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PubMed1993

Characterization of the promoter region of the membrane cofactor protein (CD46) gene of the human complement system and comparison to a membrane cofactor protein-like genetic element.

Membrane cofactor protein (MCP; CD46) is a widely expressed C regulatory protein that inhibits C activation on self-tissue. MCP binds C3b and C4b deposited on autologous cells and then serves as a cofactor for their inactivation by limited proteolytic cleavage. To characterize the DNA sequence elements responsible for controlling MCP expression, the 5' flanking region of the human MCP gene was cloned. Sequencing of 1350 nucleotides upstream from the ATG codon revealed a GC-rich region in the initial 500 nucleotides that is especially rich in the CpG dinucleotide. A CAAT box in reverse orientation, surrounded by four putative SP1 binding sites but lacking a typical TATA element, was within the first 200 nucleotides of this GC-rich region. The major transcriptional initiation site for HeLa cells, determined by primer extension and S1 nuclease protection analyses, was located 105 nucleotides from the translational start site. This overall orientation of the promoter region is characteristic of "housekeeping" genes. The MCP promoter region was further examined in HEp-2 cells by the chloramphenicol acetyltransferase (CAT) reporter gene assay, using various constructs derived from the 5' region of the MCP gene. The MCP promoter activity was confined to the GC-rich region from -624 to +96 (start site of transcription being +1). Inclusion of an AT-rich sequence from -624 to -1204 resulted in a 42% reduction in CAT activity suggesting that an inhibitor is present among the AT-rich sequences. The 5' flanking region of a highly homologous partial duplication of the MCP gene was also cloned and sequenced, and various constructs were assessed in the CAT reporter system. Many of the functionally relevant sequences seen in MCP are also found in the MCP-like 5' UT region, which is 85% homologous to MCP. The most striking difference was a 224 nucleotide deletion that was upstream from the corresponding MCP region harboring most of the promoter activity. Although expression of an MCP-like protein has not been reported, the MCP-like promoter region produced promoter activity comparable with that of MCP. These results serve as a basis for subsequent analyses of the expression of MCP in various cells and tissues and for understanding the mechanism of its modulation in inflammatory conditions. Also, through a comparison of the 5' region of MCP with other genes in the regulators of C activation gene cluster (at 1 q32), we propose a model for the evolution of the promoters in this tight linkage group.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1979

Human C4-binding protein. Association with immune complexes in vitro and in vivo.

C4-binding protein (bp), a glycoprotein with specific binding affinity for the activated form of C4 (C4b), has recently been isolated from human serum and partially characterized. This report demonstrates that C4-bp is incorporated into soluble immune complexes after complement activation in vitro. The reaction requires Ca++ ions and the presence of C4 in serum. Immunopathological studies of various forms of glomerulonephritis revealed intense C4-bp deposition in glomeruli from patients with immune-complex type of pathogenesis. C4-bp deposition was in close correlation with that of C4. These observations, together with the in vitro association of C4-bp to immune complexes, support the notion that the deposits in glomeruli represent the local accumulation of immune complexes.

Antigen-Antibody Complex↗
PubMed2000

A recombinant soluble chimeric complement inhibitor composed of human CD46 and CD55 reduces acute cardiac tissue injury in models of pig-to-human heart transplantation.

BACKGROUND: Inasmuch as complement plays a critical role in many pathological processes and in xenograft rejection, efficient complement inhibitors are of great interest. Because the membrane-associated complement inhibitors are very effective, recombinant soluble molecules have been generated. METHODS: We tested the efficacy of complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), in two models of pig-to-human xenotransplantation in which tissue injury is complement mediated. The in vitro model consisted of porcine aortic endothelial cells and human serum, and the ex vivo model consisted of a porcine heart perfused with human blood. RESULTS: In vitro, addition of CAB-2 to serum inhibited cytotoxicity and the deposition of C4b and iC3b on the endothelial cells. Ex vivo, addition of CAB-2 to human blood prolonged organ survival from 17.3 +/- 6.4 min in controls to 108 +/- 55.6 min with 910 nM (100 microg/ml) CAB-2 and 219.8 +/- 62.7 min with 1820 nM (200 microg/ml) CAB-2. CAB-2 also retarded the onset of increased coronary vascular resistance. The complement activity of the perfusate was reduced by CAB-2, as was the generation of C3a and SC5b-9. The myocardial tissues had similar deposition of IgG, IgM, and Clq; however, CAB-2 reduced the deposition of C3, C4, and C9. Hearts surviving >240 min demonstrated trace to no deposition of C9 and normal histologic architecture. CONCLUSION: These results indicate that CAB-2 can function as an inhibitor of complement activation and markedly reduce tissue injury in models of pig-to-human xenotransplantation and thus may represent a useful therapeutic agent for xenotransplantation and other complement-mediated conditions.

Animals↗
PubMed1996

DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression.

Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH+ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 - 1106, S at position 1157, and VDLL at positions 1188 - 1191. A second donor typed as C4A2, A4, B1 and was also WH+. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 - 1106, S at position 1157, and ADLR at positions 1188 - 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene.

Alleles↗
PubMed1990

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C.

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.

Antibodies, Monoclonal↗
PubMed1998

C1 inhibitor removes the entire C1qr2s2 complex from anti-C1Q monoclonal antibodies with low binding affinities.

Evidence is presented for a new C1 Inhibitor (C1 INH) function. C1 INH was capable of dislodging the entire C1qr2s2 complex from C1-activating substances that bound weakly to the globular heads of C1q. Two different mouse IgG1 monoclonal antibodies with different affinities for C1q globular heads were compared for their complement-activating properties in the presence of normal human serum. As expected the higher affinity monoclonal antibody (Qu) was more effective in binding C1q and causing C1-mediated C4b deposition. Unexpectedly, time responses of C1 (C1q) binding to immobilized 3C7 reached a peak then gradually decreased. However, C1q remained constantly bound to immobilized Qu. These results indicated that after C1 activation in human serum, the entire C1 complex (including C1q) was dislodged from 3C7, but not from immobilized Qu. The addition of purified C1 INH to purified C1, which had bound to immobilized 3C7, resulted in removal of C1 (C1q). Removal of the entire C1qr2s2 did not occur when C1 INH preparations were first neutralized by the addition of purified activated C1s. In summary, it is suggested that C1 INH plays a prominent role in dislodging the entire C1qr2s2 from immunoglobulin preparations which have a low binding affinity for the globular heads of C1q.

Animals↗
PubMed1990

Combined complete C5 and partial C4 deficiency in humans: clinical consequences and complement-mediated functions in vitro.

A family is described with two siblings who suffered at different times from a single episode of meningococcal meningitis by Neisseria meningitidis groups B and C, respectively. In the two subjects, hemolytically active fifth component of complement (C5) was not detectable and antigenic C5 was less than 0.05% and less than 0.7% of normal, respectively. Repletion of sera by purified human C5 (70 micrograms/ml) restored total complement hemolytic activities. The asymptomatic first degree family members had C5 levels compatible with a heterozygous state of C5 deficiency. C4 allotyping revealed an inherited partial deficiency (Q0) of C4A and C4B in the family with a combined C4AQ0 and C4BQ0 heterozygous condition in one and C4BQ0 heterozygosity in the other C5 deficient (C5D) subject. To our knowledge, this is the first human kindred with recognized combined C5 and C4 deficiency. No other defect of the humoral and cellular immune system was found in this family, including specific immune response to tetravalent meningococcal vaccine. The effect of partial C4 deficiency on classical pathway function was assessed by inhibition of immune precipitation (IIP) of forming bovine serum albumin (BSA)/anti-BSA immune complexes. Sera from all family members showed normal IIP values, with exception of the subject with combined partial deficiency in C4A, C4B, and complete deficiency in C5. Despite undetectable functional C5 in the C5D sera, the titration of the alternative pathway indicated intact but deficient hemolytic activities when rabbit erythrocytes (EC) were used as indicator cells in the presence of Mg2+ and EGTA in an end-point or kinetic assay. Preincubation of the two sera at 0 degrees C for 60 min with rabbit ECs reduced alternative pathway hemolytic activity by 24 and 100%, respectively. When rabbit ECs were replaced by guinea pig ECs no alternative pathway function could be measured. The results indicate that the apparent functional activity of the alternative pathway in C5D sera strongly depends on a factor(s) present in such serum and/or on the detection system used. We conclude that the two C5D individuals of the family reported here may not have sufficient C5 activity to provide efficient protection against Neisserial infections in conditions where complement functions beyond C3 opsonic activity are required in vivo.

Adolescent↗
PubMed1982

Kinetic studies of phagocytosis. III. The complement-dependent opsonic and anti-opsonic effects of normal and sle sera.

The influence of serum on phagocytosis related to the complement system was examined by means of a kinetic phagocytosis method using IgG-coated particles, isolated polymorphonuclear neutrophil leucocytes (PMNs), fresh serum, in vitro activated sera and in vivo activated sera. The previously described opsonic properties of C3b and C4b were confirmed by the enhancement of phagocytic rate by the opsonization of IgG particles with C3 and C4. An anti-opsonic effect of serum was revealed by the initial inhibition of PMN phagocytosis of IgG-coated particles in the presence of fresh serum. In vitro activated norma fresh serum and in vivo activated SLE sera mediated a prolonged or even irreversible inhibition of phagocytosis dependent on the degree of complement activation. Investigation of this anti-opsonic effect of serum, which was heat-labile, suggested that it was caused by an inhibition of the interaction between the Fc receptor and IgG mediated by the C1q component of the C1 complex.

Complement Activating Enzymes↗
PubMed1989

Subacute cutaneous lupus erythematosus. Genetic markers and clinical and immunological findings in patients.

The role of HLA and complement genes was studied in 13 patients with subacute cutaneous lupus erythematosus. Genetic markers and by combining the major histocompatibility complex class I (HLA-A, -B, and -C), class II (HLA-DR), and class III (properdin factor B [BF] and C4) phenotyping with DNA level analysis of the C4 region. Of our patients, 54% had DR2 antigens and 50% had DR3 antigens, when the frequencies in the controls were 25% and 33%, respectively. The DR3 antigen was associated with annular skin lesions that were associated with a younger age at onset, whereas the DR2 antigen was associated with papulosquamous skin lesions and an older age at onset. The frequency of C4 null alleles was 83% in the patients and 50% in the controls. The null alleles were found in both C4A and C4B loci and were not associated with any special major histocompatibility complex haplotype. The DNA studies showed that the null phenotype mostly resulted from a gene deletion. A highly increased frequency of complement C4 null alleles may be a predisposing factor for cutaneous lupus erythematosus and especially of the subacute cutaneous type.

Adult↗
PubMed1998

Possible arrangement of the five domains in human complement factor I as determined by a combination of X-ray and neutron scattering and homology modeling.

Human factor I is a multidomain plasma serine protease with one factor I-membrane attack complex (FIMAC) domain, one CD5 domain, two low-density lipoprotein receptor (LDLr) domains, and one serine protease (SP) domain and is essential for the regulation of complement. The domain arrangement in factor I was determined by X-ray and neutron scattering on serum-derived human factor I (sFI) and recombinant insect cell factor I (rFI). While the radii of gyration of both were the same at 4.05 nm and both had overall lengths of 14 nm, the cross-sectional radii of gyration were different at 1.70 nm for sFI and 1.57 nm for rFI. This difference was attributed to their different means of glycosylation which is complex-type for sFI and high-mannose-type for rFI. Homology models were constructed for the FIMAC, LDLr, and SP domains of factor I using related crystal structures, and CD5 was represented as a globular protein by referencing its electron microscopy dimensions. In these models, 38 of the 40 Cys residues in factor I were predicted to form internal disulfide bridges. The two remaining Cys residues at the N terminus of the FIMAC domain and at the center of the first LDLr domain were potentially not bridged. It was postulated that, if these two Cys residues were bridged to each other, the FIMAC, CD5, and LDLr-1 domains would form a compact triangular arrangement. This hypothesis was tested by automated scattering curve fit searches based on 9600 bilobal models, setting the FIMAC, CD5, and LDLr-1 domains as one lobe and the large SP domain as the other lobe. The searches gave a single small family of similar structures with a separation of 5.9 nm between the centers of the lobes which gave similar good X-ray and neutron fits for both sFI and rFI, despite the different glycosylations of sFI and rFI. These best-fit structures for factor I showed that this domain model is plausible, and suggested that the SP and the CD5 and LDLr-1 domains may present exposed surfaces in factor I whose roles are to interact separately with its substrates C3b and C4b and with cofactor proteins.

Amino Acid Sequence↗
PubMed1987

The superfamily of C3b/C4b-binding proteins.

The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively, which have more limited homology with the repetitive regions in this family. All available data indicate that multiple gene duplications and exon shuffling have been important features in the divergence of this family of proteins with the 60-amino-acid repeat.

Amino Acid Sequence↗
PubMed1980

The human complement system: assembly of the classical pathway C3 convertase.

The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.

Chemical Phenomena↗
PubMed1984

Gene and haplotype frequencies of the fourth component of complement (C4) in type 1 diabetics and normal controls.

C4 gene and haplotype frequencies were calculated from phenotype data of 380 unrelated Caucasian patients with insulin dependent (type 1) diabetes mellitus and were compared with analogous frequencies of 382 unrelated healthy Caucasian individuals. In diabetics, a significantly increased frequency of the rare allele C4B 3 (p less than 10(-7] and of the silent alleles C4A Q0 (p less than 10(-7] and B Q0 (p less than 0.002) was observed. Accordingly, insulin dependent diabetes is associated with partial C4 deficiency, which may contribute to the pathogenesis of the disease.

Alleles↗
PubMed1986

Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b.

Based on preliminary evidence indicating that a cell-associated protein of U937 (a human monocyte-like cell line) possessed cofactor activity and was not the C3b/C4b receptor, we sought to further characterize this protein. A sequential four-column purification procedure was devised that includes C3(H2O) affinity chromatography to isolate in reasonable yields and purity a cell-associated protein of U937 and several other human cell lines. Based on its pattern and Mr on SDS-PAGE, acidic pI, and ligand specificity, it is identical to a recently described C3(H2O) or C3b-binding membrane glycoprotein of human PBL and cell lines; having no presently identified function, it was termed gp45-70. After purifying this protein, we determined its functional capabilities and compared them to those of the other complement proteins with regulatory activity directed at components comprising the C3 convertases. This protein was the most efficient (50 times that of H) yet-described cofactor for the I-mediated first cleavage of C3b. It also was a cofactor for the first cleavage of C4b, but was not as efficient as C4bp. The second cleavage of C3b and C4b was not efficiently mediated. It had no ability to accelerate decay in the classical or alternative pathway C3 convertases. Based on this unique activity profile and ability to be surface labeled, we have renamed this molecule membrane cofactor protein (MCP). We suggest that this protein plays a major role in preventing autologous complement activation.

Antigens, CD↗
PubMed1982

Nephritic factor (NeF) of alternate pathway (NeFA) and of classical pathway (NeFc).

NeFA and NeFc were studied in various cases (115 cases). 5 cases were followed up for a long time. NeFA assay was done by the "microtest plate method". We detected the NeF activity for the first time in the cases of Sjögren syndrome (SjS) and SjS+SLE+Hashimoto's disease (Hashimoto). In some cases NeF activity disappeared after therapy, and in one case NeFA and NeFc were positive at first, but then only NeFA activity became negative following the adequate therapy. As to the antibody nature of NeF, the possibility was suggested that NeFA might be anti C3b autoantibody and NeFc might be anti C4b and/or C4b2a auto-antibody.

Complement Activation↗
PubMed1989

An update on Rodgers and Chido, the antigenic determinants of human C4.

Rodgers (Rg) and Chido (Ch) blood groups are antigenic determinants of the fourth component of human complement (C4). Nine determinants have been defined by means of hemagglutination-inhibition (HAI) with polyspecific human antiserums. The association of C4A isotypes with Rg and of C4B isotypes with Ch is strong hut not complete. Derived amino acid sequences from the C4d region of selected C4 allotypes of known antigenic expression have provided support for the previously reported complex serologic interrelationships. A structural model for antigenic determinants at four polymorphic sites, incorporating sequential and conformational epitopes, was subsequently proposed. Allotype and Rg/Ch data obtained from donors and patients, many with accompanying families, have augmented the model and revealed no exceptions. The antigenic determinants, therefore, make an important contribution to the complex polymorphism of C4.

Journal Article↗
PubMed1987

Formation of covalent complexes between the fourth component of human complement and IgG immune aggregates.

The binding properties of activated C4 to immune complexes (ovalbumin-rabbit IgG antiovalbumin) were studied by using 125I-IgG in the immune complexes or performing the C4 binding assays in the presence of 14C-iodoacetamide. High molecular weight complexes formed between C4 and IgG could be detected by the incorporation of 14C-iodoacetamide in the -SH group generated in the nascent C4b during the activation process. The same complexes with an apparent molecular weight of 180,000 daltons were detected when the immune aggregates contained 125I-IgG. Two-dimensional SDS-PAGE analysis of the C4b-IgG covalent complexes indicated: In the absence of control proteins, the complexes are formed by the alpha'-chain of C4b and the H chain of the antibody. The alpha'-H complexes are 36% sensitive to hydroxylamine and 64% resistant. This is consistent with the presence of two populations of C4, which are not equivalent in their covalent binding with immune complexes. Covalent complexes C4-C4b or C4b(like)-C4b(like) are generated during the C4 activation and they are detected as alpha-alpha' or alpha-alpha complexes, respectively. Interaction of C4b with the L chain of the antibody molecule also seems to occur, but to a lesser extent than with the H chain.

Antigen-Antibody Complex↗
PubMed1990

[Flow cytometric analysis of immunophagocytosis using sensitized fluorescent microspheres bearing C3b].

We analyzed the phagocytic activity of purified human monocytes using fluorescent latex beads sensitized with IgG or IgG.C3 by flow cytometry. To prepare IgG-sensitized latex beads (BA), BSA-coated latex beads (B) were incubated with diluted rabbit IgG anti-BSA. To bind complement components, BA were incubated with whole serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activity of factor I and C5, resulting in deposition of C1, C4b, C2a, C3b on BA (BAC). Phagocytic activity was assessed by percent phagocytosis and phagocytic index (PI). To eliminate the effects of non-phagocytosed latex beads, subtraction of the data at 4 degrees C from 37 degrees C was performed. Percent phagocytosis for 60 min. was B 5.0%, BA 18.3%, and BAC 57.5%, and PI (ingested latex beads/100 cells) was B 7.9, BA 36.8, and BAC 152.7, respectively. In addition, K-76COOH caused dose dependent inhibition on IgG.C3 mediated phagocytosis. Comparison of inhibition pattern on BAC and BA indicated that K-76COOH directly inhibited C3.C3-receptor binding.

Complement C3b↗