Covalently-bound human C4b dimers consisting of C4B isotype show higher hemolytic activity than those of C4A in the C3-bypass complement pathway.

The ability to form a covalent dimer of human C4b was investigated with purified isotypes C4A and C4B, and antibody-sensitized liposomes supplemented with C1. In this system, no C4A or C4B formed a complex with the antibody or C1. Whereas both C4A and C4B isotypes formed dimers to a similar extent, C4B formed an ester-linked dimer and C4A an amide-linked dimer. Both of these dimers served as a subunit for the C3-bypass pathway C5 convertase, since liposomes bearing Ab, C1 and a dimer of C4A or C4B, allowed the formation of C5 convertase by the addition of C2. The degree of complement-mediated liposome lysis however, was observed to be 2-3 times higher in the C4B-bearing particles than in those bearing C4A. These results indicate that the second C4b-binding site on the first C4b is different between C4A and C4B, and that in the C3-bypass pathway, C4B has a higher degree of hemolytic activity than C4A, as in the conventional classical complement pathway.

Purification and functional assessment of C3a, C4a and C5a of the common carp (Cyprinus carpio) complement.

Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5-1 nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs.

Functional properties of heterogeneous human asialo-C4 and its isotypes C4A and C4B.

The fourth component of human complement (C4) is encoded at two separate but closely linked loci within the MHC on the short arm of chromosome 6. Thus, there are two types of C4 protein in most individual and pooled normal human sera (NHS): C4A and C4B. Incubation of individual sera, pooled NHS, or purified heterogeneous C4 (C4A/C4B) with bacterial sialidase at 37 degrees C increased C-mediated hemolysis of antibody-sensitized sheep erythrocytes 1.54- to 1.93-fold. Comparative studies of Tmax of human C2, using asialo-C4 or buffer-treated C4 on EAC1gp and extrapolation to time 0 indicated a z value 4-fold higher with asialo-C4. This indicated that more hemolytically active C42 complexes are available with sialidase-treated C4 compared to untreated C4. There was no appreciable difference in the % 125I-C4 bound to EAC1gp (sialidase- or buffer-treated). Sera from two different blood donors with C4A3 phenotype (C4BQ0), two different donors with C4B1 phenotype (C4AQ0), and serum from an individual heterozygous deficient at both C4A3 and C4B1 regions (A3, AQ0; B1, BQ0) were investigated. The C4 allotypes, purified from these sera, were treated with sialidase; the C4A3 was enhanced in hemolytic assays by sialidase-treatment (1.52- to 2.3-fold), whereas the C4B1 allotype was not enhanced. Fluorometric determinations revealed that approximately the same percentage of sialic acid was released from sialidase-treated C4A3 and C4B1. Therefore, the increase in hemolytic titer observed after treatment of NHS or purified heterogeneous C4 with sialidase is a property of C4A3 but not a property of C4B1.

Genetic, structural and functional diversities of human complement components C4A and C4B and their mouse homologues, Slp and C4.

The complement protein C4 is a non-enzymatic component of the C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. The covalent binding of C4 to immunoglobulins and immune complexes (IC) also enhances the solubilization of immune aggregates, and the clearance of IC through complement receptor one (CR1) on erythrocytes. Human C4 is the most polymorphic protein of the complement system. In this review, we summarize the current concepts on the 1-2-3 loci model of C4A and C4B genes in the population, factors affecting the expression levels of C4 transcripts and proteins, and the structural, functional and serological diversities of the C4A and C4B proteins. The diversities and polymorphisms of the mouse homologues Slp and C4 proteins are described and contrasted with their human homologues. The human C4 genes are located in the MHC class III region on chromosome 6. Each human C4 gene consists of 41 exons coding for a 5.4-kb transcript. The long gene is 20.6 kb and the short gene is 14.2 kb. In the Caucasian population 55% of the MHC haplotypes have the 2-locus, C4A-C4B configurations and 45% have an unequal number of C4A and C4B genes. Moreover, three-quarters of C4 genes harbor the 6.4 kb endogenous retrovirus HERV-K(C4) in the intron 9 of the long genes. Duplication of a C4 gene always concurs with its adjacent genes RP, CYP21 and TNX, which together form a genetic unit termed an RCCX module. Monomodular, bimodular and trimodular RCCX structures with 1, 2 and 3 complement C4 genes have frequencies of 17%, 69% and 14%, respectively. Partial deficiencies of C4A and C4B, primarily due to the presence of monomodular haplotypes and homo-expression of C4A proteins from bimodular structures, have a combined frequency of 31.6%. Multiple structural isoforms of each C4A and C4B allotype exist in the circulation because of the imperfect and incomplete proteolytic processing of the precursor protein to form the beta-alpha-gamma structures. Immunofixation experiments of C4A and C4B demonstrate > 41 allotypes in the two classes of proteins. A compilation of polymorphic sites from limited C4 sequences revealed the presence of 24 polymophic residues, mostly clustered C-terminal to the thioester bond within the C4d region of the alpha-chain. The covalent binding affinities of the thioester carbonyl group of C4A and C4B appear to be modulated by four isotypic residues at positions 1101, 1102, 1105 and 1106. Site directed mutagenesis experiments revealed that D1106 is responsible for the effective binding of C4A to form amide bonds with immune aggregates or protein antigens, and H1106 of C4B catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens. The expression of C4 is inducible or enhanced by gamma-interferon. The liver is the main organ that synthesizes and secretes C4A and C4B to the circulation but there are many extra-hepatic sites producing moderate quantities of C4 for local defense. The plasma protein levels of C4A and C4B are mainly determined by the corresponding gene dosage. However, C4B proteins encoded by monomodular short genes may have relatively higher concentrations than those from long C4A genes. The 5' regulatory sequence of a C4 gene contains a Spl site, three E-boxes but no TATA box. The sequences beyond--1524 nt may be completely different as the C4 genes at RCCX module I have RPI-specific sequences, while those at Modules II, III and IV have TNXA-specific sequences. The remarkable genetic diversity of human C4A and C4B probably promotes the exchange of genetic information to create and maintain the quantitative and qualitative variations of C4A and C4B proteins in the population, as driven by the selection pressure against a great variety of microbes. An undesirable accompanying byproduct of this phenomenon is the inherent deleterious recombinations among the RCCX constituents leading to autoimmune and genetic disorders.

Elevated plasma C4a levels in multiple sclerosis correlate with disease activity.

Complement plays a pivotal role in the pathogenesis of multiple sclerosis. C4a, an activated fragment of complement component C4, has been linked to disease activity. We correlated plasma C4 and plasma and CSF C4a with clinical disease in a well-characterised cohort of patients and controls. Plasma C4 was non-significantly and CSF C4a was significantly elevated overall in patients compared to controls. Plasma C4a was raised only in acute relapse, decreasing over 2 months. Results demonstrate intrathecal and systemic activation of complement, reflected in changes in CSF and plasma C4a. The data support a role for complement activation in pathogenesis and suggest a systemic component to the disease.

Complement factors c3a, c4a, and c5a in chronic obstructive pulmonary disease and asthma.

Studies of animal models have shown that the activation of the complement system could have a role in chronic obstructive pulmonary disease (COPD) and asthma by promoting inflammation and enhancing airway hyperresponsiveness. We sought to determine whether the levels of complement factors C3a, C4a, and C5a are elevated at the site of inflammation in patients with COPD and patients with asthma. We analyzed the induced sputum of seven patients with COPD, ten patients with asthma, and twelve healthy nonsmokers. The concentrations of anaphylatoxins in the induced sputum were measured by cytometric bead array. We found significantly increased C5a/C5a desArg concentrations in supernatants of the induced sputum of patients with COPD (P = 0.007) and those with asthma (P = 0.002) compared with the control group. In patients with COPD the C5a/C5a desArg concentrations were significantly negatively correlated with lung diffusion coefficient (r = -0.71, P = 0.035). There was no significant difference in C3a/C3a desArg or C4a/C4a desArg measurements between the three groups of subjects. These in vivo results propose the involvement of complement factor C5a in the pathogenesis of COPD and asthma.

Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

A single arginine to tryptophan interchange at beta-chain residue 458 of human complement component C4 accounts for the defect in classical pathway C5 convertase activity of allotype C4A6. Implications for the location of a C5 binding site in C4.

In general, C4A allotypes of human C4 show one-fourth to one-third the hemolytic activity of C4B allotypes. An exception to this rule is C4A6 which is almost totally deficient in hemolytic activity. Previous studies have localized the defect in C4A6 to the C5 convertase stage. Of the two critical events required for C5 cleavage, namely formation of a covalent adduct between C3b and the C4b subunit of the C3 convertase (C4b2a), and binding of C5 to this C4b-C3b complex, it is a defect in the latter step that accounts for the aberrant activity of C4A6. DNA sequencing studies described in a companion paper have suggested that the sole C4A6-specific difference was a Trp for Arg replacement at beta-chain residue 458. To directly ascertain whether this single substitution was responsible for the hemolytic defect in C4A6, we have used site-directed mutagenesis to introduce this change into both C4A and C4B cDNA expression plasmids. We found that the R to W replacement totally abrogated hemolytic activity. However, irrespective of the amino acid at residue 458, the mutant proteins behaved like their wild-type counterparts with respect to covalent binding to C1-bearing targets, i.e., the C4B recombinants displayed higher binding to sheep and human red cells than did the C4A counterparts. Furthermore, the mutants were able to form covalent C4b-C3b adducts. There was, however, substantially less C5 cleavage produced by cell-bound C4boxy23b complexes made with R458W mutant C4B than with wild-type C4B. These results are consistent with the sole defect in the mutants being at the C5 binding stage and strongly suggest that Arg 458 of the C4 beta-chain contributes to the C5 binding site of the molecule.

Novel function of C4a anaphylatoxin. Release from monocytes of protein which inhibits monocyte chemotaxis.

The complement C4-derived anaphylatoxin, C4a, possesses a strong chemotaxis inhibitory capacity to blood monocytes at concentrations as low as 10(-16) mol/L. In our study, treatment with carboxypeptidase B to convert it to C4a des Arg77 decreased the inhibitory activity to less than 1/1,000. The extraordinary inhibitory capacity of C4a suggests the presence of an amplification mechanism in this inhibition. Indeed, we found that the conditioned media of peripheral blood mononuclear cells or monocyte/macrophage lineage cell lines (U937 and THP-1 cells) preincubated with 10(-16) mol/L C4a for 5 minutes or more at 37 C possessed the inhibitory capacity 100,000-fold stronger than the original activity of C4a. The monocyte-derived chemotaxis inhibitory factor seemed monocyte-specific. This cell-derived factor was sensitive to treatment with trypsin and chymotrypsin and immunologically distinct from C4a. The apparent molecular size of the monocyte factor was estimated to be approximately 20 kd by gel filtration. These results indicate that C4a anaphylatoxin induces the release from monocytes of a protein with inhibitory activity for monocyte chemotaxis.