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PubMed1993

Decreased protein S activity in sickle cell disease.

In sickle cell disease (SCD), vaso-occlusion is a complex process involving cellular, vascular and humoral factors and possibly thrombotic events. We studied three physiological inhibitors of the coagulation system, antithrombin III (AT III), protein C (PC) and protein S (PS), in three groups of subjects: 27 homozygous patients observed either in crisis or in a steady state, 23 heterozygous patients and 30 healthy subjects. PS study included the measurement of total and free PS antigen, PS activity and C4bBP antigen. In heterozygous subjects the results were similar to those of controls, but in homozygous subjects abnormalities of PS and to a lesser extent PC were observed. Values of PC were extremely variable with 10 cases lower than the normal range (2 SD of the mean) and 17 others within this range. In all cases total PS antigen was slightly reduced (77 +/- 18%, M +/- SD) with a more marked decrease of free antigen (59 +/- 17%) and normal values of C4bBP. Levels of PS activity were greatly reduced and lower than those of free antigen with a mean ratio of PS activity to free antigen of 0.6. These abnormalities were associated with significantly high concentrations of fibrinogen D-dimers. PS deficiency in SCD may be at least partly due to adsorption of free PS to aminophospholipids abnormally expressed on sickle cells membranes, microvesicles and activated platelets, while the discrepancy between PS activity and free antigen could reflect proteolytic inactivation of PS by traces of thrombin.

Adolescent↗
PubMed1993

Complement activation and immune complexes in juvenile rheumatoid arthritis.

We investigated the potential role of the complement system in juvenile rheumatoid arthritis (JRA) by examining plasma for levels of complement activation fragments C4d and Bb. We correlated findings with both disease activity and laboratory abnormalities, including immune complex (IC) levels. While there was a strong correlation between active disease and both C4d and Bb plasma levels in JRA, no strong correlations could be made between IC levels and complement activation products; weak associations were seen between complement activation fragments and erythrocyte sedimentation rate. Our data suggest that plasma complement activation is a concomitant of active disease in JRA; however, its role in the pathophysiology of JRA remains uncertain.

Antigen-Antibody Complex↗
PubMed1991

Structure of the gene coding for the alpha polypeptide chain of the human complement component C4b-binding protein.

The human gene coding for the 70-kD polypeptide of the complement regulatory component C4b-binding protein (C4BP alpha) spans over 40 kb of DNA and is composed of twelve exons. Upon transcription in liver, or in Hep-G2 cells, this gene produces a single transcript of 2,262 nucleotides, excepting the poly A tail, that presents an unusually long 5' untranslated region (5' UTR) of 223 nucleotides. The C4BP alpha gene is organized as follows: the first exon codes for the first 198 nucleotides of the 5' UTR. It is separated by a large intron from the second exon including the remaining of the 5' UTR and the coding region for the signal peptide. Each of the eight 60-amino acid repeats (short consensus repeats [SCRs]) that compose the C4BP alpha polypeptide chain is encoded by a single exon, except for the second SCR, which is split in two exons. At the 3' end of the C4BP alpha gene, the twelfth exon codes for the COOH-terminal 57 amino acids of the mature protein, which have no similarities to the SCRs, and the 245 nucleotides of the 3' UTR. Examination of the nucleotide sequence of the first exon revealed an interesting characteristic, strongly suggesting that this exon may specify a functional domain of the C4BP alpha transcript. It includes two in-phase ATG codons, in a different frame respect to that coding the C4BP alpha polypeptide, followed by an in-frame termination codon, also within the first exon. Comparison between mouse and human C4BP alpha transcripts indicates conservation of this structure within the 5' UTR. C4BP is expressed in the liver and is an acute phase protein. A computer search of the genomic sequences upstream the transcription start site demonstrates the presence of potential cis-acting regulatory elements similar to those found in the promoters of other liver-expressed and/or acute phase genes.

Amino Acid Sequence↗
PubMed1995

Human complement proteins C3b, C4b, factor H and properdin react with specific sites in gp120 and gp41, the envelope proteins of HIV-1.

Recently we reported the basic phenomenon of an interaction between the envelope glycoproteins of HIV-1 gp120 and gp41 and components of the human complement system, i.e. activated C4 (C4b) and activated C3 (C3b) and the complement regulator proteins factor H and properdin. In this study we analyze these interactions in detail. Using 46 overlapping peptides of gp120 attached to microtiter plates, binding of activated human C3 to 6 regions in gp120 was found (aa 100-129, 161-190, 231-250, 301-328, 410-449, 470-499). In competition assays with soluble peptides, representatives of four of these regions were capable to partially inhibit C3b binding to immobilized gp120. Activated human C4 interacted only with peptides covering aa 410-449, but both in direct binding assays and fluid phase inhibition studies. The multi-reactivity of gp120 with C3b was also supported by the fact that gp120 agglutinated erythrocytes coated with C3b. Guided by partial aa sequence homology of gp120 and human C4b binding protein (C4bp) as well as human properdin we detected binding of anti-properdin to aa 100-129 in gp120 and of anti-C4bp to aa 410-449 in gp120. This cross-reactivity was also confirmed by a monoclonal antibody directed against aa 416-443 of gp120, which could be shown to bind C4bp. Interestingly, aa 310-328, part of the V3-loop, were found to show an aa sequence similarity to human complement receptor type 3 (alpha-chain). Consequently, of the 4 (or possibly 6) interaction sites of gp120 with activated human C3, 3 may bind due to imitation of either properdin, CR3 or C4bp. In addition to C4b and C3b, we detected interaction of factor H with gp120; it selectively bound to aa 102-129. Using 14 overlapping peptides of gp41 attached to plates, we identified 4 areas in gp-41 (aa 561-585, 587-605, 615-635, 651-675) which bound human factor H. All of them except the first region partially inhibited factor H binding to gp41 in competition assays with soluble peptides. Properdin bound only to 2 regions (aa 584-614, 651-675). The first 3 sites in gp41 were already shown by us to share homology to sites in human C3. The region around aa 651-675 now also turned out to be similar to human C3. These data demonstrate that the interaction of both, gp120 and gp41, with the complement system is polyvalent and complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1996

Glomerular deposition of the complement C4 isotypes C4A and C4B in glomeruonephritis.

BACKGROUND: Complement C4 is a component of the classical complement pathway, which is a major mediator of inflammation in many forms of glomerulonephritis. The two isoforms of C4-C4A and C4B-differ in their physicochemical and functional properties. METHODS: The glomerular deposition of C4A and C4B was investigated in 39 cases of glomerulonephritis with classical pathway activation using isotype-specific monoclonal antibodies 99H7 (C4A) and 1288 (C4B) and indirect immunofluorescence. Complement C4 phenotypes of all patients were determined by agarose gel electrophoresis and immunoprecipitation. RESULTS: Three biopsies contained only the isotype C4B. C4 phenotyping revealed complete C4A deficiency in these three patients. Both isotypes C4A and C4B were detected in 36 biopsies. In 19 (53%) thereof staining for both isotypes was identical. In the remaining 17 (47%), staining intensity of C4A predominated over C4B. The distribution of these two staining patterns did not differ between membranous glomerulonephritis and lupus nephritis. They were also independent of C4A and C4B allotypes including the presence or absence of null alleles at either gene locus. In no case was C4B staining stronger than C4A staining. Serum creatinine and proteinuria did not differ between patients with identical and C4A-dominant C4 deposition. CONCLUSIONS: The most likely but still hypothetical explanation for predominance in glomerular deposition of C4A over C4B in many cases of immune complex-mediated glomerulonephritis is the greater affinity of C4A to protein-containing immune complexes as compared to C4B.

Adolescent↗
PubMed1990

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain.

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.

Antibodies, Monoclonal↗
PubMed1984

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. I. Isolation and characterization of factor H: the serum cofactor for the C3b/C4b inactivator (factor I).

Factor H, purified from mouse EDTA-plasma using a 4-step procedure, consists of a single polypeptide chain of Mr 150,000 on SDS-PAGE. Mouse H (Hmo) was required for the cleavage of fluid-phase mouse C3b by mouse I (Imo). The final product of degradation of fluid-phase mouse C3b was iC3b, consisting of fragments of the alpha'-chain (alpha'-70, alpha'-43) linked by disulfide bonds to an intact beta-chain. Imo alone was capable of cleavage of membrane-bound mouse C3b and of generating iC3b. The addition of Hmo nevertheless had an enhancing effect on Imo activity, but cleavage did not proceed beyond iC3b. These observations suggest that one important function of Hmo is to permit the inactivation of fluid-phase C3b, and to inhibit irreversibly its activity. The concentration of H in the plasma of male and female BALB/c mice was not significantly different. Among different inbred strains of mice, large differences were observed in the plasma levels of H, and plasma H levels were positively correlated with the plasma levels of C3. This observation, taken together with the well known role of H in the control of the activation of the alternative pathway, suggests that the turnover of C3 is controlled to some extent by H.

Animals↗
PubMedDate not supplied

[Inborn deficiency of complement C4A and C4B isotypes in persons infected with Chlamydia].

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio to detect the inherited deficiency of the isotypes. The frequency of deficiency in healthy persons blood donors was equal for C4A and C4B (0.14 for each isotype), i.e. 14% of total number (22) donors, or 28% totally. These results agree with the literary data, on which the frequency of deficiency of C4A is 0.14, and of C4B is 0.11-0.16. The inherent deficiencies of C4A and C4B for persons infected by Chlamydia were studied. For this purpose the patients (35 persons) with in blood antibodies (IgG or IgM) to Chlamydia (C. trachomatis, C. psittaci and C. pneumoniae) were investigated. The frequencies of deficiency of C4A and C4B were 0.29 and 0.46 respectively. Thus, the number of the undeficiency patients was only 25%, while among healthy persons 70-75% of individuals not having deficiencies of isotypes C4 were observed. The deficiencies of isotypes of C4 at this pathology is detected for the first time. The obtained data suggest the existence of the predisposition to the development of diseases stipulated by Chlamydia in persons with inherent deficiency of C4 component of complement.

Adult↗
PubMed1981

High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein.

Protein S, a recently described vitamin K-dependent plasma protein, is shown to exist in two forms in plasma--free protein and in complex with C4b-binding protein. C4b-binding protein is involved in the regulation of the rate of complement activation. A major proportion of C4b-binding protein in plasma is in complex with protein S. The complex is a major and previously unrecognized component of the group of plasma proteins that adsorbs to barium citrate. The complex dissociates in the presence of NaDodSO4, indicating that C4b-binding protein and protein S are held together by noncovalent bonds. Uncomplexed C4b-binding protein was purified from the supernatant after barium citrate adsorption. On NaDodSO4/polyacrylamide gels without reduction, it appeared to have a slightly faster migration rate than the C4b-binding protein dissociated from the complex with protein S. After reduction, the subunits of the two forms of C4b-binding protein appeared to have identical molecular weights. Furthermore, there is an equilibrium between free and bound protein S in plasma. The role of protein S in the complex is unknown.

Barium↗
PubMed1985

The application of DNA-cellulose chromatography in the isolation of immunoglobulin M and complement component C4b-binding protein from human serum.

By utilising the ability of some anionic serum proteins to bind to DNA-cellulose, at physiological pH and ionic strength, two such proteins have been purified and identified as immunoglobulin M (IgM) and complement component C4b-binding protein (C4BP). The method entails ammonium sulphate precipitation followed by QAE-Sephadex, DNA-cellulose and Sephacryl S-300 chromatographic steps. Fractionation of the anionic proteins eluting from QAE-Sephadex over a selected range of ionic strength has afforded IgM which was characterised electrophoretically and immunochemically. A mixture containing IgM complexed with C4BP has also been obtained by utilizing fractions eluted from QAE-Sephadex at higher ionic strength. C4BP can be isolated from this mixture by using immunoaffinity chromatography to remove IgM.

Carrier Proteins↗
PubMed1986

Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2.

Amino Acid Sequence↗
PubMed1999

Expression of the beta-chain of the complement regulator C4b-binding protein in human ovary.

Human C4b-binding protein (C4BP) is an important regulator of the complement system that also binds and inactivates the anticoagulant vitamin K-dependent protein S. These two activities are performed by two distinct polypeptides of 70 kDa and 45 kDa known as alpha and beta chains, respectively. C4BP is present in plasma in various isoforms with different alpha/beta composition. We report here that C4BPbeta, but not C4BPalpha, is expressed in adult human ovary. Expression of C4BPbeta was detected in all ovarian biopsies analyzed (n = 15), independently of age and phase of the menstrual cycle. In situ hybridization and immunostaining analyses on cryostat sections demonstrated expression of C4BPbeta in both regressing corpus luteum and corpus albicans, but not in the follicles, the corpus luteum, the ovary stroma or the vascular cells. In addition, we noted that the expression pattern of the C4BPbeta mRNA resembles that described for the connective tissue that invades the degenerating corpus luteum and causes a progressive fibrosis that gradually converts it into a scar, the corpus albicans. RT-PCR and immunostaining analyses of primary cultures derived from human ovaries demonstrated the presence of fibroblast-like cells that express C4BPbeta. As a whole, these data suggest a role for the C4BPbeta in human ovary during the healing and scar resorption processes that leads to the formation of the corpus albicans and its replacement by ovarian stroma.

Adult↗
PubMed1986

Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity.

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class-specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class-specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg-positive C4A 3a and Ch-positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101-1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion-like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.

Amino Acid Sequence↗
PubMed2000

Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein.

C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven alpha-chains and one unique beta-chain, all constructed of repeating complement control protein (CCP) modules. The beta-chain, made up of three CCPs, binds tightly to vitamin K-dependent protein S, a cofactor to anticoagulant activated protein C. When bound to C4BP, protein S loses its activated protein C cofactor function. In this study, we have mutated potentially important amino acids located at the surface of CCP1 of the beta-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta-chain and were L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants were expressed in a prokaryotic system, purified using an N-terminal His-tag, refolded using an oxido-shuffling system, and tested in several assays for their ability to bind protein S. Our data define Ile(16), Val(18), Val(31), and Ile(33) as crucial for protein S binding, with secondary effects from Leu(38) and Val(39). In addition, Lys(41) and Lys(42) contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.

Base Sequence↗
PubMed1986

Detection of both isotypes of complement C4, C4A and C4B, in normal human glomeruli.

Monoclonal antibodies reactive against the complement C4A and C4B isotypic components were used in an immunoperoxidase technique for the histological study of normal human renal tissue. Prominent staining with both antibodies was seen in the mesangial areas of all normal kidney sections investigated. Occasional staining of arteriolar walls of the same tissues, however, was also observed. In contrast, no mesangial staining was seen using monoclonal antibodies reactive against other 'early' complement components, such as C1q and C3. Specificity of the glomerular staining with the anti-C4 reagents was demonstrated in two patients possessing only the C4A serum component but lacking genetically the C4B locus products. As would be predicted, glomerular staining with the anti-C4A reagent, but not anti-C4B, was clearly demonstrable. It is concluded that both isotypes of complement C4 are present in normal human glomeruli and thus might be operative for normal mesangial function.

Antibodies, Monoclonal↗
PubMed2001

Bordetella pertussis binds to human C4b-binding protein (C4BP) at a site similar to that used by the natural ligand C4b.

Human complement regulators are important targets for pathogenic microorganisms. In one such interaction, Bordetella pertussis binds human C4b-binding protein (C4BP), a high-molecular-weight plasma protein that acts as inhibitor of the classical pathway of complement activation. At least two different B. pertussis surface components, one of which is the virulence factor filamentous hemagglutinin (FHA), contribute to the binding. We used a set of C4BP mutants and monoclonal antibodies to characterize the region in C4BP that binds B. pertussis and analyzed the salt sensitivity of the interaction. These studies indicated that positively charged residues at the interface between complement control protein modules 1-2 in the C4BP alpha-chain are important for binding, and that the site in C4BP that binds B. pertussis is very similar, but not identical, to the C4b-binding site. Bacteria-bound C4BP retained its complement regulatory function and B. pertussis selectively bound C4BP in human plasma, indicating that binding occurs also in vivo. Together, these findings indicate that B. pertussis exploits a site in C4BP, resembling that used by the natural ligand C4b.

Antibodies, Monoclonal↗