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PubMed2002

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis.

Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.

Antibodies, Monoclonal↗
PubMed2003

Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b and C4b to restrict activation of the complement cascade. Here, we report production of functional, recombinant T. cruzi CRP in mammalian cells and a one-step purification of the recombinant protein. Exchange of the crp DNA sequence encoding the carboxy-terminal GPI signal sequence with the corresponding sequence of decay accelerating factor (DAF) was necessary for recognition, cleavage, and addition of GPI in mammalian cells. CRP production was assessed in two mammalian cell lines with crp-daf gene expression driven by three different transcription control regions: Rous sarcoma virus long terminal repeat, cytomegalovirus (CMV) immediate early gene, and chicken beta-actin promoter/CMV enhancer. We present evidence that CRP produced in transfected Chinese hamster Ovary (CHO) cells was functional and protected the cells from complement-mediated lysis. To facilitate purification of the recombinant protein, a hexahistidyl tag was incorporated at 3(') end of the cDNA upstream of the GPI anchor addition sequence. An additional histidine fusion construct was made that allowed for secretion and recovery of recombinant protein from culture supernatant fluid. Both membrane and secreted forms of the protein were purified in one step by nickel nitrilotriacetic acid. The production and purification of functionally active CRP in a non-infectious expression system will allow for structure and function studies aimed at identifying the active site(s) of this protein.

Animals↗
PubMedDate not supplied

Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins.

Decay-accelerating factor (DAF), a glycoprotein that is anchored to the cell membrane by phosphatidylinositol, binds activated complement fragments C3b and C4b, thereby inhibiting amplification of the complement cascade on host cell membranes. Here, we report the molecular cloning of human DAF from HeLa cells. Analysis of DAF complementary DNAs revealed two classes of DAF messenger RNA, one apparently derived from the other by a splicing event that causes a coding frameshift near the C terminus. The apparent 'intron' sequence contains an Alu family member and encodes contiguous protein sequence. Two DAF proteins are therefore possible, having divergent C-terminal domains which differ in their hydrophobicity. Both mRNAs are found on polysomes, suggesting that both are translated. We propose that the major (90%) spliced DAF mRNA encodes membrane-bound DAF whereas the minor (10%) unspliced DAF mRNA may encode secreted DAF and we present expression data supporting this. The deduced DAF sequence contains four repeating units homologous to a consensus repeat found in a recently described family of complement proteins.

CD55 Antigens↗
PubMed1999

Enhancement of lectin pathway haemolysis by immunoglobulins.

We recently reported that indicator sheep erythrocytes (E) coated with mannan and sensitized with mannan-binding lectin (MBL) (E-M-MBL) are lysed by human serum in the absence of calcium via the lectin pathway of complement activation by a process which requires alternative pathway amplification and is associated with increased binding of and control by complement regulatory proteins C4 bp and factor H. In the present study, we investigated the effect of immunoglobulin (Ig) on this haemolysis. Co-sensitization of indicator E with anti-E haemolysin led to threefold enhancement of lectin pathway haemolysis in the absence of calcium, associated with increased binding of C3 and C5. Lysis was enhanced approximately twofold when E-M-MBL were chemically or immunologically coated with IgM or IgA, and fourfold when coated with IgG, prior to lysis in human serum-Mg-ethyleneglycol tetraacetic acid. The presence of haemolysin did not reduce the binding or inhibitory activity of C4 bp, and the enhancing activity of haemolysin was retained in serum depleted of C4 bp. By contrast, binding of factor H was greatly reduced in the presence of haemolysin, which had no enhancing effect in serum depleted of factor H. These experiments demonstrate the ability of IgG, IgM and IgA to enhance lectin pathway cytolysis, and that this enhancement occurs by neutralization of the inhibitory activity of factor H. Immunoglobulin enhancement of lectin pathway cytolysis represents another interaction between the innate and adaptive systems of immunity.

Animals↗
PubMed1998

Stable transfection of Trypanosoma cruzi epimastigotes with the trypomastigote-specific complement regulatory protein cDNA confers complement resistance.

Trypanosoma cruzi blood stage trypomastigotes are highly resistant to complement-mediated killing in normal serum. A previously described trypomastigote surface glycoprotein was shown to have binding affinity for human complement components C3b and C4b and restrict activation of the complement cascade, thus preventing lysis of the parasites. Insect stage epimastigotes do not produce detectable levels of this 160-kDa complement regulatory protein (CRP) and are highly sensitive to the lytic effects of complement. Epimastigotes were stably transfected with a T. cruzi expression vector carrying the trypomastigote CRP cDNA and produced fully functional recombinant CRP. The recombinant CRP had binding affinity for C3b, and the transfected epimastigotes were protected from complement-mediated lysis. These results demonstrate for the first time that a developmentally regulated gene of T. cruzi trypomastigotes can be expressed in noninfectious epimastigotes and that production of CRP by epimastigotes is sufficient to confer a virulence-associated trait. Furthermore, these studies demonstrate the critical role that trypomastigote CRP plays in the protection of parasites from the deleterious effects of complement, thus establishing the protein as a virulence factor of T. cruzi.

Animals↗
PubMed2000

[Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes].

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.

Animals↗
PubMed1992

C4B deficiency is not associated with meningitis or bacteremia with encapsulated bacteria.

The two isotypes of the fourth complement component are C4A and C4B. C4B forms ester bonds more efficiently than C4A and so, in theory, is more likely than C4A to bind to polysaccharide capsules of encapsulated bacteria. Two studies have reported homozygous C4B deficiency in patients with meningitis or bacteremia caused by encapsulated organisms. In the present study the association between C4B deficiency and these disorders was evaluated in four groups: patients with bacteremia, those with meningitis, those who developed Haemophilus influenzae type b (Hib) disease after Hib polysaccharide vaccination, and patients less than 1 year old with meningitis. Healthy adults served as controls. Of the 257 patients, 2.3% had homozygous C4B deficiency compared with 3.7% of 349 controls. According to these data, there is no increase in homozygous C4B deficiency among patients with bacteremia or meningitis caused by encapsulated bacteria.

Bacteremia↗
PubMed1996

Prognostic significance of complement alleles Bf and C4 in early rheumatoid arthritis.

The prognostic significance of class III major histocompatibility complex complement components, factor B (Bf), C4A and C4B, were studied in a 3-year prospective study of 73 patients with early RA. Patients with C4B null allele had higher disease activity with more radiological progression than patients with C4A null allele or patients without null allele. C4B null allele also associated with increased susceptibility to side effects from antirheumatic treatment. The Bf phenotypes did not associate with the severity of RA. C4B null allele may have prognostic significance in determining a special subgroup of RA patients with a more complicated course of the disease.

Adult↗
PubMed1987

Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rodgers/Chido determinants: application to the study of C4 null alleles.

Definitive restriction fragment length polymorphisms (RFLPs) representing the exact locations responsible for isotypicity between the human complement components C4A and C4B, and their generally associated major Rodgers (Rg1) and Chido (Ch1) antigenic determinants, have been designed. By means of C4d-specific genomic probe for Southern blot analysis, a C4A gene can be defined by the presence of the 276 bp and 191 bp N1a IV fragments, while a C4B gene can be defined by a single 467 bp N1aIV fragment. In addition, an Rg1-expressing C4 gene can be represented by a 565 bp EcoO 109 fragment, and a Ch1-expressing C4 gene by a 458 bp EcoO 109 fragment, under the same conditions. All these polymorphic restriction fragments can be unambiguously and conveniently detected. In combination with the Taq I polymorphic patterns specific for the C4 loci and for the neighboring 21-hydroxylase genes, the nature and structure of the tandem C4,21-hydroxylase gene complex can be elucidated. In this study, it is inferred that the null allele of the HLA haplotype B44 DR6 C4A3 C4BQO is not a C4B allele, but probably encodes another C4A 3 allotype at the second C4 locus.

Alleles↗
PubMed1991

High doses of intravenous Ig inhibit in vitro uptake of C4 fragments onto sensitized erythrocytes.

We have recently reported that intravenous Ig (IVIg) inhibits uptake of activated C3 fragments onto antibody-sensitized red blood cells (RBCs). To elucidate the mechanism by which IVIg exerts its effect on the complement system, we examined the possible interference with the C4 step of the classical complement cascade. We examined the capacity of autologous serum containing high concentrations of human IVIg to deposit C4 fragments onto model targets (guinea pig and/or human erythrocytes sensitized with rabbit anti-guinea pig/human erythrocytes IgG antibody). C4 binding was quantified with radiolabeled anti-C4. Guinea pig serum with added IVIg suppressed C4 uptake onto IgG-sensitized guinea pig erythrocytes at all time points (0, 5, 15, and 30 minutes). Using sera of guinea pigs treated with increasing doses of IVIg, this effect was shown to be dose-responsive. Serum from a patient treated with IVIg showed reduced C4 uptake onto sensitized homologous RBCs. In comparison with the serum from the same patient before IVIg therapy was administered, levels were decreased almost to background. C4 functional titers in those two samples were not different. C3 uptake was studied in parallel with C4 to compare the degree of inhibition using sera with increasing doses of IVIg in both the human and guinea pig system. C3 and C4 inhibition curves completely overlapped. Our findings suggest that IVIg is an effective inhibitor of deposition of early complement activation products (C4b, C3b) onto target surfaces and may indicate interference of IVIg with multiple sites of complement activation.

Animals↗
PubMed1998

The complement cofactor protein (SBP1) from the barred sand bass (Paralabrax nebulifer) mediates overlapping regulatory activities of both human C4b binding protein and factor H.

We have previously shown that serum of the teleost fish barred sand bass (Paralabrax nebulifer) cleaves the alpha'-chain of human C4b and C3b. The proteins that participate in these reactions were purified, and a specific protease and a single cofactor protein were identified. Functional characterization of the recombinantly expressed sand bass cofactor protein (SBP1) and truncated forms containing short consensus repeats (SCRs) 1-2, 1-3, 1-4, 1-5, and 12-17 revealed that SBP1 and SCRs 1-4 mediate the functional activities of the human plasma regulatory protein C4bp and factor H. They form a complex with C4b, inhibit the formation, and accelerate the decay of the classical pathway C3 convertase and display cofactor activity for the cleavage of C4b. In contrast, the interaction of SBP1 and SCRs 1-4 with human C3b in all these activities was limited. This difference is due to species-specific incompatibilities between the cofactor protein and human C3b. SBP1 and SCRs 1-5 displayed full binding and cofactor activity for methylamine-treated C3 from trout, a species closely related to the sand bass. The presence of only one cofactor in the fish plasma that combines the functional activities of C4bp and factor H demonstrates that the sand bass cofactor protein is the ancestral precursor to the two complement regulatory proteins in human plasma.

Animals↗
PubMed1991

Evidence that C4b-binding protein (proline-rich protein) is synthesized by hepatocytes.

C4b-binding protein (C4bp), a glycoprotein involved in regulating the classical pathway of the complement system, binds the activated form of C4b and accelerates the decay rate of the C4b, C2a complex. Recently, sequence analysis of the cDNA for proline-rich protein (PRP) demonstrated that PRP is identical with C4bp. We measured the concentration of C4bp in serum by single radial immunodiffusion in patients with various liver diseases. Concentration of C4bp was significantly lower in hepatic cirrhosis (P = 0.001) and higher in fatty liver (P = 0.0002) than the control values, after adjusting for age, sex, and concentration of total cholesterol, triglyceride, and C-reactive protein. Significant positive correlations were observed between the concentration of C4bp in serum and total protein, albumin, cholinesterase level, and lecithin-cholesterol acyltransferase activity. Immunohistochemical analysis of human liver with specific antiserum to human C4bp demonstrated reaction endproducts in the hepatocytes around the central veins. These observations provide evidence that C4bp is synthesized by hepatocytes.

Animals↗
PubMed1990

C4b-binding protein and a 60,000-Dalton plasma protein share antigenic determinants with membrane cofactor protein of complement.

Membrane cofactor protein (MCP) of the C system is a widely distributed regulatory molecule with C3b/C4b binding and factor I-dependent cofactor activity. A rabbit polyclonal antibody was raised against purified human MCP, and it was found to also immunoprecipitate C4b-binding protein (C4bp). Other related complement regulatory proteins, factor H, C3b/C4b receptor, and decay-accelerating factor, were not recognized by this polyclonal antibody to MCP. The cross-reactive epitope was sensitive to reduction with 2-ME and about 3% of the anti-MCP antibody reacted with C4bp. The amino-terminal 48,000-Da, chymotryptic fragment of C4bp was recognized by the antibody to MCP. This fragment of C4bp contains a seven-amino acid peptide that is identical, in its sequence and its location in the third short consensus repeat, to one found in MCP. Two polyclonal antibodies to C4bp, one raised to native and the other to reduced C4bp, did not cross-react with MCP. In addition to this one-way cross-reaction with C4bp, a protein with a m.w. of approximately 60,000 (p60) was found in two of three C4bp preparations that also cross-reacted with antiserum to MCP. p60 was present in trace quantities in the C4bp preparation and was successfully isolated from plasma by C3b affinity chromatography. Its Mr was distinct from that of MCP and other known C3b/C4b binding proteins. Furthermore, p60 was isolated by two different procedures and such material possessed no detectable cofactor activity. Based on these results, p60 is a plasma C3b-binding protein that shares epitopes with C4bp and MCP, and is probably not a soluble form of MCP.

Antigens, CD↗
PubMed1988

Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

Antigen-Presenting Cells↗
PubMed1992

Functional properties of heterogeneous human asialo-C4 and its isotypes C4A and C4B.

The fourth component of human complement (C4) is encoded at two separate but closely linked loci within the MHC on the short arm of chromosome 6. Thus, there are two types of C4 protein in most individual and pooled normal human sera (NHS): C4A and C4B. Incubation of individual sera, pooled NHS, or purified heterogeneous C4 (C4A/C4B) with bacterial sialidase at 37 degrees C increased C-mediated hemolysis of antibody-sensitized sheep erythrocytes 1.54- to 1.93-fold. Comparative studies of Tmax of human C2, using asialo-C4 or buffer-treated C4 on EAC1gp and extrapolation to time 0 indicated a z value 4-fold higher with asialo-C4. This indicated that more hemolytically active C42 complexes are available with sialidase-treated C4 compared to untreated C4. There was no appreciable difference in the % 125I-C4 bound to EAC1gp (sialidase- or buffer-treated). Sera from two different blood donors with C4A3 phenotype (C4BQ0), two different donors with C4B1 phenotype (C4AQ0), and serum from an individual heterozygous deficient at both C4A3 and C4B1 regions (A3, AQ0; B1, BQ0) were investigated. The C4 allotypes, purified from these sera, were treated with sialidase; the C4A3 was enhanced in hemolytic assays by sialidase-treatment (1.52- to 2.3-fold), whereas the C4B1 allotype was not enhanced. Fluorometric determinations revealed that approximately the same percentage of sialic acid was released from sialidase-treated C4A3 and C4B1. Therefore, the increase in hemolytic titer observed after treatment of NHS or purified heterogeneous C4 with sialidase is a property of C4A3 but not a property of C4B1.

Asialoglycoproteins↗
PubMed1993

Association of C4B deficiency (C4B*Q0) with erythema nodosum in leprosy.

A considerable number of studies have postulated significant associations between susceptibility to the different clinical manifestations of leprosy and the MHC. In this investigation, the association between the MHC class III complement proteins C2, BF, C4A and C4B and leprosy in a patient population of Southern Brazil was studied. A total of 109 non-related leprosy patients was investigated; 73 presented with lepromatous leprosy (LL), 46 of them had the immunopathological reaction of erythema nodosum (ENL), the remaining 36 were tuberculoid, borderline and indeterminate leprosy (TIBL) patients. The control group included 172 healthy individuals matched with the patients according to their ethnic and geographical origin. C2, BF, C4A and C4B allotypes were determined by standard technologies including Western blots for C2 and C4 variant alleles with monoclonal and polyclonal antibodies. Non-expressed ('silent') C4 alleles in hemizygously deficient individuals were estimated semiquantitatively on the basis of the C4A and C4B isotype ratio and by the MASC ('minimal chi-square') method. The results showed a significantly elevated presence of the non-expressed C4B allele (C4B*Q0) in the LL and ENL patient groups in comparison with the controls. The most significant difference was observed in the ENL group when compared with the controls. In addition, all patients who were homozygously C4B-deficient had ENL, and most of them had the BF*F1 allele. The comparison between LL patients with and without ENL also showed a statistically significant difference in the presence of C4B*Q0, indicating that C4B deficiency itself is associated with ENL. The relative risk of LL patients with the C4B*Q0 allele suffering from ENL was 5.3 compared with LL patients without C4B*Q0. Since immune complexes (IC) are considered to be the pathogenic cause of ENL, our findings indicate that C4B deficiency may play an important role in the abnormal immune response against Mycobacterium leprae and in the lack of IC clearance, leading to ENL reactions. Individuals with this allele seem to be at a higher risk of developing pathological immune reactivity in lepromatous leprosy.

Adult↗
PubMed1986

Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein.

Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.

Animals↗