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PubMed1989

Interaction of fluid phase C1/C1q and macrophage membrane-associated C1q with gram-negative bacteria.

Many gram-negative bacteria are killed after treatment with normal non-immune sera and directly bind and activate C1 in the absence of antibodies. For the immediate killing of such serum-sensitive bacteria, like R-forms of Salmonella strains, all serum complement components are essential. When purified serum C1 to C9 are used, further activation of the cascade requires an additional serum factor. This glycoprotein differs from antibody and mediates the attachment of C4b to the bacterial cell surface. The antibody-independent interaction with C1 occurs via C1q, which binds to LPS. In addition outer membrane proteins bind C1q and C1. The association of these porins with LPS may potentiate the antibody-independent C1q and C1 binding to serum-sensitive bacteria. Porins can contribute to complement activation mainly through the classical pathway. LPS and porins from bacterial cell walls are also involved in the binding of gram-negative bacteria to macrophages. This antibody-independent attachment and ingestion of gram-negative bacteria is mediated by endogenous macrophage-membrane associated C1q.

Animals↗
PubMed1989

[Major complement inhibiting factors from the venom of the Central Asian cobra Naja naja oxiana].

The properties of two anticomplementic factors isolated by CM-Sepharose chromatography from the basic non-adsorbed on DEAE-Sepharose fraction of the Central Asian cobra Naja naja oxiana venom, were studied. Of these three factors (CFB-I, CFB-II and CFB-III) the latter had been characterized earlier. CFB-I was shown to be a protein with an N-terminal Asp and a molecular mass of about 39 kDa (data from gel chromatography); its content in the venom is 3.6 mg/g of dry venom. The protein inhibits mainly the classical pathway of the complement activation, being bound to component C4 (Ki = 9 nM). CFB-I seems to be analogous to the CI inhibitor from the venom of the Naja haje cobra. An analysis of the N-terminal sequence of CFB-II showed it to be identical to the earlier characterized cytotoxin I. CFB-I inhibits the formation of C3 convertase with Ki = 2.2-2.8 microM by way of binding to C4b and thus interfering with the component C2 sorption.

Animals↗
PubMed1990

[Lupus and protein deficiencies of the classical complement pathway].

Deficiencies in proteins of the classic complement pathway are particularly frequent in patients with autoimmune diseases, notably systemic lupus erythematosus (SLE). The C4 component is a polymorphous glucoprotein coded by two closely linked genes, C4A and C4B, located within the HLA complex. C4, and in particular the C4A isotype plays a major role in maintaining immune complexes in solution. Fifty percent of patients with SLE are homozygous or heterozygous to the silent allele C4 AQO. Hereditary CE deficiency is often complicated by lupus-related diseases which may be associated with repeated infections. The biological particularity of SLE associated with complement protein deficiencies is the frequency of anti-SSA (Ro) antibodies.

Complement C2↗
PubMed2000

Two clusters of acidic amino acids near the NH2 terminus of complement component C4 alpha'-chain are important for C2 binding.

Previous work has indicated a role for the NH2-terminal segment of the C3 alpha'-chain in the binding interactions of C3b with a number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the NH2 termini of their respective alpha'-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases, as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the NH2-terminal segment of the C4 alpha'-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic peptide (C4 residues 740-756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cell-associated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 alpha'-chain contributes importantly to the interaction of C4b with C2.

Amino Acid Sequence↗
PubMed1987

Association between restriction fragment length variants of the complement C4 genes and MHC haplotypes.

DNA polymorphism of the human major histocompatibility complex (MHC)-linked complement C4 genes was studied using restriction enzymes XbaI and TaqI, and Southern hybridization. The results show that some, but not all, C4 phenotypes can be divided into subtypes. Analysis of MHC haplotypes indicates that in each extended MHC haplotype, i.e. in the haplotypes having a particular combination of HLA and complotype phenotypes in significant linkage disequilibrium, the C4 genes always produce just one 'conserved' restriction enzyme fragment pattern, while in the other haplotypes the C4 genes are more heterogeneous. Furthermore, the findings provide preliminary evidence for a C4B gene duplication, and suggest that the C4 genes may often be 'corrected' alike.

Chromosome Mapping↗
PubMed2000

Assessment of the interaction of human complement regulatory proteins with group A Streptococcus. Identification of a high-affinity group A Streptococcus binding site in FHL-1.

Group A Streptococcus (GAS), the most frequent bacterial cause of suppurative infections in humans, expresses on the cell surface M proteins with capacity to bind factor H, FHL-1 and C4b binding protein (C4BP). This has been interpreted as a mechanism developed by this pathogen to decrease phagocytosis by macrophages and polymorphonuclear cells. We report the analysis of the capacity to bind factor H, FHL-1 and C4BP of 69 clinical isolates from 19 different serotypes. We show that strains binding complement regulators (30/69) belong to specific M serotypes. Of these, M18 strains are relatively frequent and interact with all three complement regulators simultaneously. However, the most virulent M1 and M3 strains did not bind complement regulators in our assays. The relevance of the interaction between complement regulators and S. pyogenes was analyzed using different approaches with the conclusion that under physiological conditions only FHL-1 and C4BP bind to streptococci. We show that FHL-1 presents a higher binding affinity for S. pyogenes than factor H because it carries a hydrophobic, high-affinity, GAS binding site in addition to the heparin binding site in SCR7. Using synthetic peptides we provide evidence that the high-affinity GAS binding site in FHL-1 involves the hydrophobic tail (Ser-Phe-Thr-Leu) that distinguishes FHL-1 from factor H.

Amino Acid Sequence↗
PubMed1990

Association of C3 and C4A complement types with familial amyloidotic polyneuropathy.

A mutant variant of the serum protein transthyretin (TTR-met30) appears to be a necessary but not sufficient condition for the development of familial amyloidotic polyneuropathy (FAP). We have studied a number of serum protein markers (alpha 1-antitrypsin, properdin factor B, C3, C4A, C4B, haptoglobin, transferrin and group-specific component) in FAP patients and healthy controls in an attempt to identify additional pathogenic factors which may influence the risk for developing FAP in male and female patients as well as the age of onset of the disease. Statistically significant associations were found in the complement systems C3 and C4A. The C3F variant was significantly increased in all FAP patients with a relative risk (RR) of 2.0, more pronounced in female patients (RR = 2.6) and patients with an early onset of the disease (RR = 4.5). In the FAP patients only the variants A3 and A4 were found in the C4A system. C4A3 was found in all patients, which was significantly higher than in the controls. The remaining serum protein systems showed no statistically significant associations with FAP. The results suggest that genetic variants of complement factors C3 and C4A may interact with the mutant TTR-met30 by modifying the expression and onset of FAP.

Age Factors↗
PubMed2001

Complement factor I is upregulated in rat hepatocytes by interleukin-6 but not by interferon-gamma, interleukin-1beta, or tumor necrosis factor-alpha.

Complement factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b and thus prevents the assembly of the C3 and C5 convertases. We have investigated the proinflammatory cytokines IL-6, IL-1beta, TNF-alpha and IFN-gamma for their potential role in the regulation of FI expression. Of the investigated cytokines, only IL-6 increased the FI-specific RT-PCR signal in isolated hepatocytes, in the two rat hepatoma-derived cell lines FAO and H4IIE or in HUVECs. Quantitative competitive RT-PCR showed an IL-6 induced upregulation of FI-specific mRNA by about ten-fold. These data are in accord with Northern blot analyses in which the FI-mRNA was upregulated by IL-6 between five- and seven-fold. IL-6, but not IL-1beta, TNF-alpha or IFN-gamma also increased FI-protein levels in cell culture supernatants by about five-fold as determined by a semiquantitative immunoblot using a novel monoclonal antibody specific for rat FI.

Amino Acid Sequence↗
PubMed2005

Emerging roles and new functions of CD46.

In the past 20 years, our understanding of the workings of complement regulatory protein, CD46 (membrane cofactor protein), has grown as has the impressive list of pathogens interacting with this membrane-bound complement inhibitor. Referred to as a "pathogen magnet," CD46 serves as a receptor for seven human pathogens. Initially discovered as a widely expressed C3b- and C4b-binding protein, it was subsequently shown to be a cofactor for the serine protease factor I to inactivate by limited proteolysis these two opsonins and components of the convertases. The involvement of CD46 in reproductive processes continues to be an emerging story. It is a protector of placental tissue, but it may also play a more direct role in reproduction through its expression on the inner acrosomal membrane of spermatozoa. Cross-linking CD46 with antibodies or natural or pathogenic ligands induces rapid turnover and signaling events. In this regard, much attention is currently focused on generating human T lymphocyte regulatory cells by cross-linking CD46. Finally, highlighting its importance in protecting cells against excessive complement activation is the discovery that even a heterozygous deficiency of CD46 predisposes to hemolytic uremic syndrome.

Amino Acid Sequence↗
PubMed1993

HLA antigens and complement C4 allotypes in patients with chronic biologically false positive (CBFP) seroreactions for syphilis: a follow-up study of SLE patients and CBFP reactors.

We report a follow-up of our previous study of HLA markers in 118 unrelated patients: 49 with definite systemic lupus erythematosus (SLE) (group 1), 32 with definite or probable SLE and chronic biologically false positive (CBFP) seroreactions for syphilis (group 2), and 37 CBFP reactors (group 3). Definite SLE was confirmed in 28 (90.3%) of the patients in group 2, equally in HLA B8- and HLA B7-positive patients. Three of the CBFP reactors developed SLE, two (40%) out of five HLA B8-positive as compared to one (6.6%) out of 15 HLA B7-positive CBFP reactors (P = 0.07). Fourteen patients died (groups 1 and 2). Eight of the 24 HLA B8-positive patients died in contrast to one of the 20 HLA B7-positive patients (P < 0.02). Of the CBFP reactors, 70.9% had complement C4 null alleles as compared to 47.9% in controls (P = 0.05) and 50% had C4A null alleles as compared to 17.8% in controls (P < 0.05). C4B null alleles were found in 28.6% (28.6% in controls, P is not significant). The null alleles for C4A were not solely in a linkage disequilibrium with the HLA B8 DR3 haplotype. CBFP reactors with C4A null alleles had a higher risk of developing SLE, lupus-like disease or symptoms such as photosensitivity, cutaneous vasculitis and/or autoantibodies than did those with no C4A null alleles (P < 0.02).

Adolescent↗
PubMed1984

MHC-linked class III genes. Analysis of C4 gene frequencies, complotypes and associations with distinct HLA haplotypes in German Caucasians.

The class III complement components, C4, C2 and factor B (BF), are encoded in the human major histocompatibility complex (MHC). The two genes determining C4 (C4A and C4B) display considerable polymorphism and, thus, are important markers for HLA. In combination with alleles of C2 and BF they can be grouped into unique complotypes. We have analyzed the C4 alleles in a panel of 204 unrelated German Caucasians and studied their segregation with HLA haplotypes in 24 normal families. Inclusion of the class III markers with the class I and II alleles provides a more refined picture of the genetic structure of the MHC in these families. When charted according to the HLA-B locus specificities the MHCs can be clustered into groups showing distinctly homogenous or heterogenous complotypes. The identification of such groups is valuable for the selection of genetic material to analyze the molecular genetics of the human MHC.

Complement C2↗
PubMed1999

Structure-activity relationships within the N-terminal short consensus repeats (SCR) of human CR1 (C3b/C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the classical and alternative pathways of complement activation.

Genes coding for between one and four short consensus repeats (SCR) of the N-terminal region of human complement receptor 1 (CR1) were synthesized from oligonucleotides and those encoding SCR(1-2), SCR(1-3), SCR(1-4), SCR3 and SCR(3-4) were expressed as inclusion bodies in Escherichia coli. Following solubilization in urea, the proteins were partially purified and refolded and the activity of each protein was assessed in both classical and alternative pathway complement assays. All fragments showed a varying degree of activity with the general order being SCR(1-3) = SCR(1-4) > SCR(1-2). Addition of SCR3 to SCR(1-2) significantly improved potency, whereas the addition of SCR4 conferred no additional benefit. This observation, coupled with the ability of the single-domain SCR3 to inhibit classical pathway mediated lysis with an IH50% (inhibition of hemolysis by 50%) of 4.8 microM, demonstrates that SCR3 provides key binding interactions with activated complement components. SCR(1-3) was able to inhibit both classical and alternative pathways of complement activation, showing that the N-terminal SCR of CR1 retain the ability to interact with C3b. Assays for CR1-like cofactor activity for factor I using C4b-like C4 or C3b-like C3 as substrates showed that SCR(1-3) possessed such cofactor activity and that C4b-like C4 was a better substrate. When compared to full-length (30 SCR) soluble CR1 (sCR1), SCR(1-3) was significantly less potent in accord with a model involving multi-valent binding of C3b/C4b to CR1.

Animals↗
PubMed1989

Sequence of the gene for murine complement component C4.

The gene for murine complement component C4 lies in the S region of the murine major histocompatibility (H-2) complex; in this paper, we report the nucleotide sequence of this gene. The present sequence extends from a SmaI restriction enzyme cleavage site near the 5' end of the gene to a KpnI restriction enzyme cleavage site 569 nucleotides 3' of the polyadenylation site. The sequence spans 15,956 base pairs and together with previously reported data provides a complete sequence extending from the site of transcriptional initiation to the polyadenylation site. The sequence reveals that the C4 gene has 40 introns which range from 75 to 1089 base pairs in length and which include three murine B1 middle repetitive elements, a MT repeat element, and an apparently novel repeat sequence that is also found in noncoding regions of the murine beta-glucuronidase, lymphotoxin (TNF-beta), and rat alpha-crystallin genes. An intron splits the protein coding sequence precisely at the site of proteolytic activation of C4 by complement protease C1s; however, except for this one case, the intron positions show no striking relationship to the structural features of the C4 protein. The length of the murine C4 gene relative to the isotypic C4A and C4B genes in man suggests the independent loss of a 6-kilobase intron from both murine and human C4 genes.

Animals↗
PubMed1991

Phospholipid-anchored and transmembrane versions of either decay-accelerating factor or membrane cofactor protein show equal efficiency in protection from complement-mediated cell damage.

Decay-accelerating factor (DAF) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that protects cells from complement-mediated damage by regulation of the C3 convertase. To investigate the role of the GPI anchor in the function of DAF, the cDNA encoding human DAF was expressed by transfection in Chinese hamster ovary (CHO) cells. Testing of these DAF transfectants in an antibody plus human complement-mediated cytotoxicity assay demonstrated that DAF protects these cells from cytotoxicity, and that the level of protection increases with expression of surface DAF. A cDNA construct encoding a transmembrane version of DAF (DAF-TM) protects CHO transfectants from cytotoxicity with equal efficiency to DAF. This DAF-TM construct used the TM and cytoplasmic domains of membrane cofactor protein (MCP); an alternate TM version of DAF constructed with the TM and cytoplasmic domains of HLA-B44 showed equivalent protection. The protection from cytotoxicity involved a decrease in the deposition of C3 on the cell, consistent with the effect of DAF on the C3 convertase. A second pair of anchor variants, MCP and a GPI-anchored construct, MCP-PI, were also equivalent in their complement protection. The equivalent function of GPI-anchored and TM versions of a protein was not expected based on the hypothesized increased lateral mobility of GPI-anchored proteins, which should confer a functional advantage in contacting ligand, in this case, C3b or C4b, on the cell surface. These data suggest either that GPI-anchored and TM versions of a protein have equal lateral mobility in the membrane, or else that increased lateral mobility is not advantageous to DAF or MCP in carrying out their complement inhibitory roles. Furthermore, DAF and MCP demonstrated approximately equal protection of cells from complement-mediated cytotoxicity, suggesting that DAF and MCP provide overlapping levels of protection to cells against damage mediated by the complement system.

Animals↗
PubMed1994

Characterisation of the novel gene G11 lying adjacent to the complement C4A gene in the human major histocompatibility complex.

Twelve transcriptional units have now been located in a 160 kb segment of DNA that includes the genes encoding members of the serum complement system C2, Factor B (Bf) and C4 within the class III region of the human major histocompatibility complex (MHC). The common arrangement of these genes is tel-C2-Bf-RD-G11-C4A-[P450c21A-YA-XA]-C4B-[P450c21B-YB ]-+ ++TNX-cen. Characterisation of cDNA and genomic clones corresponding to the novel gene G11 has revealed that the gene spans approximately 9.1 kb of DNA and is split into 7 exons. The 5' end of the gene is associated with a CpG-island while the 3' end of the gene lies 611 bp from the transcriptional start site of the C4A gene. The approximately 1.4 kb G11 mRNA, which is expressed in a number of different cell types including monocytes, hepatocytes, epithelial cells, T and B lymphocytes, encodes protein products of 254 or 258 amino acids due to differential use of two splice sites lying 12 bp apart at the end of exon 3. These polypeptides share homology with a limited number of proteins including human cytochrome P450XIB1 and the tyrosine kinase transforming protein from fujinami virus. Duplication of the C4/P450c21 transcriptional unit occurred by a nonhomologous recombination event. Sequence analysis of a 1.5 kb segment of DNA flanking the C4B gene has revealed that 914 bp of the 3' end of the G11 gene also lies 611 bp from the transcriptional start site of the C4B gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B.

Mannose-binding lectin (MBL) is a serum protein that has been demonstrated to activate the classical complement pathway and to function directly as an opsonin. Although MBL deficiency is associated with a common opsonic defect and a predisposition to infection, the role of the protein in bacterial infection remains unclear. We have investigated MBL binding to Neisseria meningitidis serogroup B1940 and three isogenic mutants, and the subsequent activation of the two major isoforms of C4 (C4A and C4B) by an associated serine protease, MASP. The mutants lacked expression of the capsular polysaccharide (siaD-), the lipo-oligosaccharide (LOS) outer core that prevented LOS sialylation (cpsD-), or both capsule and LOS outer core (cps-). Using flow cytometry, it was possible to detect strong MBL binding to the cps- and cpsD- mutants over a wide range of concentrations. In contrast, minimal or no MBL binding was detected on the parent organism, with binding to siaD- only at higher MBL concentrations. C4 was activated and bound by mutants that had previously bound MBL/MASP, but there was no significant difference in the amounts of C4A and C4B bound. When sialic acid residues were removed from the parent organism by neuraminidase treatment, the binding of both MBL and C4 increased significantly. Our results suggest that MBL may bind to and activate complement on these encapsulated organisms, and the major determinants of these effects are the LOS structure and sialylation.

Bacterial Proteins↗