The chemical structure of the C4d fragment of the human complement component C4

The complete amino acid sequence of the C4d fragment (380 residues long) of the human complement component C4 is presented. Most of the sequence was determined by analysis of CNBr peptides and tryptic peptides obtained from S-carboxymethylated protein. The sequence of the amino terminal 88 residues [Campbell R. D., Gagnon J. and Porter R. R. (1981) Biochem. J. 199, 359-370] and a 106 residue polymorphic segment of C4d [Chakravarti D. N., Campbell R. D. and Gagon J. (1983) FEBS Lett. 154, 387-390] was extended. Some overlaps not provided by the protein sequence analysis were obtained from the amino acid sequence predicted by the nucleotide sequence [Belt K. T., Carroll M. C. and Porter R. R. (1984) Cell 36, 907-914]. The present protein sequence data provide information for the isolation of all the CNBr and succinylated tryptic peptides of C4d. In addition to the polymorphism previously described, two other sets of polymorphic amino acid residues at positions 153 (Ile/Ser) and 154 (Gln/Ala) have been identified. The major site of glycosylation has been shown to be an asparagine residue located in the sequence -Asn-Val-Thr- in the carboxy terminal end of C4d. A remarkable difference in the predicted secondary structure of C4d arising from one set of four polymorphic residues in a stretch of six residues and another single polymorphic residue suggests a structural basis for the origin of the different chemical reactivities of the C4 isotypes (C4A and C4B) and their serological difference in the expression of Rodgers or Chido blood group antigens. Possible non-covalent membrane attachment sites have been suggested from the hydropathy profile. Comparison of the C4d sequence with human C3, C5 and alpha 2-macroglobulin revealed extended stretches of sequence similarity (between 19 and 38% homology) with the corresponding regions of these proteins.     

Revised nomenclature for human complement component C4*2

This note describes the recommended designations for allotypes of human complement component C4, which were approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).     

Reference typing report for complement component C4

During the 7th Complement Genetics Workshop, Mainz, Germany, May 1998, a complement component C4 typing exercise took place with the aim of applying present technologies to the definition of reference C4 alleles/phenotypes and the recognition of nonexpressed (Q0) C4 alleles within expressed haplotypes. Eleven samples were submitted from 3 laboratories and tested by 14 participating laboratories with basic protein-typing technologies; in addition, each laboratory contributed data from local expertise. The samples were introduced to the reference typing for one or more characteristic allotype or for partial or total nonexpression of one isotype. The blinded samples were centrally evaluated and the results discussed among the participants at a plenum meeting. From the results, the samples could be classified into a group of common, easy to diagnose pheno-/allotypes, less common but still unanimously recognised variants, and a third group with difficult pheno-/allotypes. Within the latter group, the allotypes were either new (C4A '92'; C4B '93') and/or showed partial or total reversed antigenicity and unusual Rodgers/Chido (Rg/Ch) PCR subtypes (C4A '92'; C4A 12; C4B '35'; C4B '13'). Semiquantitative C4-alpha-chain estimates of relative isotype levels correlated well with the number of alleles seen at each locus by agarose gel electrophoresis, and were superior to other isotype quantitation methods. From the evaluation of the reference typing it was concluded that the recognition of rare, aberrant or hybrid C4 alleles with partial or total reversed Rg/Ch antigenicity or monoclonal reactivity is still difficult in most instances; besides isotype-dependent lysis, relative migration values, immunoblots with Rg- and Ch-specific monoclonal antibodies, Rg/Ch PCR typing, side-by-side comparison with already described allotypes will ultimately be required. The recognition of nonexpressed alleles within C4A and C4B expressed phenotypes remains the major obstacle in C4 genetic typing. Finally, a conclusive interpretation of DNA typing results will be achieved only in the context of complete allotyping results at the protein level, and at present cannot replace conventional protein allotyping.     

Polymorphism of the fourth complement component in the dog

The polymorphism of the fourth complement component was studied in 118 unrelated dogs from different breeds and 73 unrelated beagles. In the mixed breed population, five different variants were observed with the following gene frequencies: C4*1 = 3.9%; C4*2 = 13.6%; C4*21 = 6.0%; C4*3 = 9.0% and C4*4 = 51.1%. In beagles, only four variants could be identified with gene frequencies of C4*2 = 24.2%; C4*21 = 1.4%; C4*3 = 20.6% and C4*4 = 46.3%. In both populations analyzed positive linkage disequilibria between certain DLA antigens and C4 variants were recognized. The biochemical analysis of the canine C4 revealed a double-banding pattern for the C4 alpha chain which gives preliminary evidence for two gene loci encoding for the fourth complement component in the dog.     

Engineering of human complement component C3 for catalytic inhibition of complement

As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies.     

Nomenclature for human complement component C2*2

This note describes the designations for variants of the human complement component C2, which were approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).     

A solid phase fluorescent immunossay for the quantitation of the C4 component of human complement.

A non-competitive method for the determination of the C4 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C4 in serum in the range of 10 mg/dl to 170 mg/dl within 2 h. C4 levels as low as 150 ng/ml can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C4 levels in healthy adults. Good agreement was found between the two methods.     

Receptors on guinea-pig erythrocytes specific for cell-bound fourth component of human complement (C4).

Guinea-pig erythrocytes have receptors for heterologous (human and rabbit) complement activated by the classical pathway on cell surfaces. This was shown in the present study by rosette-forming reactions of guinea-pig erythrocytes and human lymphocytes or sheep erythrocytes pre-treated with antibody and human R3 complement. The binding is temperature-dependent and is enhanced by treating the guinea-pig erythrocytes with neuraminidase. The receptors were shown to be specific for C4 by inhibition tests employing a range of anti-human complement antibodies (including anti-Clq, -Cl inhibitor, -C4, -C2, -C3 and -C3b inactivator). Of these reagents, only anti-C4 inhibited the receptor activity, indicating that the guinea-pig erythrocyte C4-receptors differ from those on lymphocytes, monocytes, polymorphonuclear leucocytes and human erythrocytes which are reported to react with both C3b and C4b. In contrast to the strong affinity observed for heterologous C4, guinea-pig erythrocytes appear to react very weakly, if at all, with homologous C4.     

Polymorphism of C3 component of complement in the Polish population. II. Rare phenotypes in C3 system

In a sample of the Polish population including 4741 adults, 15 phenotype variants were found in 22 of them. These variants are determined by 12 rare alleles of the mean frequency 0.0023. Family studies of several probands with C3 phenotype variants have confirmed their genetic determination. They have been observed to be heterozygotes in which beside one common gene, rare codominant alleles are situated. Studies on polymorphism of C3 component carried out on numerous populations, allowed the discovery of new phenotype variants within the C3 group system. Family studies have confirmed their hereditary character and that they are determined by the alleles codominant in relation to the commonly occurring C3S and C3F. The paper presents the results of studies on the rare C3 phenotype variants encountered in the Polish population.     

Difference in the biological properties of the two forms of the fourth component of human complement (C4).

The two forms of human C4 were compared in their haemolytic activity and in their capacity to mediate inhibition of immune complex precipitation in human C4 deficient sera. Whereas haemolysis by C4B was 3.2 fold more efficient than by C4A, C4A was 1.7 fold more efficient at inhibiting immune precipitation than C4B. Thus the biological properties of the two forms of human C4 are different and it is suggested that C4A is mainly involved in immune complex clearance reactions.     

Polymorphism of C3 component of complement in the Polish population. I. Population and family studies

In a sample of the Polish population numbering 4741 subjects, the three common types C3S, C3F and C3FS and 15 phenotype variants were found with frequencies 0.0046. The frequencies of C3S and C3F genes determining the common types were 0.8227 and 0.1750, respectively. Examination of 40 newborns and their mothers has revealed that C3 types are formed during the fetal life. The results of studies on 76 families with 157 children and 2332 mother-child pairs have confirmed that 3C3 types are determined by a single genetic locus in which codominant autosomal alleles are situated.     

Synthesis and regulation of the fourth component of complement (C4) in the human monocytic cell line U937: comparison with that of the third component of complement (C3).

Production of the fourth component of complement (C4) by the human monocytic cell line U937 and its regulation were investigated in comparison with the production of the third component of complement (C3) in a cell culture system. Although no detectable C4 was produced by U937 without stimulation, U937 was induced by recombinant interferon-gamma (IFN-gamma) to synthesize C4 in a dose- and time-dependent fashion. The production of C4 was reversibly inhibited by cycloheximide, indicating that it resulted from de novo synthesis. The C4 synthesized by U937 cells was functionally active as assessed by haemolytic assay. SDS-PAGE following biosynthetic labelling showed that subunit structure of C4 synthesized by U937 cells was identical with that of plasma C4 but that molecular weight of alpha-chain was greater than that of plasma C4. We compared the regulation of C4 synthesis with that of C3 synthesis. Although C3 synthesis by U937 cells was enhanced by IFN-gamma, lipopolysaccharide (LPS) and phorbol myristate acetate (PMA), C4 synthesis was induced only by IFN-gamma. LPS and IFN-gamma induced a synergistic increase in C3 synthesis by U937 cells. U937 cells incubated with LPS and IFN-gamma synthesized a greater amount of C4 than those incubated with IFN-gamma alone. Thus it was demonstrated that the synthesis of C3 and C4 was independently regulated. This study shows that the U937 cell line provides a useful model for studies on the synthesis of complement proteins and on the regulation of complement production.     

Electrophoretic polymorphism of the fourth component of human complement (C4) in paired maternal and foetal plasmas

The polymorphism of the fourth component of complement (C4) as demonstrated by antigen—antibody crossed electrophoresis was used to study paired samples of maternal and cord plasma. Curve analysis was employed to analyse the qualitative and quantitative composition of the C4 patterns. Among the fifty-two paired samples studied, the findings in nine could not be explained by placental passage of C4 from mother to foetus, and evidence for foetal synthesis of C4 was found in five cases. Family studies supported the genetic basis for the electrophoretic polymorphism of C4.     

The architectural transition of human complement component C9 to poly(C9).

Several regions of C9 including three cysteine-rich modules homologous to those in thrombospondin (TS), the low density lipoprotein receptor (LDL), the epidermal growth factors (EDGF), as well as two middle sections of the polypeptide chain were expressed in bacteria. Antibodies derived from these segments were used to probe the relative exposure of epitopes in C9 and poly(C9) using ELISAs. The results indicated that the TS and LDL modules are fully exposed in both monomer and polymer; however, the middle region of the polypeptide chain is buried in the monomer but external in the polymer. Using specified conditions, Fab fragments to the TS and LDL modules did not block C9 polymerization, but those to the middle region of the polypeptide chain and to some extent to the EDGF module did so. Immuno-electron microscopy of poly(C9) indicated that the C9 polypeptide chain assumes a 'U' shape, in which the TS and LDL modules are located on the upper rim. The EDGF module is located on the lower edge of the upper rim, and midsection of the polypeptide chain constructs the barrel of the tubule. Computer assisted contrast enhancement of select electron micrograph images of poly(C9) allowed the clear visualization of each subunit. These were seen to have a volute shape. The upper rim is composed of whorls that are apparently not in lateral contact. It is concluded that the TS and LDL modules do not participate directly in polymerization but cover the hydrophobic central region of the polypeptide chain in the monomer. As a consequence of circular polymerization the midsection of the polypeptide chain becomes exposed as each C9 lengths to fashion a volute form. reserved.