Rodney R. Porter

The structural basis of the multiple forms of human complement component C4

cDNA clones of human complement components C4A and C4B alleles were prepared from mRNA obtained from the liver of a donor heterozygous at both loci. cDNA from one C4A allele was sequenced to give the derived complete amino acid sequence of 1722 amino acid residues of the C4 single chain precursor molecule and the estimated sequences of the three peptide chains of secreted C4. Comparison with partial sequences of a second C4A allele and a C4B allele has led to the tentative identification of some class differences in nucleotide sequences between C4A and C4B and of allelic differences between C4A alleles in this highly polymorphic system.

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.

Deletion of complement C4 and steroid 21-hydroxylase genes in the HLA class III region.

Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21‐hydroxylase genes (21‐OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28‐30 kb that included both the C4A and 21‐OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21‐OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one‐half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross‐over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross‐over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21‐OHA gene is discussed.

Molecular characterization of the HLA-linked steroid 21-hydroxylase B gene from an individual with congenital adrenal hyperplasia

21‐Hydroxylase deficiency which causes congenital adrenal hyperplasia is one of the most common defects of adrenal steroidogenesis. There are two 21‐hydroxylase genes in man, A and B, and these have been mapped to the HLA class III region. Only the 21‐hydroxylase B gene is thought to be active. To understand the molecular basis of congenital adrenal hyperplasia in a patient with the salt‐wasting form of the disease, we cloned and characterized his single 21‐hydroxylase B gene. The nucleotide sequence of this gene and a 21‐hydroxylase B gene from a normal individual have been determined. Comparison of the two sequences has revealed 11 nucleotide alterations, of which two are in the 5′ flanking region, four are in introns, one is in the 3′ untranslated region and four are in exons. Two of the differences in exons cause codon changes, with Ser‐269 and Asn‐494 in the normal 21‐hydroxylase B gene being converted to Thr and Ser, respectively. These amino acid substitutions may give an insight into those residues necessary for 21‐hydroxylase enzymatic activity. We have also confirmed that the 21‐hydroxylase A gene is a pseudogene due to three deleterious mutations in the exons. In addition, comparison of the 21‐hydroxylase B gene sequence with other published sequences indicates that this microsomal cytochrome P‐450 may be polymorphic.

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.

Deletion of the steroid 21-hydroxylase and complement C4 genes in congenital adrenal hyperplasia.

DNA was analysed from 20 patients with congenital adrenal hyperplasia due to cytochrome P-450 steroid 21-hydroxylase deficiency. Using probes recognising sequences in both the 21-hydroxylase gene and the adjacent fourth component of complement (C4), one patient was found to have a homozygous deletion of DNA which encompassed the C4B and 21-hydroxylase B genes. Evidence is presented for this deletion arising by recombination between homologous regions of 21-hydroxylase A and B. Seven patients appeared to be heterozygous for the same deletion, but no detectable alteration in the 21-hydroxylase gene could be demonstrated in others.

The purification and properties of some less common allotypes of the fourth component of human complement

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4 Al, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4A and C4-B proteins, respectively, apart from the switch in their antigenic properties.

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.

The activation of complement components by aggregates of antibodies and their fragments

Abstract The relative consumption of the complement components C1, C2, C3 and C4 from guinea pig serum on addition of rabbit antibody-antigen aggregates, papain digested aggregates, heat aggregated rabbit IgG and Fc has been measured. It has been found that antibody-antigen aggregates are much more efficient with respect to consumption of all four components than heat aggregated IgG which in turn is more efficient than heat aggregated Fc. In the latter two preparations the inefficiency is most marked with C2 and C3 consumption. The results suggest that splitting of C2 may depend on prior association with bound C4b which yields a bound C42 complex to the Fab section of the aggregated antibody.

Amino acid sequence around the proposed thiolester bond of human complement component C4 and comparison with the corresponding sequences from C3 and α2-macroglobulin

The amino acid sequence around the proposed thiolester bond in human complement component C4 has been determined. The sulphydryl and reactive acyl groups involved in the intrachain thiolester bond are contained in a short section of sequence which shows almost complete identity with the equivalent sequence from alpha 2-macroglobulin and C3. Other regions of homology between the three proteins near the proposed thiolester bond are also apparent.