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PubMed1984

C-reactive protein inhibits pneumococcal activation of the alternative pathway by increasing the interaction between factor H and C3b.

We previously studied two alternative pathway activators, Streptococcus pneumoniae and positively charged liposomes, which react with C-reactive protein (CRP). Binding of CRP to these surfaces initiates classical pathway but blocks alternative pathway activation. In this study, we investigated the mechanism of this inhibition using S. pneumoniae, R36a. R36a were pretreated with CRP (CRP-R36a) or buffer and incubated with C2-deficient human serum to which 125I-labeled C3 had been added. The amount of specific 125I-C3 binding was decreased from 8200 mol/CFU on R36a to 2200 mol/CFU on CRP-R36a. In contrast, when the same experiment was performed with purified factors B, D, P, and C3, in the absence of regulatory proteins, specific 125I-C3 uptake was slightly lower on R36a (6100 mol/CFU) than on CRP-R36a (8100 mol/CFU). The ability of the fixed C3b to inactivate factor B in the presence of factor D was equivalent on the two surfaces. The binding of the regulatory factor H to C3b fixed to R36a and CRP-R36a was compared by using purified 125I-labeled factor H. The ratio of factor H bound to C3 bound was twofold greater on CRP-R36a than on R36a. This increase was found by using C2-deficient serum or purified factors B, D, P, and C3 to fix C3b to the surfaces. The ability of CRP to inhibit C3 binding to R36a was restored by the addition of factors H and I to factors B, D, P, and C3. These results indicate that CRP inhibits alternative pathway activation by increasing regulation of bound C3.

Binding, Competitive↗

PubMed1987

Anti-idiotypic antibodies in antisera against human C3 and factor H and their application in the enrichment of antibodies specific for H-binding domains of C3.

Antisera separately raised against C3 and factor H should contain antibodies against the binding domains by which these factors interact. Because these interacting domains are likely to be sterically complementary to each other, antibodies specific for these domains should also be sterically complementary or of anti-idiotypic specificity. To test this hypothesis, rabbit anti-human C3 IgG antibodies which bound to Sepharose-coupled rabbit anti-factor H IgG (anti-H-binding anti-C3) were separated by affinity chromatography. In a control experiment, the anti-human factor H column was found to bind 10 times more anti-C3 antibodies than a Sepharose column coupled with nonimmune rabbit IgG. The binding specificity of the control eluate was indistinguishable from the original anti-C3 preparation, whereas the anti-H-binding anti-C3 was shown to be enriched in regard to antibodies directed against the 42 kd fragment of the C3 alpha-chain of C3c, as demonstrated by immunoblotting of electrophoretically separated polypeptides of C3c and C3d in polyacrylamide gels. In competition binding studies, factor H was 25 times more potent as inhibitor of the anti-H-binding anti-C3 than of the original anti-C3 preparation. This similarity in binding specificity also paralleled a functional similarity in that anti-H-binding anti-C3 in the presence of factor I effected a 20% degradation of the alpha'-chain of C3b into fragments of 65, 45, and 42 kd which are the normal degradation products in the presence of factors I and H. These results suggest that the affinity procedure described resulted in the enrichment of anti-C3 antibodies with selective specificities for factor H-binding domains of C3, from a polyclonal antibody source.

Antibody Specificity↗

PubMed1984

C3b covalently bound to IgG demonstrates a reduced rate of inactivation by factors H and I.

We have prepared C3b covalently linked to IgG via a hydroxylamine-sensitive bond between the C3b alpha' chain and sites predominantly, but not exclusively, located in the IgG heavy chain. This C3b species displays relative resistance to inactivation by factors H and I when compared with free C3b. This resistance appears to be due entirely to reduced affinity of C3b-IgG for factor H. Resistance to inactivation is not conferred on C3b by binding to another serum glycoprotein of similar size, ceruloplasmin, and may be a special property of IgG. C3b-IgG demonstrates an enhanced capacity to consume serum C3 relative to C3b. These alterations of the behavior of C3b when bound to IgG may in part explain the augmentation of alternative pathway activity by IgG. In addition, IgG-induced protection of C3b might influence both complement-mediated killing and phagocytosis of bacteria, as well as modify the in vivo handling of IgG-containing soluble immune complexes.

Ceruloplasmin↗

PubMed1985

Factor I co-factor activity of CR1 overcomes the protective effect of IgG on covalently bound C3b residues.

We have shown previously that C3b resides in a protected site when it is covalently bound to IgG (C3b-IgG). Such C3b displays a reduced affinity for factor H, with consequent enhanced survival in the presence of factors H and I and increased capacity for promoting alternative pathway consumption of C3. Because erythrocyte CR1 may be a major co-factor for factor I-mediated inactivation of immune complex-borne C3b in blood, we have examined the effect of covalently bound IgG on the C3b-CR1 interaction. Binding of monomeric C3b and C3b-IgG to human erythrocyte CR1 demonstrates identical ionic strength dependence for both species. Identical numbers of binding sites with indistinguishable affinities are detected by both ligands. Cleavage of the alpha'-chain of C3b and the alpha'-heavy chain of C3b-IgG proceeds at the same rate when erythrocyte CR1 serves as co-factor for factor I. Unlike factor H, CR1 supports a second cleavage of fluid-phase iC3b alpha'1 chain (free or bound to IgG) that generates C3c and a 33,000 m.w. fragment, which bears antigenic markers characteristic of C3g. Inactivation of C3b and C3b-IgG by CR1 and factor I also occurs at physiologic ionic strength, but proceeds very slowly relative to rates attainable with sub-physiologic inputs of factor H. CR1 does not recognize IgG-bound C3b as being in a protected site but, because of low binding affinity at physiologic ionic strength, is probably highly dependent on multivalent ligand-receptor interactions to efficiently exert its co-factor functions. Thus, inactivation of C3b-IgG heterodimers or small immune complexes bearing limited numbers of C3b residues may remain largely factor H-dependent in vivo, with resultant enhanced C3b survival.

Antigen-Antibody Complex↗

PubMed1986

Murine protein H is comprised of 20 repeating units, 61 amino acids in length.

A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized with this cDNA probe. Ten positives were colony-purified, and the largest plasmid cDNA insert, MH8 (4.4 kb), was sequenced by the dideoxy chain termination method. MH8 contained the complete coding sequence for the precursor of murine complement protein factor H (3702 bp), 100 bp of 5'-untranslated sequence, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length. Similar repetitive units are present in the C4b binding protein, the C3b-receptor (CR1), complement factor B and C2, and in beta 2-glycoprotein I and the interleukin 2 receptor. This finding suggests a common evolutionary origin for regions of these proteins.

Amino Acid Sequence↗

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↗

PubMed1995

Characterization of binding of human beta 2-glycoprotein I to cardiolipin.

beta 2-Glycoprotein I-cardiolipin complexes are reported to be a target antigen for the binding of a subset of anti-phospholipid antibodies. The characteristics of binding of beta 2-glycoprotein I to cardiolipin are reported in this paper. Binding at neutral pH is specific, saturable, dependent on ionic strength and independent of bivalent cation. Binding at low pH is qualitatively different from that at neutral pH, and is not dependent on ionic strength. Denaturation of beta 2-glycoprotein I by heat inactivation and reduction/alkylation indicates that beta 2-glycoprotein I-cardiolipin interaction does not require the native three-dimensional structure of beta 2-glycoprotein I, implying that a linear sequence motif may be responsible. Modification of amino acid residues by KCNO treatment completely destroys binding capacity, indicating crucial involvement of lysine residues in binding of beta 2-glycoprotein I to cardiolipin. Complement factor H, which has some similar highly charged linear sequence motifs to beta 2-glycoprotein I and is composed of the same type of protein module, was found to bind to cardiolipin and inhibit the binding of beta 2-glycoprotein I to cardiolipin. Three different lysine-rich segments of the fifth domain of beta 2-glycoprotein I may be involved in binding to cardiolipin.

Amino Acid Sequence↗

PubMed1995

Human factor H and C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b.

Human factor H in the complement (C) system has been characterized as a decay-accelerator for the alternative C pathway C3 convertase and a cofactor for factor I-mediated inactivation of C3b. The current concept is that it does not serve as a C4b-inactivating cofactor. In the present study, we demonstrated that in fluid-phase, factor H and Factor I can cleave methylamine-treated C4(C4ma), a C4b analogue, to C4d, regardless of its isotype. The buffer pH and ionic strength were critical factors for the C4ma cleavage, which proceeded at around pH 6.0 and low conductivity around 3.0 mS. Similar results were obtained with fluid-phase C4b. Cell-bound C4b, however, did not undergo factor I-mediated inactivation by factor H. Hence, all of the human cofactors reported to date can mediate factor I-mediated cleavage of both C3b and C4b at least in the fluid-phase.

Carrier Proteins↗

PubMed1995

Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b.

Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes.

Binding Sites↗

PubMed1987

Characterization of the human erythrocyte complement receptor CR1 (C3b receptor) by epitope mapping.

Monoclonal antibodies and an anti-idiotypic serum against human complement receptor CR1 (C3b receptor, immune adherence receptor) were used to identify CR1 and some of its proteolytic fragments by an immunoblotting technique. The anti-idiotypic serum had a specificity for the C3b-binding site, as could be shown by its cross-reactivity with complement factor H. The monoclonal antibodies GARP-4 and GARP-37 were specific for epitopes located nearby the ligand-binding site, because they blocked the immune adherence reaction. For the immunoblotting technique, it was essential to use non-reducing conditions, since reduction of CR1 destroyed the epitopes. Therefore, mainly large (disulphide-linked) fragments of CR1 were obtained. A chymotryptic fragment of Mr 56,000 identified by GARP-4, was the smallest cleavage product to be associated with the C3b-binding domain. Different proteases gave CR1 degradation products of similar Mr, indicating the presence of distinct domains, three of which had a Mr approximately 38,000. A schematic model of CR1 substructure was deduced from the epitope mapping data.

Antibodies, Monoclonal↗

PubMed1989

Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with cryptococci opsonized with the reconstituted pathway compared with that observed when yeast cells were opsonized with normal serum.

Complement Activation↗

PubMed1982

A general method for affinity purification of complement component C3b using factor H-sepharose.

Complement component C3b has been purified from human, rabbit and bovine serum by affinity chromatography on human factor H-Sepharose after preliminary fractionation by poly(ethylene glycol) and DEAE-Sepharose. The yields are high (35--40%) and the whole process is rapid (3 days). Binding of C3b to factor H-Sepharose is equimolar, has a sharp optimum pH at 7.6 and is quite sensitive to ionic strength.

Animals↗

PubMed1983

Inhibition of classical C5 convertase in the complement system by factor H.

This paper described the influence of factor H on the haemolytic activity of the classical C5 convertase. Factor H showed little effect on the interaction of C5 with EAC1,4b,2a,3b cells bearing low numbers of C3b sites, but displayed the inhibitory effect on the interaction of C5 with the intermediate cells bearing high numbers of C3b sites. The higher the number of C3b sites on the cells, the greater the degree of the inhibition by factor H. The inhibition by factor H was accompanied by the inhibition of consumption of C5 from the fluid phase, indicating that factor H inhibits the activity of C5 convertase, not the binding of activated C5 to the cells.

Animals↗

PubMed1991

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Amino Acid Sequence↗

PubMed1995

Synthesis of complement proteins in amnion.

The amnion is a metabolically active tissue that has been identified as a site of synthesis of numerous products. We report that amnion tissue explants and amnion-derived epithelial cells synthesize and secrete six proteins of the complement system, C1r, C1s, C1 inhibitor, factor B, C3, and factor H. Synthesis of C2 was minimal and variable, and C5 was not detected. The six synthesized proteins had size and subunit composition characteristic of proteins synthesized in HEp2, a long term cell line derived from malignant epithelial cells. Constitutive and regulated synthesis of five of the six proteins was similar in amnion tissue and cells. However, synthesis of factor B was different in tissue and cells; constitutive synthesis was 12-fold higher in tissue than in cells, and interleukin-1 did not alter synthesis in tissue, but increased synthesis by 11.7-fold in cells. These results indicate that amnion may be a source of complement proteins present in the amnion fluid and may contribute to local host defense along with endometrial glandular epithelial cells, which synthesize C3. Furthermore, our results suggest that amnion tissue is stimulated in vivo to synthesize factor B and cannot respond to interleukin-1 with a further increase in the synthesis rate.

Adult↗

PubMed1993

Molecular modelling of the domain structure of factor I of human complement by X-ray and neutron solution scattering.

Factor I is a typical multidomain protein of the complement system. It regulates complement activation by proteolytic degradation of C3b or C4b in the presence of factor H, complement receptor type 1, membrane cofactor protein or C4b-binding protein as cofactor. It is constructed from five presumed independently folded domains, namely a factor I module, a CD5-like domain, two low-density-lipoprotein receptor type A domains and a serine-proteinase domain. X-ray and neutron solution scattering was used to study the arrangement of these domains in factor I. Factor I was determined to be monomeric in solution, with an A280(1%,1cm) of 12.3-14.1. Its radius of gyration (RG) was 3.96 nm by X-rays in a high positive solute-solvent contrast, and 3.84 nm by neutrons at infinite solute-solvent contrast. The cross-sectional radius of gyration (RXS) was likewise found to be 1.64 nm by X-rays and 1.55 nm by neutrons. The RG data were not noticeably dependent on the solute-solvent contrast, whereas the RXS data showed a small dependence. The maximum dimension of factor I was determined to be 12.8 nm from the RG and RXS data, and 14-15 nm from the X-ray and neutron distance distribution functions. This length is too short to account for a linear arrangement of the domains in factor I. Small sphere models were developed for factor I in which the largest domain was modelled from the crystal structure for beta-trypsin. The attachment of either an elliptical cylinder or a two-armed V-shaped structure to this domain to represent the remaining four small domains gave good scattering curve-fits for factor I, and were compatible with experimental sedimentation coefficients. The non-extended domain models for factor I imply that the steric accessibility of each domain will be reduced, and this may be important for its functional activity.

Carrier Proteins↗

PubMed1985

Cigarette smoke can activate the alternative pathway of complement in vitro by modifying the third component of complement.

Cigarette smoking is associated with significant increases in the number of pulmonary mononuclear phagocytes and neutrophils. A potent chemoattractant for these cells is C5a, a peptide generated during complement (C) activation. We, therefore, investigated the possibility that cigarette smoke could activate the complement system in vitro. Our results show that factor(s) (mol wt less than 1,000) present in an aqueous solution of whole, unfiltered cigarette smoke can deplete the hemolytic capacity of whole human serum in a dose-dependent manner. The particle-free, filtered gas phase of cigarette smoke is inactive. The smoke factor(s) do not activate serum C1, but do deplete serum C4 activity. Treatment of purified human C3 with whole smoke solution modifies the molecule such that its subsequent addition to serum (containing Mg/EGTA to block the classical pathway) results in consumption of hemolytic complement by activation of the alternative pathway. Smoke-modified C3 shows increased anodal migration in agarose electrophoresis, but this is not due to proteolytic cleavage of the molecule as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to methylamine-treated C3, C3 treated with smoke is only partially susceptible to the action of the complement regulatory proteins Factors H and I. In addition, smoke-modified C3 has diminished binding to Factor H as compared with methylamine-treated C3. Finally, smoke-modified C3 incorporates [14C]methylamine which suggests that the thiolester bond may be intact. These data indicate that aqueous whole cigarette smoke solution can modify C3 and activate the alternative pathway of complement, perhaps by a previously unrecognized mechanism. Should this occur in vivo, complement activation might partly account for the extensive pulmonary leukocyte recruitment observed in smokers.

Complement Activation↗

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↗