfere with the binding of H to C3b or vice versa only to a smaller extent but recognized a determinant still accessible on H bound to C3b and was able to agglutinate EAC14o23b-H in an indirect Coombs test. All three antibodies were shown to bind to tonsil cells and Raji cells in an indirect cell ELISA. The membrane-associated molecule detected by these antibodies had an apparent m.w. of 140,000 D in SDS-PAGE. All three antibodies partially inhibited the binding of EAC14o23b to tonsil lymphocytes and, in the presence of 0.1 mM DFP, to Raji cells; binding of EAC14o23bi and EAC14o23d to tonsil cells was not affected. We conclude that MAH-3 recognizes a determinant distinct from the ones recognized by MAH-1 and MAH-2, the latter possibly defining identical epitopes that are located close to the binding site for C3b. The fact that these two distinct epitopes could be detected on a 140,000-D membrane-associated protein from lymphoid cells strongly suggests that this molecule is at least antigenically related to serum H and shares with H a region carrying the binding site for C3b. The rosette inhibition studies imply that this structure is important for the binding of C3b-coated particles to lymphoid cells.

Animals↗

PubMed1991

Human complement factor H: molecular cloning and cDNA expression reveals variability in the factor H-related mRNA species of 1.4 kb.

Previously, we have shown that three different mRNA species of 4.3 kb, 1.8 kb and 1.4 kb, related to human complement factor H, are constitutively expressed in the human liver. Probing with our cDNA clone H-46 which represents 920 bp of the 3' end of the 4.3 kb mRNA of factor H on human liver RNA, we always detected the 4.3 kb and the additional, abundantly expressed mRNA species of 1.4 kb in length, indicating that the 1.4 kb transcript is highly homologous to the 3' end of the classical factor H mRNA of 4.3 kb. Using H-46 as a probe, several cDNA clones were isolated from a liver cDNA library and sequenced. The open reading frame of the novel mRNA species encodes a peptide consisting of five internal short consensus repeat motifs (SCR), identifying the translational product to be a member of the SCR family. Sequence comparison with cDNA clones derived from liver RNA of a different donor provided evidence for variability in the factor H related proteins encoded by the 1.4 kb mRNA species. Interestingly, this variability was found to be restricted to the three carboxyterminal SCR domains. Expression data indicate that our variant is not recognized by the monoclonal antibody 3D11.

Amino Acid Sequence↗

PubMed1979

Metabolism of human beta 1H: studies in man and experimental animals.

Metabolic studies were performed with a purified, functionally-active preparation of human beta 1H. In seven normal human subjects, the half-life ranged from 66--87 hr with fractional catabolic rates (FCR) of 1.04--1.63%/hr. Synthesis rates were 0.22--0.57 mg/kg/hr and extravascular distribution ratios were 0.34--0.67. There was evidence of extra-vascular catabolism in each subject. In sixteen patients with immunological disease four showed hypercatabolism of beta 1H. However, three patients with C3 mephritis factor (NeF) had normal beta 1H turnover despite profound reduction in C3 concentration; it is suggested that the reaction of beta 1H with the C3b. Bb convertase exposes it to a catabolic site and that in the NeF patients the NeF stabilized convertase prevents such exposure. Studies of the acute phase response were carried out in nine patients following elective surgery, with C-reactive protein (CRP) used as the control protein: six patients showed no rise in beta 1H levels and three showed a small (20%) rise whereas all exhibited a gross rise in CRP. Pre-incubation of 125I- beta 1H with NHS, with NHS in the presence of NeF and with C3b+C3b 1NA caused no change in beta 1H turnover in animals despite demonstrable total C3 conversion with the NeF.

Animals↗

PubMed1990

Quantitation of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays.

Sensitive enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies have been developed to specifically detect components of the alternative pathway of complement in human blood plasma. Normal values of the factor B split products Ba (1.01 +/- 0.30 micrograms/ml, mean +/- SD), Bb (0.65 +/- 0.23 micrograms/ml), of the C3-fragments C3b/iC3b/C3dg (17.9 +/- 5.7 micrograms/ml), native factor B (238 +/- 48 micrograms/ml), factor D (1.05 +/- 0.27 micrograms/ml), and factor H (702 +/- 292 micrograms/ml) were determined in the EDTA-plasma of healthy probands (n = 55). The simultaneous quantitation of the main cleavage products and of control proteins in the plasma samples permits precise analysis of the activation of the alternative pathway of complement in various disease states. In addition, we describe a method for the specific depletion of factor B prior to fragment-specific assays utilizing monoclonal antibodies conjugated to paramagnetic beads. The latter should permit the quantitation of other complement split products.

Animals↗

PubMed1983

Surface modulation of classical pathway activation: C2 and C3 convertase formation and regulation on sheep, guinea pig, and human erythrocytes.

We examined the formation of the early classical complement (C) pathway enzymes on sheep (Es), guinea pig (Egp), and human (Eh) erythrocytes (E). Each species' E were sensitized with sufficient IgM or IgG anti-E Ab to establish equal numbers of C1-fixing sites on all E. After sensitization with 100 C1-fixing sites of Ab and excess C1, uptake of C4 was equivalent on all three cell types, judged by anti-C4 binding (for guinea pig C4) or by direct uptake of radiolabeled protein (for human C4). With equal numbers of cell-bound C1 and C4, however, there were marked differences in C2 convertase activity on Es, Egp, and Eh. Sheep EAC14 utilized C2 at least 20 times faster than Egp and Eh bearing the same number of C1 and C4 molecules. C3 cleavage was even further depressed on Egp and Eh, and was not changed by the substitution of oxyC2 for normal human C2. In whole guinea pig serum (GPS), 300 times more C1-fixing sites were required on Egp than on Es to achieve similar amounts of lysis; however, equivalent C3 uptake on Egp and Es was associated with equal extents of lysis, demonstrating that GPS lysis of these cells was regulated by early steps in classical pathway (CP) activation. Incubation of E bearing radiolabeled C4b with Factor I demonstrated that C4b on Egp was highly resistant to cleavage compared to the same protein bound to Es or Eh. Studies with partially purified C3 convertase decay-accelerating factors from Eh stroma demonstrated that these membrane proteins could not account for the observed surface regulation of CP activity because these proteins do not affect the rate of C2 cleavage by EAC14. We conclude that E surface molecules have an important role in modulation of CP activation. This surface-associated CP regulation occurs at the level of cell-bound C4b.

Animals↗

PubMed2000

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.

Adjuvants, Immunologic↗

PubMedDate not supplied

Differential modulation of complement factor H and C3 expression by TNF-alpha in the rat. In vitro and in vivo studies.

The biosynthesis of alternative regulatory complement protein factor H was investigated using both an in vivo rat model and an in vitro rat hepatocyte culture system, and compared to that of C3 component. Subcutaneous injection of a single dose of 20 micrograms of recombinant murine tumor necrosis factor-alpha (rmTNF-alpha) had no effect on factor H liver mRNA levels, while it increased C3 mRNA levels. In correlation with this, serum factor H levels remained unchanged after rmTNF-alpha injection, whereas C3 levels were increased. In contrast in vitro studies showed that rmTNF-alpha had no effect on factor H and C3 expression by rat hepatocytes. Recombinant human interleukin-1 alpha (rhIL-1 alpha) did not alter the expression of factor H, whereas it increased C3 expression, and recombinant human interleukin-6 (rhIL-6) stimulated expression of both proteins. This study shows that TNF-alpha is not directly responsible for the increased levels of factor H observed in vivo during induced inflammation in the rat. Its in vivo effect on C3 secretion might be secondary to the TNF-alpha-induced release of IL-1 and/or IL-6.

Animals↗

PubMed1988

Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1.

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.

Cell Line↗

PubMed1990

Competition for binding sites on C3b by CR1, CR2, MCP, factor B and factor H.

The reaction of radiolabeled C3b-binding proteins with C3b-coated particles has been investigated. CR1 binding was inhibited by factor H and factor B (in the presence of properdin), but not by properdin alone. CR2 and MCP binding were also inhibited by factor H. Therefore factor H, factor B, CR1, CR2 and MCP probably comprise a group of mutually competitive proteins with similar or overlapping binding sites on C3b. These results correlate with their structural homology and suggest that they all evolved from a single C3b-binding molecule. Factor H, CR1 and MCP are also cofactors for the factor-I-mediated cleavage of C3b. A species incompatibility between rat factor I and human CR1 for the cleavage of human C3b suggests the possibility that cofactors may also function by interacting directly with factor I.

Animals↗

PubMed1984

Simplified method for purification of mouse beta 1H.

A simple three-step method was described for purification of murine beta 1H, one of the essential regulatory proteins of complement system. The method consists of heparin-Sepharose affinity chromatography; gel filtration on a Sepharose 6B column, and DNA-cellulose affinity chromatography. By this method over 10 mg of beta 1H can be purified by more than 200-fold from 100-ml of EDTA serum of various strains. Overall yield of beta 1H was about 45%. The purified beta 1H was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. The purified mouse beta 1H showed physicochemical properties very similar to those described for human beta 1H: mouse beta 1H is a beta-globulin consisting of a single polypeptide chain of molecular weight of 160,000. Purified mouse beta 1H retained its functional activity as the essential cofactor for the cleavage of fluid-phase human C3b by the human C3b inactivator. Immunization of rabbits with the purified mouse beta 1H resulted in the production of the potent and monospecific antibody.

Animals↗

PubMed1987

Isolation of bovine complement factor H.

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.

Animals↗