Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

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 on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella 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 with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the 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.     

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.     

The isolation and structure of C4, the fourth component of human complement.

The fourth component of complement, C4, was isolated from human serum in good yield, and in confirmation of previous reports was shown to be formed from three peptide chains, alpha, beta and gamma, with apparent mol.wts. 90 000, 80 000 and 30 000 respectively. Preparative methods are described for the isolation of the three peptide chains and their amino acid analyses reported. Component C4 contains 7.0% carbohydrate, alpha-chain 8.6% and the beta-chain 5.6%. The N-terminal amino acid sequences are given for 12 residues of the alpha-chain, eight of the beta-chain and 19 of the gamma-chain.     

A structural model for the location of the Rodgers and the Chido antigenic determinants and their correlation with the human complement component C4A/C4B isotypes

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12 000–14 000, in contrast to initial studies which showed the relative mass to be 33 000. Using polymorphic Ly-6-specific antibodies, the 33 000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2⁺, Ly-6A.2^– strains and a Ly-6A.2^– mutant cell line BW(Thy-1^–e). This clearly demonstrated that 33 000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2⁺ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12000–14000, although a minor 33 000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipilate the 12000–14000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12 000–14 000 molecules were only immunoprecipitated from Ly-6A.2⁺ cells, whereas 33 000 molecules were precipitated from both Ly-6A.2⁺ cells and Ly-6A.2^– cells. These findings suggest that the 33 000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.     

Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation.

The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface-bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site-directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B-like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A-like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group-containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s-1 (t1/2 of 10.3 s) for C4A and 1.08 s-1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

The binding of human complement component C4 to antibody-antigen aggregates.

The binding of human complement component C4 to antibody-antigen aggregates and the nature of the interaction have been investigated. When antibody-antigen aggregates with optimal C1 bound are incubated with C4, the C4 is rapidly cleaved to C4b, but only a small fraction (1-2%) is bound to the aggregates, the rest remaining in the fluid phase as inactive C4b. It has been found that C4b and th antibody form a very stable complex, due probably to the formation of a covalent bond. On reduction of the C4b-immunoglobulin G (IgG) complex, the beta and gamma chains, but not the alpha' chain, of C4b are released together with all the light chain, but only about half of the heavy chain of IgG. The reduced aggregates contain two main higher-molecular-weight complexes, one shown by the use of radioactive components to contain both IgG and C4b and probably therefore the alpha' chain of C4b and the heavy chain of IgG, and the other only C4b and probably an alpha' chain dimer. The aggregates with bound C1 and C4b show maximal C3 convertase activity, in the presence of excess C2, when the alpha'-H chain component is in relatively highest amounts. When C4 is incubated with C1s in the absence of aggregates, up to 15% of a C4b dimer is formed, which on reduction gives an alpha' chain complex, probably a dimer. The apparent covalent interaction between C4b and IgG and between C4b and other C4b molecules cannot be inhibited by iodoacetamide and hence cannot be catalysed by transglutaminase (factor XIII). The reaction is, however, inhibited by cadaverine and putrescine and 14C-labelled putrescine is incorporated into C4, again by a strong, probably covalent, bond. It is suggested that a reactive group, possibly an acyl group, is generated when C4 is activated by C1 and that this reactive group can react with IgG, with another C4 molecule, or with water.     

The primary structure of the fourth component of human complement (C4)-C-terminal peptides

C-terminal CNBr peptides of the three polypeptide chains of C4 were obtained and sequenced. These results supplement previously obtained data, notably the protein sequence derived from cDNA sequencing of pro-C4 (Belt KT, Carroll MC & Porter RR (1984) Cell 36, 907-914) and the N-terminal sequences of the three polypeptides (Gigli I, von Zabern I & Porter RR (1977) Biochem. J. 165, 439-446), to define the complete primary structure of the plasma form of C4. The beta (656 residues), alpha (748 residues), and gamma (291 residues) chains are found in positions 1-656, 661-1408, and 1435-1725 in the pro-C4 molecule.     

Polymorphism of human complement component C4

An assessment has been made of the polymorphism of human complement component C4 by comparing derived amino acid sequences of cDNA and genomic DNA with limited amino acid sequences. In all, one complete and six partial sequences have been obtained from material from three individuals and include two C4A and two C4B alleles. Differences were found between the 4 alleles from 2 loci in only 15 of the 1722 amino acid residues, and 12 lie within one section of 230 residues, which in 1 allele also contains a 3-residue deletion. In three variable positions, an allelic difference in one C4 type was common to the other types. Three nucleotide differences were found in four introns. In spite of marked differences in their chemical reactivity, the many allelic forms appear to differ in less than 1% of their amino acid residue positions. This unusual pattern of polymorphism may be due to recent duplication of the C4 gene, or may have arisen by selection as a result of the biological role of C4, which interacts in the complement sequence with nine other proteins necessitating conservation of much of the surface structure.     

Isolation of the fourth component (C4) of rat complement.

The fourth component of rat complement was purified to homogeneity by sequential chromatography of rat plasma in benzamidine on QAE-A50, SP-C50, hydroxyapatite, and gel filtration on Bio-Gel A 1.5. The final material was homogeneous on SDS-PAGE analysis and had a calculated m.w. of 198,000. A monospecific antibody against rat C4 was obtained from immunized rabbits. The concentration of rat C4 in the plasma of normal 4-month-old Wistar rats was 190 +/- 34 microgram/ml (mean +/- 1 S.D.).     

Restriction fragment analysis of duplication of the fourth component of complement (C4A)

The two genes encoding the fourth component of complement (C4A and C4B) reside between HLA-B and HLA-DR on human chromosome 6. Two kilobases downstream from each C4 gene lies a 21-hydroxylase gene (CA21HA and CA21HB, respectively). Utilizing the method of Southern blotting and a 5'-end 2.4-kb BamHI/KpnI fragment of the C4 cDNA, we have analyzed TaqI-digested DNA from four pedigrees with one or more extended haplotypes containing a C4A duplication, as demonstrated by protein electrophoresis and segregation analysis. Two C4A protein duplications (C4A*2,A*3,C4B*QO and C4A*3,A*5,C4B*QO) segregated with two large TaqI DNA restriction fragments (7.0 and 6.0). In pedigree Fi, one individual homozygous for HLA-A3,B35,C4,DR1,DQ1,BFF,C2C,-C4A2,3,C4BQO had TaqI 7.0- and 6.0-kb restriction fragments with equal hybridization intensities as measured by two-dimensional densitometry (7.0/6.0 kb = 0.83, SD = 0.12, N = 7). A hybridization probe for the 21-hydroxylase gene also demonstrated equal gene dosage (CA21HA/CA21HB = 1.01). DNA from another individual (Ma I-2) with a different C4A gene duplication (C4A*3,A*5,C4B*QO) also had equal densitometry measurements (7.0/6.0 kb = 1.07). We conclude that two extended haplotypes from unrelated pedigrees have two C4 genes and both C4 genes encode separate C4A alleles. These findings are compatible with a gene conversion event of C4B to C4A.     

Molecular genetics of the fourth component of human complement

The fourth component of complement in humans is coded for by two closely linked loci, i.e., C4A and C4B, that have been positioned within the class III region of the human major histocompatibility complex along with the genes for C2, Bf, and steroid 21-OH. Both C4 loci are highly polymorphic and certain alleles, particularly the nulls, are associated with susceptibility to autoimmune disease. About one-half of the null alleles are due to a large deletion that includes both a C4 and flanking 21-OH gene. Despite the near identity of the products of the two loci, the proteins differ dramatically in their efficiency of covalent binding to antigen. The amino acid substitutions responsible for the functional differences have been identified and they are clustered relatively near the covalent binding site within the C4d region of the alpha chain. These observations support the hypothesis that the susceptibility to autoimmune disease is related to the structural variation of the C4 protein.     

The purification and characterization of bovine C4, the fourth component of complement.

The fourth component of complement, C4, was isolated from bovine plasma in high yield, by using simple purification techniques. The protein, like human component C4, is a beta-globulin with a mol.wt. of about 200 000 and consists of three polypeptide chains, alpha, beta and gamma, with apparent mol. wts. of 98 000, 82 000 and 32 000 respectively. The chains of C4 have been separated by methods previously used for human C4. Their amino acid compositions are very similar to those of the human component, but differences in carbohydrate distribution have been observed. The haemolytic activity of bovine C4 is totally destroyed by incubation with bovine C1s, the activated subcomponent of the first component of complement. Component C4, treated in this way, was shown to be cleaved in the alpha chain, which was decreased in mol.wt. by about 9000, corresponding to the removal of subcomponent C4a.     

Chido and Rogers antigenic determinant on the fourth component of human complement

The blood group substances Chido (Cha) and Rogers (Rga) represent two electrophoretic variants of human C4. Based on the observation that anti-Cha and anti-Rga antisera agglutinated human red blood cells prepared in sucrose-activated autologous serum (LIS cells) at 37 degrees C, it has been assumed that the Cha and Rga antigenic determinants reside in the C4d fragment of C4. Here, we present evidence indicating that C4d is not present on those cells. In order to identify structurally the C4 fragments deposited, LIS cells were prepared at 37 degrees C and 4 degrees C in autologous serum to which 125I-C4 was added. Membranes of LIS cells were solubilized and analyzed by SDS-PAGE in 5 to 15% gradient gels followed by autoradiography. C4d was not deposited on LIS cells prepared at 37 degrees C, whereas C4c (beta, gamma, alpha 3 alpha 4) was. Cells prepared at 4 degrees C carried C4d (alpha 2) and C4c. Anti-Cha and anti-Rga antisera agglutinated both cell types, although C4d was not present on the cells prepared at 37 degrees C. Purified C4, C4c, C4d, and alpha-, beta- and gamma-chains of C4, as well as alpha 3 and alpha 4, were used to neutralize these antisera. C4 and the alpha-chain C4d and alpha 4 fragment of C4c, but neither the alpha 3 fragment nor the beta- or gamma-chains, were capable of neutralizing anti-Cha and anti-Rga antisera. These results strongly suggest that C4d and alpha 4 share an antigenic determinant, both of which are recognized by anti-Cha and anti-Rga antisera.     

The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.     

Modulation of human B cell immunoglobulin secretion by the C3b component of complement

The human C3b component of complement was found to inhibit the differentiation of human B lymphocytes into immunoglobulin-secreting cells in vitro. Pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses were inhibited by C3-coated zymosan particles and by purified human C3b. C3b inhibited the PWM-driven responses in a dose-dependent fashion, and it was necessary for C3b to be present in the early phases of the cultures. C3b acted directly on B cells rather than on helper T cells because it inhibited the PFC responses of MNC depleted of T cells and subsequently stimulated with a T cell-independent Epstein Barr virus mitogen. Furthermore, C3b failed to stimulate the generation of suppressor lymphocytes and/or monocytes that might have been responsible for the inhibition of B cell responses. Our results indicate that C3b or its fragments exert negative modulatory effects on human B lymphocyte responses.