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PubMed2005

Identification of complement regulatory domains in vaccinia virus complement control protein.

Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.

CD55 Antigens↗
PubMed1983

Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: importance of null alleles of C4A and C4B in determining disease susceptibility.

The families of 29 patients with systemic lupus erythematosus and 42 normal subjects were studied to determine the inheritance of the HLA-A, B, C, and DR antigens and also the complement polymorphisms for C2, C4A, C4B, and Bf, which are encoded in the same region of the sixth chromosome. Null (silent) alleles for C4A, C4B, or C2 were found in 24 of the 29 (83%) patients compared with 18 of the 42 (43%) normal controls. HLA-DR3 was present in 20 (69%) of the patients and seven out of 39 (18%) of the normal controls. There was strong linkage disequilibrium between DR3 and the null alleles for C4A and C4B. The data did not permit the relative contributions of DR3 and null factors of C4A and C4B as genetic risk factors to be distinguished. The known association of systemic lupus erythematosus with uncommon inherited and acquired deficiencies of complement components suggests, however, that the presence of null alleles for C4A and C4B, as well as C2, found in most of the patients, is relevant to their genetic susceptibility to this disease.

Adolescent↗
PubMed1988

C4 complement allotypes in juvenile dermatomyositis.

Twenty probands with juvenile dermatomyositis and their relatives were studied to determine the inherited segregation patterns of class I, II, and III HLA region markers including C4A, C4B, Bf, and C2 complement polymorphisms. The extended haplotype B8, DR3, C4A*Q0, C4B*1, C2*C, and Bf*S was present in 13 of the 20 probands. Three other probands also carried a haplotype with a null allele for C4A and two further probands carried a null allele for C4B; only two probands had no detectable C4 null allele. These data confirm previous studies showing high frequencies of B8 and DR3 in patients with juvenile dermatomyositis, but show that there is a higher association with null alleles of C4. This suggests that the C4 genes are either themselves the disease-susceptibility genes or are in very strong linkage disequilibrium with such genes.

Adolescent↗
PubMed1992

Allotypes of the fourth component of complement in Koreans.

The analysis of genetic polymorphism in C4 was performed on EDTA-plasma from 169 healthy unrelated Koreans. Plasma samples were subjected to high-voltage agarose gel electrophoresis followed by immunofixation. C4B allotypes were further detected by a hemolytic overlay method. The allele frequencies of C4A and C4B were as follows; for C4A, C4A*3 = 0.6099, C4A*4 = 0.1702, C4A*Q0 = 0.1525, C4A*2 = 0.0461, and C4A*R = 0.0213; for C4B, C4B*1 = 0.6406, C4B*2 = 0.2740, C4B*5 = 0.0569, C4B*Q0 = 0.0178, and C4B*R = 0.0107. C4A*3 and C4B*1 were among the most common alleles at each locus. C4A*6 was not detected in this study, but this allele is relatively common in both Caucasoid and Negroid populations. C4B*5 is a common allele in Asian, which is rare in Caucasoids and Negroids. C4B*5 appeared to be a characteristic allele of Oriental. In the C4A locus, five individuals with duplicated allotypes (three C4A 3,3 + 2, one C4A 4,3 + 2, and one C4A 3,3 + 3) were observed, and in the C4B locus, one individual with duplicated allotype (C4B 1,1 + 1) was detected.

Alleles↗
PubMed1995

Ig-binding surface proteins of Streptococcus pyogenes also bind human C4b-binding protein (C4BP), a regulatory component of the complement system.

Streptococcus pyogenes, an important human pathogen, expresses several proteins that interact with the immune system of the host. Among the proteins isolated from different bacterial strains are antiphagocytic M proteins, Ig Fc-binding proteins and exotoxins that act as superantigens. Here we report a novel interaction between S. pyogenes and the human immune system, the ability of most S. pyogenes strains to bind human C4BP (C4b-binding protein), a 570-kDa serum protein that inhibits the classical pathway of complement activation. Molecular analysis of three different streptococcal strains demonstrated that C4BP binds to protein Arp or protein Sir, two Ig-binding cell surface molecules that are members of the M protein family. These bacterial proteins have separate high affinity binding sites for Ig and for C4BP, as demonstrated by inhibition tests and binding assays with purified components. A single streptococcal cell surface molecule, Arp or Sir, therefore combines the abilities to bind Ig and C4BP, two high m.w. components of the immune system. Two bacterial strains expressing Arp or Sir were shown to selectively bind C4BP in whole human serum, suggesting that S. pyogenes also binds C4BP in the infected host. When bound to streptococcal cells, C4BP retained its ability to act as a cofactor in the degradation of C4b by factor I. These results indicate that many strains of S. pyogenes interfere with the classical pathway of complement activation by binding C4BP to the bacterial cell surface.

Antigens, Bacterial↗
PubMed2003

A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b.

The erythrocyte type one complement receptor (E-CR1) mediates erythrocyte binding of complement-opsonized immune complexes (IC), and helps protect against random deposition of circulating IC. Two linked CR1 polymorphisms occur in binding domains, at I643T and Q981H. In Caucasians, the variant alleles (643T, 981H) are associated with low constitutive E-CR1 expression levels. This study was conducted to determine if these polymorphisms affect ligand binding, and if so, represent risk factors for the autoimmune IC disease, systemic lupus erythematosus (SLE). In an ELISA comparing relative ligand binding differences, E-CR1 from individuals homozygous for the variant residues (643TT/981HH) exhibited greater binding to C4b, but not C3b, than homozygous wild-type E-CR1. Analysis of single-binding domain CR1 constructs demonstrated that the 981H residue imparted this enhanced C4b binding. No differences were observed in the 981H allele frequency between Caucasian controls (0.170, n = 100) and SLE patients (0.130, n = 150, P = 0.133), or between African American controls (0.169, n = 71) and SLE patients (0.157, n = 67). In a subset of individuals assessed for CR1 size, excluding from this analysis those expressing at least one B allele revealed a trend for over-representation of the 981H allele in Caucasian controls (0.231 frequency, n = 26) versus SLE patients (0.139, n = 83, P = 0.089), but again no difference between African American controls (0.188, n = 24) and SLE patients (0.191, n = 34). These data suggest that the 981H residue compensates for low constitutive expression of E-CR1 in Caucasians by enhancing C4b binding. This may contribute protection against SLE.

Black People↗
PubMed2004

The Kaposi's sarcoma-associated herpesvirus complement control protein mimics human molecular mechanisms for inhibition of the complement system.

Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O. B., Blackbourn, D. J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.

Amino Acid Motifs↗
PubMed1996

Effect of immunoglobulin variable region structure on C3b and C4b deposition.

Many of the biological activities of immunoglobulins, including interaction with the complement system, are attributed to the structure of the heavy chain constant domains. However, previous studies indicated that immune complexes formed with independently derived isotype-matched pairs of monoclonal antibodies vary with respect to their capacity to activate complement and to serve as targets for C3b and C4b deposition. The goal of the present study was to provide a structural basis for explaining how variable domains influence C3b and C4b deposition on immunoglobulins. Heavy and light chain variable domains from a pair of IgG2a antibodies previously shown to differ in terms of complement activation and C3b and C4b deposition were cloned and sequenced. The two clones utilize distinct heavy and light variable region genes and the translated amino acid sequence reveals several residues that could serve as potential targets for complement deposition which differs between the two antibodies. Molecular modeling suggests that many of the relevant differences between the two antibodies are located in solvent exposed portions of the heavy and light chain variable domains and that some of the relevant sites are located within the complementarity determining regions. Differences in antibody affinity do not provide an explanation for the previously observed role of variable domains on interactions with the complement system. These data suggest that sequence variations within solvent-exposed variable domain residues may play a key role in C3b and C4b deposition on immunoglobulins.

Amino Acid Sequence↗
PubMed1987

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system.

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The effect of residue 1106 on the thioester-mediated covalent binding reaction of human complement protein C4 and the monomeric rat alpha-macroglobulin alpha 1 I3.

The histidine at position 1106 of the C4B isotype of human complement is involved in catalyzing the covalent binding of the thioester to glycerol and water. By replacing the histidine with other residues, it was found that tyrosine is also capable of mediating the reaction. We propose that they act as nucleophiles by first attacking the thioester, upon activation, to form acyl intermediates, which subsequently react with the hydroxyl groups of glycerol or water. The monomeric alpha-macroglobulin, alpha 1I3 of the rat, was also studied. Unlike alpha 2-macroglobulin, which is a tetramer, alpha 1I3 has binding properties similar to those of C4A.

Amino Acid Sequence↗
PubMed1998

Regulation of complement-mediated swine endothelial cell lysis by a surface-bound form of human C4b binding protein.

BACKGROUND: Human C4b-binding protein (C4bp) functions as a cofactor for factor I in the degradation of C4b and C3b and, in addition, accelerates the rate of decay of the C4b2a complex. METHODS: In this study, we constructed a surface-bound form of human C4b-binding protein (C4bp-PI) consisting of a short consensus repeat 1-8 of the alpha-chain of C4bp and a glycosyl phosphatidylinositol (GPI) of the decay-accelerating factor (CD55) and established stable swine endothelial cell (SEC) lines expressing C4bp-PI by transfection of cDNA. Amelioration of complement-mediated lysis by the transfectant molecules was tested as an in vitro hyperacute rejection model of swine to human discordant xenograft, using the lactate dehydrogenase assay. RESULTS: Flow cytometric profiles of the stable SEC lines with C4bp-PI showed a high level of expression of this molecule. The cell lysate of the SEC line with C4bp-PI showed strong cofactor activity in not only C4b but also C3b, whereas the activity of plasma C4bp to bind to C3 was very weak. Approximately 150 x 10(4) molecules of C4bp-PI per SEC blocked human complement-mediated cell lysis by approximately 75%. CONCLUSIONS: The results suggest that the surface-bound form of C4bp will be very useful in clinical xenotransplantation.

Animals↗
PubMed1994

Quantitative analysis of C4b dimer binding to distinct sites on the C3b/C4b receptor (CR1).

The complement receptor CR1 (CD35) is a transmembrane protein composed in its extracellular portion of short consensus repeats (SCR 1-30) organized into four long homologous repeats (LHR-A, LHR-B, LHR-C, and LHR-D). Each LHR, except LHR-D, contains a binding site for C3b and/or C4b within its first four SCR. The binding reaction between CR1 and soluble dimers of C4b (C4b2) was analyzed using the native receptor on human erythrocytes and full-length recombinant CR1 expressed in stably transfected Chinese hamster ovary (CHO) cells. CR1 mutants expressed similarly were used to determine the SCR of LHR-A required for C4b2 binding and the potential of C3b binding sites in CR1 to bind C4b2. Erythrocyte CR1, CHO cells expressing full-length recombinant CR1 (ABCD), constructs ACD, and SCR(1-4)D each bound C4b2 with similar affinities (Kd, approximately 4 x 10(-7) M). Construct SCR(1-2)D bound C4b2 with lower affinity (Kd, 1.4 x 10(-6) M) indicating that SCR(1-4) are required for a fully functional C4b2 binding site. Construct SCR(15-18)D, which contains a C3b site, also bound C4b2 with lower affinity (Kd 1.2 x 10(-6) M) than its binding to C3b dimers. Constructs SCR(15-16)D and D did not bind C4b2. Each CR1 construct that bound C4b2 functioned as a cofactor for factor I-mediated cleavage to C4d.

Animals↗
PubMed1993

Studies on the mechanism of complement-mediated inhibition of antibody binding to HIV gp41.

We have previously demonstrated that HIV envelope gp41 binding to specific antibodies decreases after preincubation of fluid-phase gp41 in normal human serum. This inhibition is proven to be mediated by the classical complement pathway. In this study recombinant gp41 (rgp41) and/or synthetic peptides were preadsorbed to solid phase, and then complement (normal human serum/heated human serum/purified Clq/heated Clq) and anti-gp41 antibodies were added either after each other or simultaneously, and the amounts of bound antibody, and deposited C3b, C4b and Clq were measured. Complement-dependent inhibition of antibody binding to solid-phase rgp41 was found, and Clq seems to be at least partially responsible for this phenomenon. Heating of Clq did not affect this process. Higher amounts of anti-gp41 antibodies significantly and dose-dependently enhanced C4b and C3b fixation to solid-phase rgp41. In the case of synthetic peptides corresponding to the immunodominant region of gp41, significant antibody binding to the solid-phase peptides was also detected, and pretreatment of peptides preadsorbed to solid phase with normal human serum almost totally abolished the antibody binding.

Amino Acid Sequence↗
PubMed2003

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins.

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

Cloning, Molecular↗
PubMed2006

Expression of the peptide C4b-binding protein beta in the arthritic joint.

BACKGROUND: C4b-binding protein (C4BP) is a plasma oligomeric glycoprotein that participates in the regulation of complement and haemostasis. Complement-regulatory activity depends on the C4BPalpha-polypeptide, whereas the C4BPbeta-polypeptide inactivates protein S, interfering with the anti-coagulatory protein C-dependent pathway. OBJECTIVE: To investigate the expression of C4BPbeta in the rheumatoid joint. METHODS: Expression of C4BP was studied in synovial explants from patients with rheumatoid arthritis, osteoarthritis and healthy controls, using immunohistochemistry and in situ hybridisation. C4BP isoforms and free C4BPbeta were studied in synovial effusions from patients with rheumatoid arthritis, osteoarthritis and microcrystalline arthritis (MCA) by immunoblotting; total and free protein S levels were studied by enzyme immunoassay. RESULTS: C4BPbeta was overexpressed in the synovial membranes of patients with rheumatoid arthritis, in close association with the severity of synovitis and the extension of interstitial fibrin deposits. As many as 85% fluids from patients with rheumatoid arthritis contained free C4BPbeta, whereas this unusual polypeptide was present in 50% fluids from patients with MCA and 40% fluids from patients with osteoarthritis. Free protein S at the effusions was pathologically reduced in patients with rheumatoid arthritis and MCA, and remained normal in patients with osteoarthritis. CONCLUSION: C4BPbeta is expressed by the inflamed synovial tissue, where it can participate in processes of tissue remodelling associated with invasive growth.

Adult↗
PubMed1993

Complement polymorphism in herpes gestationis: association with C4 null allele.

BACKGROUND: Herpes gestationis (HG) is a rare, pregnancy-related skin disease characterized by the production of an autoantibody to a component of the hemidesmosome. It is associated with the class II antigens HLA-DR3 and HLA-DR4, but its potential association with the "class III antigens" C2, C4, and factor B has not previously been studied. OBJECTIVE: Our purpose was to study complement polymorphism in HG. METHODS: Using electrophoresis and immunofixation techniques, we determined the allele frequencies of C4A, C4B, C3, and factor B in 42 patients with a history of HG. RESULTS: Ninety percent of patients carried a C4 null allele (C4*QO). No statistically significant association with C3 or factor B alleles was seen. CONCLUSION: HG is associated with the presence of a C4*QO. Whether the C4*QO is the primary genetic association, or whether the C4*QO is related to its linkage disequilibrium with DR3 and DR4 has yet to be determined.

Adult↗
PubMed2000

Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins.

Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP alpha-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the alpha-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.

Amino Acid Substitution↗
PubMed1996

Ligand-binding renders the 160 kDa Trypanosoma cruzi complement regulatory protein susceptible to proteolytic cleavage.

It has been previously shown that a surface complement regulatory protein (CRP) of Trypanosoma cruzi trypomastigotes binds human complement components C3b and C4b, and inhibits C3 convertase formation, thus contributing to the resistance of the bloodstage parasites to complement-mediated lysis. The blood stage parasites rapidly and spontaneously release a limited set of membrane glycoproteins, including CRP, and the modulation of release of CRP following ligand binding was investigated. Incubation of the parasites with C3b results in the release of CRP at a reduced apparent molecular mass. To determine if proteolytic processing was responsible for the reduction in apparent molecular mass of the released CRP, the proteolytic activity present in trypomastigote membrane preparations was examined. In addition to a well described cysteine protease, a novel 75 kDa protease was identified in tissue culture-derived trypomastigotes and axenically-derived metacyclic trypomastigotes membrane preparations. This protease was inhibited by aprotinin and leupeptin, but not L-trans-epoxysuccinyl-leucylamide-(4-guanidinobutane), N-[N-L-3-transcarboxyirane-2-carbonyl-L-leucinyl]-agmatine (E64). Treatment of the parasites with protease inhibitors did not affect spontaneous shedding of proteins, however, protease inhibitors abrogated the effect of C3b-binding on CRP degradation. These results indicate that binding of C3b to the CRP renders the CRP susceptible to cleavage by a parasite protease, possibly as a means of removing the CRP-C3b from the parasite surface. This process may represent an additional immune evasion mechanism which allows the parasite to avoid both complement-mediated lysis and clearance.

Animals↗