A monoclonal antibody which can distinguish between the two isotypes of human C4.

A monoclonal antibody to Human C4 (L003) has been shown to bind to the polymorphic C4d fragment of the alpha-chain of C4. Another monoclonal antibody (L001) was shown to bind to the beta-chain. L003 reacts differently with the two isotypes of C4, C4-A and C4-B. The equilibrium constant of L003 for C4-B is approx. 7-fold higher than that for C4-A, while L001 has similar affinity for both C4 types. Also, L003 binding to C4-A is very much more pH dependent than is its binding to C4-B. These observations explain the very useful property of L003 in being able to separate the two isotypes by affinity chromatography.

The internal thioester and the covalent binding properties of the complement proteins C3 and C4.

The covalent binding of complement components C3 and C4 is critical for their activities. This reaction is made possible by the presence of an internal thioester in the native protein. Upon activation, which involves a conformational change initiated by the cleavage of a single peptide bond, the thioester becomes available to react with molecules with nucleophilic groups. This description is probably sufficient to account for the binding of the C4A isotype of human C4 to amino nucleophiles. The binding of the C4B isotype, and most likely C3, to hydroxyl nucleophiles, however, involves a histidine residue, which attacks the thioester to form an intramolecular acyl-imidazole bond. The released thiolate anion then acts as a base to catalyze the binding of hydroxyl nucleophiles, including water, to the acyl function. This mechanism allows the complement proteins to bind to the hydroxyl groups of carbohydrates found on all biological surfaces, including the components of bacterial cell walls. In addition, the fast hydrolysis of the thioester provides a means to contain this very damaging reaction to the immediate proximity of the site of activation.

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.

A monoclonal antibody to C1q which appears to interact with C1r2C1s2-binding site.

A monoclonal antibody (SB-4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 10(10) M-1. It has been shown that the antibody binds to the A-B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of performed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2-C1s2 complex.

The effect of residue 1106 on the thioester-mediated covalent binding reaction of human complement protein C4 and the monomeric rat alpha-macroglobulin alpha 1 I3.

The histidine at position 1106 of the C4B isotype of human complement is involved in catalyzing the covalent binding of the thioester to glycerol and water. By replacing the histidine with other residues, it was found that tyrosine is also capable of mediating the reaction. We propose that they act as nucleophiles by first attacking the thioester, upon activation, to form acyl intermediates, which subsequently react with the hydroxyl groups of glycerol or water. The monomeric alpha-macroglobulin, alpha 1I3 of the rat, was also studied. Unlike alpha 2-macroglobulin, which is a tetramer, alpha 1I3 has binding properties similar to those of C4A.

A monoclonal antibody raised against human beta-factor XIIa which also recognizes alpha-factor XIIa but not factor XII or complexes of factor XIIa with C1 esterase inhibitor.

A monoclonal antibody (mAb 2/215) against human beta-factor XIIa (beta-FXIIa), was shown by equilibrium binding studies to have a high affinity for alpha-factor XIIa (alpha-FXIIa) (Kd 1.8 nM) and beta-FXIIa (Kd 0.65 nM) but no detectable reaction with FXII zymogen or alpha-FXIIa:C1 esterase inhibitor (C1-INH) complex. Surface plasmon resonance studies showed that the mAb 2/215 bound to immobilized alpha-FXIIa with high affinity (KD 3.93 +/- 1.46 x 10(-11) M). Western blots employing mAb 2/215 indicated that human plasma contained small amounts of alpha-FXIIa but no beta-FXIIa. mAb 2/215 did not inhibit the amidolytic activity of beta-FXIIa and protected beta-FXIIa from inhibition by C1-INH. The recovery by ELISA,employing mAb 2/215 as the capture antibody, of alpha-FXIIa added to plasma was 11.3%, 42% after inhibition of alpha-FXIIa with 3:4dichloroisocoumarin, and 82% when 0.5% Triton-X100 was added to the assay. Gel filtration showed that the majority of plasma alpha-FXIIa existed as a complex (Mr approximately 170,000). This distinctive mAb increases the capacity to study the contact system in health and disease.

Isolation and initial characterisation of complement components C3 and C4 of the nurse shark and the channel catfish.

Complement components C3 and C4 have been isolated from the serum of the nurse shark (Ginglymostoma cirratum) and of the channel catfish (Ictalurus punctatus). As in the higher vertebrates, the fish C4 proteins have three-chain structures while the C3 proteins have two-chain structures. All four proteins have intra-chain thioesters located within their highest molecular mass polypeptides. N-terminal sequence analysis of the polypeptides has confirmed the identity of the proteins. In all cases except the catfish C3 alpha-chain, which appears to have a blocked N-terminus, sequence similarities are apparent in comparisons with the chains of C3 and C4 from higher vertebrates. We have confirmed that the activity/protein previously designated C2n is the nurse shark analogue of mammalian C4. This is the first report of structural evidence for C4 in both the bony and cartilaginous fish.

The phylogeny and evolution of the first component of complement, C1.

Extensive study of the phylogeny and evolution of the complement system has always been hampered by the difficulties involved in functional assays. These tests rely on the compatibility of the components from different species. Whereas all mammalian species appear to have almost identical classical, alternate and lytic pathways, non-mammalian vertebrates show minor differences. The source of the immunoglobulin molecule used to demonstrate classical pathway activation has also been shown to be crucial. The use of antibodies directed against complement components has been beneficial in the study of relationships, but cross-reactivity with non-complement proteins limited their use. The isolation and purification of complement components and the biochemical characterisation including amino acid analysis and peptide sequencing resulted in an enormous increase in our knowledge of the evolution of the complement system. Amino acid sequence analysis together with gene cloning of all human complement components finally revealed a number of sofar unknown features concerning the domain structure of the components and the relation of certain motifs and repeats to other proteins. The use of cDNA probes in Southern blot analysis of chromosomal DNA from various species enabled an extension of our scope of the phylogeny of complement. In this article we summarized the data on the phylogeny and evolution of the first component of complement and the associated molecules. We provide evidence for the conservation of the classical pathway and C1q in particular which appears to predate the divergence of the cartilaginous fish from the higher vertebrates. The possibility that the classical pathway could predate the alternate pathway is discussed.

The phylogeny and evolution of the thioester bond-containing proteins C3, C4 and alpha 2-macroglobulin.

The complement system is an effector of both the acquired and innate immune systems of the higher vertebrates. It has been traced back at least as far as the echinoderms and so predates the appearance of the antibodies, T-cell receptors and MHC molecules of adaptive immunity. Central to the function of complement is the reaction of the thioester bond located within the structure of complement components C3 and C4. The structural thioester first appeared in a protease inhibitor, alpha 2-macroglobulin, in which it is involved in the immobilisation and entrapment of proteases. An important development in the C3 molecule has been the acquisition of a catalytic His residue which greatly increases the rate of reaction of the thioester with hydroxyl groups and with water.

Structural basis of the binding specificity of the thioester-containing proteins, C4, C3 and alpha-2-macroglobulin.

We have previously noted a large difference in the specificity of the covalent binding reaction of human C4-A and C4-B. Here we report data on three other thioester-containing proteins. Human C3 is unreactive with glycine but its reactivity with glycerol (k'/ko = 23.0 M-1) is similar to that of human C4-B (k'/ko = 15.5 M-1). Human alpha 2-macroglobulin reacts with glycine (k'/ko = 206 M-1) in a manner similar to C4-B (k'/ko = 119 M-1) but its reactivity with glycerol (k'/ko = 1.2 M-1) is C4-A like (k'/ko = 1.3 M-1). Mouse C4 is C4-B like in its reaction with both glycine (k'/ko = 136 M-1) and glycerol (k'/ko = 26.0 M-1). Of these proteins, only C4-A shows a very high rate of reaction with glycine (k'/ko = 13,400 M-1). The comparison of the primary structures of these proteins has allowed us to propose the Leu Asp:Ile His substitutions at positions 1105 and 1106 in the human pro-C4 molecule as the residues largely responsible for the binding specificities of these proteins. The Leu:Ile change would not markedly affect the reactivity of these proteins, but may be necessary for allosteric reasons. The Asp in C4-A and His in C4-B seem likely to be the major specificity-defining residues.

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 thioester and isotypic sites of complement component C4 in sheep and cattle.

The region inclusive of the thioester and the isotype-determining sites of the sheep C4 genes from a single animal was amplified by polymerase chain reaction (PCR). Two bands, at 880 base pairs (bp) and 1000 bp, were resolved by agarose gel electrophoresis. Four different clones were obtained for the 880 bp (type 1) product and two from the 1000 bp (type 2) product. Two of the type 1 clones (type 1H) and both type 2 clones (type 2H) code for the PCPVIH sequence at the isotypic site whereas the other two type 1 clones (type 1D) code for the PFPVMD sequence. By restriction mapping and Southern blot analysis, there appears to be four C4 gene loci for the sheep: two type 1H, one type 1D, and one type 2H. The type 1H and type 2H genes are likely to code for proteins with C4B-like properties whereas the type 1D genes for proteins with C4A-like properties. The same region of the sheep C4 genes of nine other breeds of sheep are also amplified by PCR and analyzed by restriction mapping and Southern hybridization. Each of the sheep has type 1H, type 2H, and type 1D genes and appears to have four C4 gene loci except for the Orkney, which may have five. A single band of 880 bp was obtained from the PCR product from the genomic DNA of a single cow. Five different clones were identified, two of which code for the PFPVMD sequence and three for the PCPVIH sequence at the isotypic site, which is consistent with previous finding that C4 proteins with A- and B-like activities could be purified from the plasma of the same animal. Comparison of the nucleotide sequences of the isotype-determining region of the sheep and cattle C4 genes with those of the primates and mouse suggests that the C4A-like genes evolved independently in the primates and the 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 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-A1, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4-A and C4-B proteins, respectively, apart from the switch in their antigenic properties.

The origin of the very variable haemolytic activities of the common human complement component C4 allotypes including C4-A6.

The human complement component C4 occurs in many different forms which show big differences in their haemolytic activities. This phenomenon seems likely to be of considerable importance both physiologically and pathologically. C4 is coded by duplicated genes between HLA-D and HLA-B loci in the major histocompatibility complex in man. Several fold differences in haemolytic activity between products of the two loci C4-A and C4-B have been correlated with changes of six amino acid residues in this large protein of 1722 residues and with differences of several fold in the covalent binding of C4 to antibody-antigen aggregates. Some allotypes of one locus also differ markedly, notably C4-A6 which has 1/10th the haemolytic activity of other C4-A allotypes. A monoclonal antibody affinity column has been prepared which is able to separate C4-A from C4-B proteins and, using serum from an individual expressing only the C4-A6 allele at the C4-A locus, C4-A6 protein has been prepared. Investigation has shown C4-A6 to have the same reactivity as other C4-A allotypes except in the formation of the complex protease, C5 convertase. This protease is formed from C4, C2 and C3 and if C4-A6 is used it has approximately 1/5th the catalytic activity compared with other C4-A allotype. Allelic differences in sequence identified in C4 proteins so far are few and it is probable that the big difference in catalytic activity of C5 convertase is caused by very small changes in structure.

Inhibition of the covalent binding reaction of complement component C4 by penicillamine, an anti-rheumatic agent.

D(-)-Penicillamine [D(-)-beta beta-dimethylcysteine] is an anti-arthritic drug, but its use is limited by adverse side effects, which include problems in immune-complex clearance. Complement is important as a source of inflammatory mediators in rheumatoid arthritis and is also involved in immune-complex clearance. Thus inhibition of the complement cascade would be likely to contribute to both the therapeutic and the toxic effects of penicillamine. It is shown that penicillamine and cysteine are potent inhibitors of the covalent binding of activated complement component C4 to immune complexes. [35S]Cysteine itself becomes covalently bound to C4b through the thioester site. Penicillamine and cysteine are more reactive with the C4A isotype than with the C4B isotype of the HLA class III protein C4. The limited amino acid sequence differences between C4A and C4B include a cysteine/serine interchange, and it is suggested that the cysteine residue in C4A contributes to the increased rate of reaction of C4A with the alpha-amino-beta-thiol compounds.

A comparison of the properties of two classes, C4A and C4B, of the human complement component C4.

A remarkable difference has been observed between the reactivity of the two forms of human complement component C4. C4B binds twice as effectively as C4A to antibody-coated red cells, but the reverse occurs with protein-antigen complexes. C4B reacts much more effectively with hydroxyl groups than C4A and this is reversed for reaction with amino groups in spite of the very small difference in amino acid sequence between the two forms of C4. No other differences in stability, activation or inactivation were observed. These findings emphasise the biological advantage of the duplication of the C4 gene in its reaction with a wide range of antigenic structures. The correlation of the presence of different forms of C4 with susceptibility to autoimmune diseases may be explicable by these big differences in binding reactivity.