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PubMed1978

Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator.

We recently described the isolation from human serum of a high molecular weight protein with specific binding affinity for fluid-phase activated C4. We show here that the C4-binding protein (C4-Bp) functions as an essential cofactor in the proteolysis of C4b in the presence of C3b-inactivator (C3bINA). C4-bp, together with C3bINA, cleave the alpha'-chain of C4b into three fragments called alpha2, alpha3, and alpha4, with mol wt of 47,000, 25,000, and 17,000 daltons, respectively. The alpha2 fragment was dissociated from C4b without reduction, whereas the alpha3 and alpha4 fragments were disulfide bonded the other chains of C4b. The reaction did not occur when either C4-bp or C3bINA were omitted, nor in the presence of either protein in combination with beta1H. Native C4 was not affected by C3bINA aand C4-bp. C4b was not cleaved when incubated in serum of a patient with genetic deficiency of C3bINA. However, when purified C3bINA was added, the alpha'-chain of C4b was cleaved and fragments with the same molecular weight as alpha2, alpha3, and alpha4 were generated.

Carrier Proteins↗

PubMed1987

Allo-anti-Chido in a Ch-positive patient.

Allo-anti-Chido (Ch) was detected in a patient whose red cells typed as Ch+. The C4 allotype of the patient was A4,B2 which associates strongly with the Ch phenotype Ch:1,-2,3,4,-5,6. Anti-Ch2 + Ch5 were the Ch specificities identified. Absence of only Ch2 and Ch5 determinants on the C4B protein allowed this unique immune response to blood transfusion.

Aged↗

PubMed1996

Complement C4 phenotypes in patients with end-stage renal disease.

The phenotypes of complement C4 were determined by agarose gel electrophoresis in 130 patients with end-stage renal failure of various causes and compared with those of 140 healthy controls. C4 allotype frequencies did not differ between patients and controls. Null alleles of both isotypes C4A and C4B were increased, but also without reaching significance. In type 1 diabetics an increased frequency of C4AQ0 (25 vs. 11.8%, p < 0.05) was found. Patients with two null alleles were far more frequent in the group with insulin-dependent diabetes mellitus (25 vs. 3.6%, p < 0.01). We confirmed the presence of a previously described uremic variant of C4B1. Additional uremic variants of C4 were detected in uremic patients homozygous for C4A3, B2 and B3. The relative electrophoretic migration values of the uremic variants of C4A3, B1, B2 and B3 were 132.1 +/- 2.9, 35.8 +/- 1.5, 70.4 and 73.9. These variants appear early in the course of chronic renal failure and disappear after successful renal transplantation. Uremic variants are the only acquired C4 phenotypes known so far. How uremia causes these variants remains unclear, but probably involves carbamylation of the C4 molecule.

Alleles↗

PubMed1998

A monomeric human C4b-binding protein (C4bp) more efficiently inactivates C3b than natural C4bp: participation of C-terminal domains in factor I-cofactor activity.

We designed a cDNA construct encoding an artificial membrane molecule consisting of all 8 short consensus repeats (SCRs) of human monomeric C4b-binding protein (C4bp) followed by DAF's GPI anchor, named mC4bp, and expressed the protein on swine endothelial cells (SEC). At the same level of expression, mC4bp protected host cells as effectively as DAF, the most potent complement (C) regulator on the membrane. This result was unexpected from the reported functional properties of natural multimeric C4bp. Here, we investigated the mechanism whereby mC4bp has potent cell-protective activity. Our results were as follows: (1) mC4bp serves more efficiently as a methylamine-treated C3 (C3ma)-inactivating factor I-cofactor than natural C4bp and as efficiently as MCP as a methylamine-treated (C4ma)-inactivating cofactor by fluid-phase cofactor assay: (2) the potency of C3ma inactivation by mC4bp and factor I is quite high compared to those of other cofactors: (3)blocking studies using mAbs against C4bp suggested that both the 48 kDa N-terminal fragment and the C-terminal domain near the portion responsible for bundle formation participate in the high C3ma-inactivating capacity of mC4bp. Thus, acquiring high C3ma-inactivating capacity secondary to monomeric alteration leads to high C regulatory activity of mC4bp. These results infer that mC4bp differs from C4bp in its potent factor I-cofactor activity and is a good candidate as a safeguard against hyperacute rejection of xenografts.

Complement C3b Inactivator Proteins↗

PubMed1994

Differential susceptibility of immune complexes to release from the erythrocyte CR1 receptor by factor I.

This study was designed to explore the role of Factor I in the release of immune complexes (IC) from human erythrocytes (E). The interactions between E and IC constructed with murine monoclonal antibodies were examined using, as a complement source, autologous plasma, plasma depleted of Factor I by > 90%, or Factor I-depleted plasma reconstituted with purified Factor I. Striking differences were observed in the interactions between E and different types of IC in Factor I-depleted plasma. The release of IC constructed with IgG1, IgG3, IgM or IgA antibodies was abolished by Factor I depletion whereas IC containing IgG2a or IgG2b antibodies were still released from E in Factor I-depleted plasma. Moreover, when IC containing IgG2a antibodies were incubated briefly in Factor I-depleted plasma, under conditions in which the IC were bound but not released, and then resuspended in the presence or absence of Factor I, as little as 5% of the normal physiologic level of Factor I released the IC from E. Thus, IC containing IgG2a antibodies appear to be exquisitely susceptible to release from E by Factor I. Additional differences in the susceptibility of IC containing IgG1, IgG3, IgM or IgA antibodies to release from E were revealed when Factor I-depleted plasma was reconstituted with Factor I. Under these conditions, the relative susceptibility of IC to release was: IC containing IgG1 or IgA antibodies > IC containing IgM antibodies > IC constructed with IgG3 antibodies. While isotype was critical in determining susceptibility to release, some clonotypic differences between isotype-matched pairs of IC were also evident. Differences in IC release from E by Factor I may reflect antibody matrix-mediated differential susceptibility of IC-bound C3b and/or C4b to cleavage by Factor I and may have implications for immunoregulation, host effector cell mechanisms and the pathophysiology of IC diseases.

Antigen-Antibody Complex↗

PubMed1988

Structure-function relationships in the inhibitory effect of heparin on complement activation: independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Blood Coagulation↗

PubMed1994

Expression of a hybrid complement regulatory protein, membrane cofactor protein decay accelerating factor on Chinese hamster ovary. Comparison of its regulatory effect with those of decay accelerating factor and membrane cofactor protein.

C activation on the cell surface is supposedly regulated by membrane cofactor protein (MCP) and decay accelerating factor (DAF). These are complementary in function: MCP acts as a cofactor in factor I-mediated C3b and C4b inactivation, thus preventing the assembly of C3 convertases, whereas DAF accelerates spontaneous decay of the assembled C3 convertase. In this report, a hybrid MCP-DAF was expressed on Chinese hamster ovary cells by transfecting cDNA, and its regulatory activity was compared with those of MCP and DAF transfectants and with a transfectant expressing both MCP and DAF (MCP + DAF). The C3 deposition on sensitized CHO cells through activation of the classical pathway was blocked to a different degree with these transfectants, the order being MCP + DAF > DAF > hybrid MCP-DAF > MCP. Likewise, the C3 deposition via the alternative pathway was blocked efficiently in the order hybrid > MCP + DAF > MCP. The C-mediated cytolysis of CHO cells virtually reflected the degree of C3 fragment deposition. The MCP-DAF transfectant acquired additive protective activity against alternative pathway-mediated C3 deposition and cytolysis but was less potent in circumventing classical pathway attack than cells that expressed DAF alone or DAF + MCP. Hybrid MCP-DAF may be useful for alleviating C-mediated cell damage, especially via the alternative pathway.

Animals↗

PubMed1989

Complotypes and extended haplotypes in laboratory medicine.

The region on the short arm of the sixth human chromosome encoding class I and class II histocompatibility antigens involved in immune recognition also encodes a group of molecules unrelated to HLA termed class III which includes C2, C4, and factor B. The four genes encoding the complement proteins occupy about 120 kb of genomic DNA between HLA-B and HLA-DR and are closer to HLA-DR. The four genes are inherited as a single unit, without observed crossover, called a complotype, designated by its BF, C2, C4A, and C4B alleles. There are about fifteen complotypes with frequencies of 0.01 or higher on normal caucasian chromosomes. Analysis of linkage disequilibrium between HLA-B, HLA-DR alleles, and complotypes reveals that about 30% of normal caucasian chromosomes consist of fixed sets called extended or fixed haplotypes. There are over a dozen such extended haplotypes defined by their HLA-B, DR and complotype alleles. They appear to contribute most of the previously described linkage disequilibrium between HLA-A/HLA-B and HLA-B/HLA-DR allelic pairs as well as most of the known HLA marker-disease associations. It is postulated that extended haplotypes consist of fixed DNA over at least the 10(6) base pairs of the HLA-B-DR interval, and independent examples in apparently unrelated individuals are thus identical or nearly identical over this interval. A practical consequence of this concept is the possible prediction of successful tissue transplantation donor-recipient pairs.

Chromosome Mapping↗

PubMed2001

Structure/function of C5 convertases of complement.

C5 convertases are serine proteases that cleave both C3 and C5. Alternative pathway C3/C5 convertases formed with monomeric C3b (C3b,Bb) because of their weak interaction with C5 primarily cleave C3 thereby opsonizing the cell surface with C3b. In contrast, C3/C5 convertases formed with a high density of C3b/cell exhibit higher affinities for C5 as indicated by Km values well below the physiological concentration of C5 in blood. These C3/C5 convertases bind C5 efficiently and cleave it at a velocity approaching Vmax thereby switching the enzyme from C3 cleavage to production of the cytolytic C5b-9 complex. Studies of the structure of C3/C5 convertases have postulated that C4b-C3b and C3b-C3b dimers from high affinity C5 binding sites while indel studies have shown two binding sites in C5 for the convertase in addition to the C5 cleavage site. Together, these studies indicate that with increasing deposition of C3b on the surface, C3b complexes are formed which through multivalent attachment bind the substrate C5 with higher affinities, thereby converting the low affinity C3/C5 convertases to high affinity C5 convertases. The process underlying the formation of high affinity C5 convertases during complement activation is discussed.

Animals↗

PubMed1990

Participation of C3 and its ligands in complement activation.

C3, the most abundant complement protein in blood, plays a central role in the activation sequence of the complement system as well as in host defense. Expression of the multiple functions of C3 requires its cleavage by highly specific enzymes termed C3 convertases. C3 in a conformationally altered form, C3H2O, resulting from the slow spontaneous hydrolysis of the internal thioester bond of native C3, initiates the assembly of a C3 convertase which continuously cleaves C3 in the blood at slow rates generating a constant supply of small amounts of C3b. When an activator of the alternative complement pathway is present, C3b becomes covalently attached to its surface via an ester or amide bond. Activator surface-bound C3b initiates the assembly of an "amplification" C3 convertase, C3bBb(P), which can efficiently activate C3 and generate additional convertase complexes on the surface of the activator. C3b generated by an amplification or classical pathway C3 convertase can also bind covalently to the noncatalytic subunit, C3b or C4b, respectively, resulting in the generation of a C5 convertase, an enzyme catalyzing the cleavage/activation of C5. In terms of participation in host defense, several fragments of C3, including C3a, C3b, iC3b, and C3dg, mediate a number of important functions such as increased vascular permeability, enhancement of phagocytosis, elimination of immune complexes, and perhaps also proliferative responses and/or differentiation of B cells.

Complement Activation↗

PubMed1992

Molecular heterogeneity of the fourth component of complement (C4) and its genes in vitiligo.

In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*Q0 allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo.

Adrenal Hyperplasia, Congenital↗

PubMed1981

Isolation and properties of a complement inhibitor from Naja haje venom, distinct from known anticomplementary factors in cobra venom.

A complement inhibitor (CI) has been isolated from cobra (Naja haje) venom which is distinct from the two known anticomplementary factors in cobra venom [1], in functional properties as well as structure. CI is a small (mol. wt 26,000, determined by sodium dodecyl sulphate gel electrophoresis), heat-labile glycoprotein; the amino acid composition is that of a globular protein. CI interferes at various steps of the complement sequence, including reactions of the classical and alternative pathway. No effect was observed on C4 fixation and on the assembly of the membrane attack complex from C6-9 (minor inhibiting effects, if present, have not been excluded). Initiation of the alternative pathway is inhibited by CI already at the stage of cleavage of factor B. CI binds to C4, C4b, C3 and C3b; since the major inhibitory action of CI is lost after washing of cell intermediates, complex formation and, as a consequence, steric hindrance may be responsible for the inhibiting effects of CI. CI also interferes with binding of C3b to C3b receptors on human erythrocytes. CI is non-toxic in mice when given intraperitoneally in doses of 5 microgram/g.

Amino Acids↗

PubMed1984

Structure and organization of the C4 genes.

This 200 000 Mr serum protein is coded for by at least two separate loci, C4A and C4B, which map in the HLA Class III region on chromosome 6 in man. Both loci are highly polymorphic with more than 30 alleles, including null alleles assigned to the two loci. The complete nucleotide sequence of a full length C4A cDNA clone and a substantial part of a C4b cDNA clone has shown class differences which can be used to synthesize nucleotide probes specific for C4A and C4B. Three C4 loci of approximately 16 kilobases each spaced by 10 kilobases have been identified in DNA from one individual and aligned 30 kilobases from the factor B gene by overlapping cloned genomic fragments from a cosmid library. Characterization of these genes by restriction mapping, nucleotide sequence analysis and hybridization with C4A and C4B specific synthetic oligonucleotides show that these genes are very similar.

Alleles↗

PubMed1993

Identification of a surface structure in the fourth component of human complement, C4, which becomes hidden upon activation by C1(-)s.

Treatment of complement component C4 with C1(-)s and methylamine induces a series of conformation changes such as to generate functional binding sites. A monoclonal antibody (mAb), Al 121/6, which does not inhibit the haemolytic activity of C4 was found to bind to native C4 and C4d, but not to C4b and methylamine-treated C4, unless these C4 derivatives were denatured. These results suggested that a linear epitope for mAb Al 121/6 in the C4d domain is originally located at the surface of C4 and becomes hidden as a result of conformational changes induced by C1(-)s or methylamine treatment. The hidden linear epitope was exposed again upon further cleavage of C4b into C4c and C4d. Trypsin digestion of C4d and its chemical modification with phthalic anhydride suggested that the epitope is located at the C-terminal 13 kDa region of C4d and that lysine residues are involved in the epitope. There is a single lysine residue at 1259 in the 13 kDa C-terminal side of C4d and the synthetic undecapeptide Leu1254-Asp1264 was found to inhibit the binding of C4 to mAb Al 121/6, suggesting that the epitope for mAb Al 121/6 is involved in the sequence. The N-terminal portion of the peptide is partly overlapping, with a highly hydrophobic amino acid sequence spanning residues Ala1249-Leu-Leu-His-Leu-Leu-Leu1255. The surface hydrophobicity of C4 has been reported to decrease upon treatment with C1(-)s and methylamine. So it appears that the hydrophobic sequence spanning Ala1249-Leu1255 may be hidden, together with the linear epitope, into the inner region of C4 upon treatment with C1s and methylamine.

Antibodies, Monoclonal↗

PubMed1990

Structural and functional correlation of the human complement receptor type 1.

Human CR1 is widely distributed in the circulation as a surface receptor as well as in soluble form in the plasma. It mediates a variety of functions that include phagocytosis and regulation of the complement cascade. This receptor has been cloned and the primary structure reveals that the cell-bound molecule is an integral membrane protein with typical transmembrane and cytoplasmic domains. Its extracellular portion is composed entirely of 30 short consensus repeats (SCR) each having 60 to 70 amino acids. This type of motif is the common structural element of the superfamily of complement regulatory and receptor proteins on chromosome 1. The amino-terminal 28 SCR of CR1 is uniquely organized into four tandem long homologous repeats (LHR) with sequence homologies among corresponding SCR as high as 99%. Each LHR encodes approximately 45 kD and each except the one that is proximal to the cell surface contains a separate binding site for C3b or C4b. Analysis of the genomic structure of CR1 reveals that these LHR are results of intragenic duplication of 20- to 30-kb segments of DNA. The structural allotypes of CR1 that vary in the lengths of the polypeptides are encoded by alleles that contain different numbers of LHR. Their predicted structures would have different numbers of C3b binding sites, perhaps resulting in molecules with different capacities to bind immune complexes.

Alleles↗

PubMed1996

The reaction mechanism of the internal thioester in the human complement component C4.

A key step in the elimination of pathogens from the body is the covalent binding of complement proteins C3 and C4 to their surfaces. Proteolytic activation of these proteins results in a conformational change, and an internal thioester is exposed which reacts with amino or hydroxyl groups on the target surface to form amide or ester bonds, or is hydrolysed. We report here that the binding of the human C4A isotype involves a direct reaction between amino-nucleophiles and the thioester. A two-step mechanism is used by the C4B isotype. The histidine at position 1,106(aspartic acid in C4A) first attacks the thioester to form an acyl-imidazole intermediate. The released thiol then acts as a base to catalyse the transfer of the acyl group to amino- and hydroxyl-nucleophiles, including water.

Amino Acid Sequence↗

PubMed1974

Fourth component of human complement: description of a three polypeptide chain structure.

The fourth component of human complement (C4) was shown to be composed of three distinct polypeptide chains linked by disulfide bonds and noncovalent forces. The sum of the molecular weights of the chains equalled that of the intact molecule. The mol wt of the alpha-, beta-, and gamma-chains were respectively, 93,000, 78,000, and 33,000 daltons. Action of C1s on C4 affected only the alpha-chain, reducing its mol wt to 87,000 daltons. The size of the activation peptide. C4a, is therefore estimated to be 6,000 and that of the major fragment C4b, 198,000 daltons. Periodic acid-Schiff-stained SDS polyacrylamide gels of reduced C4 revealed carbohydrate to be associated with all three chains. A modification of the original method of isolation of C4 is presented.

Chemical Phenomena↗

PubMed1983

Gene order and gene distances in the HLA region studied by the haplotype method.

The present report describes a method to establish gene order and gene distances in chromosomal regions where several genes are located closely together. The method is applied to the study of the complement loci in the HLA complex on chromosome 6 in man. The method is based on allelic association, i.e. alleles of closely linked loci are nonrandomly associated on haplotypes. A haplotype which yields information has a frequency higher than would be expected from the frequencies of each of its alleles. They occur, moreover, with a frequency which makes them the main source of the least frequent of the alleles in the combinations. Other haplotype combinations involving this allele (these alleles) are most likely the results of recombinational events involving a main haplotype. Such crossovers may therefore, on certain conditions, be used for gene mapping purposes. Some basic rules for the use of the method are given. A total of 701 haplotypes involving the short arm of chromosome 6 have been studied. Typings have been performed with regard to HLA-A, -B, -C, -D/-DR, C4, C2 and Bf. The study confirms previous localization of the complement loci between HLA-D and -B. The investigation suggests the order HLA-D-Bf-C4-C2-HLA-B. There is, moreover, slight evidence in favour of a localization of the C4A gene on the HLA-B side of C4B. Given an HLA-A-HLA-B distance of 0.8 cM, suggested relative distances are: HLA-D-Bf:0.44 cM, Bf-C4:0.04 cM, C4-C2:0.11 cM, and C2-HLA-B:0.12 cM.

Alleles↗