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.Deceased     

DNA polymorphism of human HLA-linked complement C4 allotypes, including C4 null alleles, in the Finnish population

Human HLA-linked complement C4 gene products, C4A and C4B, show extensive genetic polymorphism. In both loci, an allele without a gene product, C4 null, is also observed. We have performed a restriction enzyme analysis of genomic DNA samples from individuals having all common (frequency over 1%) C4 protein allotypes observed in the Finnish population. Only one allotype-specific RFLP marker was observed. With some enzymes a DNA polymorphism was observed, which was not detectable by C4 protein typing. Analysis of 10 different C4B null haplotypes and 4 C4A null haplotypes suggested that only one haplotype, HLA-B8 C4A0 B1, carried a C4A gene deletion. This was observed in all 4 unrelated individuals homozygous for this haplotype.     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.     

The molecular architecture of human complement component C6

The molecular architecture of human complement component C6 was elucidated at several levels of structural organization. The entire primary structure of C6 was determined by sequencing C6 cDNA that was cloned from a human liver lambda gt11 library. The polypeptide chain of C6 contains 913 amino acids. The protein is homologous with the other terminal components of complement, C7-C9. Specifically, C6 has 29% of its residues identical with C7, and 55 of the 56 cysteines found in C7 match those in C6. The C6 polypeptide chain is cross-linked by 32 disulfide bonds, and most of the cysteines are located in short (34-77 amino acids) discrete segments that exhibit homology with a wide variety of other proteins such as thrombospondin, the low density lipoprotein receptor, epidermal growth factor, and complement factors H and I. C6 is a glycoprotein, and it has two oligosaccharide groups attached to asparagines located near the amino and the carboxyl termini of the molecule. The organization of secondary structural elements in C6 was elucidated using circular dichroism spectroscopy and an empirical method based on sequence analysis. C6 has an estimated 12% alpha-helix, but is comparatively richer in beta-sheet (29%) and beta-turns (21%). Most of the predicted alpha-helical structure resides in a portion of the polypeptide chain that is free of cysteine and which shares homology with C9 and perforin. The tertiary structure of the C6 molecule was visualized by transmission electron microscopy; it has a sickle shape with dimensions of 144 x 66 A. The combined results are discussed and comparisons made with the other late acting components of complement and perforin.     

Structural basis for the C4d.1/C4d.2 serologic allotypes of murine complement component C4

The C4d.1 antigenic specificity was first defined serologically in 1959 as an H-2-associated cellular alloantigen first designated "G," later H-2.7. It was subsequently shown to be an allotype of component C4 of the C system, with the antigenic determinant carried on the C4d proteolytic fragment of the alpha-chain, thus the designation C4d.1. Alloantisera defining an antithetical Ag, C4d.2, were also prepared. Previous studies in our laboratory showed that the structural difference between the two specificities resides in a single tryptic peptide of C4d. As an efficient approach to definition of the amino acid difference(s) involved, genomic clones covering the C4d regions from two H-2 haplotypes of the C4d.1 type have been prepared and sequenced, and compared with two sequences already available for C4d.2-type molecules. The results indicate that the rather striking serologic difference between C4d.1 and C4d.2 is attributable to the single amino acid substitution of arginine in C4d.2 for glutamine in C4d.1. The substituted residue is in a highly hydrophilic region of the C4 molecule, at a position homologous to one that contributes to the Chido/Rodgers serologic difference of human C4 molecules. This substitution also determines a new Pst I site in C4d.1 strains. A HindIII restriction fragment length polymorphism between C4d.1 and C4d.2 has also been observed.     

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.     

The complement component C4 of mammals.

Human complement component C4 is coded by tandem genes located in the HLA class III region. The products of the two genes, C4A and C4B, are different in their activity. This difference is due to a degree of 'substrate' specificity in the covalent binding reactions of the two isotypes. Mouse also has a duplicated locus, but only one gene produces active C4, while the other codes for the closely related sex-limited protein (Slp). In order to gain some insight into the evolutionary history of the duplicated C4 locus, we have purified C4 from a number of other mammalian species, and tested their binding specificities. Like man, chimpanzee and rhesus monkey appear to produce two C4 types with reactivities similar to C4A and C4B. Rat, guinea pig, whale, rabbit, dog and pig each expresses C4 with a single binding specificity, which is C4B-like. Sheep and cattle express two C4 types, one C4B-like, the other C4A-like, in their binding properties. These results suggest that more than one locus may be present in these species. If this is so, then the duplication of the C4 locus is either very ancient, having occurred before the divergence of the modern mammals, or there have been three separate duplication events in the lines leading to the primates, rodents and ungulates.     

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 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.     

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.     

X-ray crystal structure of the C4d fragment of human complement component C4

C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.     

Phenotyping of human complement component C4, a class-III HLA antigen.

The plasma complement protein C4 is encoded at two highly polymorphic loci, A and B, within the class-III region of the major histocompatibility complex. At least 34 different polymorphic variants of human C4 have been identified, including non-expressed or 'null' alleles. The main method of identification of C4 polymorphic allotypes is separation on the basis of charge by agarose-gel electrophoresis of plasma. On staining by immunofixation with anti-C4 antibodies, each C4 type gives three major bands, but, since individuals can have up to five allotypes, the overlapping banding pattern is difficult to interpret. We show that digestion of plasma samples with carboxypeptidase B, which removes C-terminal basic amino acids, before electrophoresis, produces a single, sharp, distinct band for each allotype and allows identification of the biochemical basis of the multiple banding pattern previously observed in C4 phenotype determination.     

The sequence and topology of human complement component C9.

A partial nucleotide sequence of human complement component C9 cDNA representing 94% of the coding region of the mature protein is presented. The amino acid sequence predicted from the open reading frame of this cDNA concurs with the amino acid sequence at the amino-terminal end of three proteolytic fragments of purified C9 protein. No long stretches of hydrophobic residues are present, even in the carboxy-terminal half of the molecule which reacts with lipid-soluble photoaffinity probes. Monoclonal antibody epitopes have been mapped by comparing overlapping fragments of C9 molecule to which the antibodies bind on Western blots. Several of these epitopes map to small regions containing other surface features (e.g., proteolytic cleavage sites and N-linked oligosaccharide). The amino-terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cysteine-rich domains. A model for C9 topology based on these findings is proposed.     

Degradation of human complement component C4b in the presence of the C4b-binding protein-protein S complex.

Vitamin K-dependent protein S and the higher-molecular-weight form of C4b-binding protein (C4bp-high) interact, forming a 1:1 complex with a KD of approx. 1 X 10(-7) M [Dahlb?ck (1983) Biochem. J. 209, 847-856]. In the present study the effect of protein S on the degradation of C4b by Factor I (C3b inactivator) and C4bp was investigated both in fluid phase and on cell surfaces, with the use of highly purified components. Fluid-phase degradation of C4b was monitored on sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, and the effect on surface-bound C4b was estimated by haemolytic assay. No effect of protein S could be demonstrated in any of the systems used. Thus, although bound to C4bp, protein S is neither involved in, nor does it affect, the interaction between C4bp and C4b. This indicates that the binding sites on the C4bp molecule for protein S and for C4b are independent and different.     

Function of the factor I modules (FIMS) of human complement component C6.

In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex.     

The unactivated form of the first component of human complement, C1.

The first component of complement, C1, was isolated unactivated from human serum by repeated additions of di-isopropyl phosphorofluoridate during isolation. The unactivated subcomponents were also isolated, and evidence is given that the three subcomponents C1q, C1r and C1s account wholly for the activity of component C1 in serum. No evidence could be found for a fourth subcomponent, C1t. The approximate molar proportions of the subcomponents in serum are C1q/C1r/C1s = 1:2:2. Optimum activity by haemolytic assay was found at approximate molar proportions C1q/C1r/C1s of 1:4:4. No activity was found when subcomponents were assayed singly or in pairs, except for subcomponents C1q and C1s, which in molar ratio 1:4 gave 15-20% of the activity of the mixture C1q + C1r + C1s. The proteolytic activity of the isolated subcomponent C1s varied according to the method of activation used. Subcomponents C1q + C1r + C1s and C1q + C1s in the presence of antibody-antigen aggregates were activated and inactivated simultaneously, showing a peak of activity and subsequent loss of activity. Both reactions are probably due to proteolysis, and analysis of the peptide bonds split will be necessary to distinguish these two phenomena.     

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.