Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

The inhibition of covalent binding of the nascent complement component C4b to its target

The inhibition of covalent binding of the nascent C4b fragment of the human complement component to its natural target, immunoglobulin G, was studied. To this end, an immunoenzyme system was developed. In this ELISA method, the complement was activated on the sorbed IgG molecules and the resulting nascent C4b fragment acylated IgG or interacted with a competitive inhibitor added to the system. The inhibition constants for binding of the nascent C4b to its target were determined for immunoglobulins G1, G2, G3, G4, M, and A1, as well as for ferritin, yeast mannan, capsid polysaccharides of theNeisseria meningitidis A, B, and C serotypes, diphtheria anatoxin, epinephrine, and salicylic acid. On the basis of the experimental data, the immunoglobulin role at the activation stage of the complement regulation cascade, the relationship between the antigen immunogenicity and its ability to interact with C4b, and the direct effect of a number of therapeutic agents on the complement system were discussed. Lectins of various specificities were shown to inhibit the enzymic activation of C4 by the first complement component and the subsequent C4b sorption by its target, which allowed us to suggest that some oligosaccharide fragments of the C1s and C4 molecules are spatially close to the C1s active site and to the thioester bond of C4.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

Inhibition of binding of activated compliment component C4b with its target]

The inhibition of covalent binding of the nascent C4b fragment of the human complement component to its natural target, immunoglobulin G, was studied. To this end, an immunoenzyme system was developed. In this ELISA method, the complement was activated on the sorbed IgG molecules and the resulting nascent C4b fragment acylated IgG or interacted with a competitive inhibitor added to the system. The inhibition constants for binding of the nascent C4b to its target were determined for immunoglobulins G1, G2, G3, G4, M, and A1, as well as for ferritin, yeast mannan, capsid polysaccharides of the Neisseria meningitidis A, B, and C serotypes, diphtheria anatoxin, epinephrine, and salicylic acid. On the basis of the experimental data, the immunoglobulin role at the activation stage of the complement regulation cascade, the relationship between the antigen immunogenicity and its ability to interact with C4b, and the direct effect of a number of therapeutic agents on the complement system were discussed. Lectins of various specificities were shown to inhibit the enzymic activation of C4 by the first complement component and the subsequent C4b sorption to its target, which allowed us to suggest that some oligosaccharide fragments of the C1s and C4 molecules are spatially close to the C1s active site and to the thioester bond of C4.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

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.     

Covalent binding properties of the C4A and C4B isotypes of the fourth component of human complement on several C1-bearing cell surfaces

In a previous study we demonstrated that the thioester-mediated transacylation of the human C4B isotype onto sheep erythrocytes (ES) was approximately fourfold more efficient than that of C4A. Moreover, although C4B formed predominantly ester linkages, C4A displayed a preference for amide bond formation. We therefore suggested that the relative functional activity observed for the two isotypes would be a combined reflection of their nucleophilic preference and the surface composition of the C1-bearing target. The present study tests this hypothesis. Chemical modification of amino groups on Es with ethylacetimidate produced a twofold decrease in the C1-dependent binding of C4A isotype, while having a negligible effect on C4B binding. Furthermore, with human erythrocytes and two human leukocyte cell lines, K562 and U937, the C4B to C4A deposition ratio decreased from greater than 4 with ES to between 1.5 and 2. Irrespective of the target, C4A and C4B maintained their preference for forming amide and ester bonds, respectively. Interestingly, SDS-PAGE profiles of radiolabeled C4A and C4B, which had been covalently deposited on the various cells, suggested a further degree of transacylation specificity, as the two isotypic alpha-chains sometimes bound to different membrane components. These differences were not easily accounted for by simple differences in the abundance of the preferred nucleophile for each isotype on a given surface constituent, nor were they due to the preferential binding of one isotype to the sensitizing antibody. We speculate that nascent C4B may contain a substrate binding site that facilitates productive attack on the thioester carbonyl by molecules containing the class of nucleophile preferred by each isotype.     

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.     

The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.     

Complement Activation Products C3a and C4a as Endogenous Antimicrobial Peptides

All vertebrate species are constantly challenged by infectious agents and pathogens. In order to fight these infectious agents the human host has developed a sophisticated and powerful immune defense. The complement system, which represents the first defense line of innate immunity is activated immediately, within seconds. The activated immune system recognizes and damages an invading microbe, coordinates the host immune response and further orchestrates the adaptive immune response. Activation of the complement system leads to a rapid and amplified response which includes the generation of small peptides like C3a and C4a that display antimicrobial, anti-fungal and anaphylactic activity. Here we report how these antimicrobial peptides are generated during the immune response and summarize the functional mechanisms of these intrinsically generated anti microbial peptides.     

Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.     

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.     

Adiponectin binds C1q and activates the classical pathway of complement

The adipose-specific protein adiponectin binds to a number of target molecules, including damaged endothelium and the surface of apoptotic cells. However, the significance of this binding remains unclear. This study demonstrates the binding of purified C1q to recombinant adiponectin under physiological conditions, and the dependence of this upon Ca⁺⁺ and Mg⁺⁺. Binding was enhanced by metaperiodate-mediated destruction of glucosylgalactosyl sugars on adiponectin. Adiponectin was bound by the globular domain of the A chain of collagenase-digested C1q, and C1q binding induced deposition of C4 and C3 through activation of the classical complement pathway. After Western blotting, affinity-purified adiponectin from human serum bound C1q, whereas adiponectin in whole serum did not, unless pre-treated with metaperiodate. These results suggest adiponectin is member of the pattern-recognition family of defence collagens, able to bind target molecules and activate complement. It may therefore play an important role in innate immunity and autoimmune phenomena.     

Measurement by ELISA of Complement Factor 4 (C4) in the Rat Brain: Necessity for Removal of Cerebrovascular Proteins

Assessment of complement 4 (C4) levels in experimental animals is used as a marker for activation of the classical complement pathway. The objective of this study was to develop a method for measuring C4 concentrations in the rat brain. An ELISA (sensitivity = 0.5 ng C4/ml) was used to measure C4 in regional brain homogenates from Fischer rats cardiac-perfused with phosphate buffered saline to remove cerebrovascular contents, and from sham-perfused rats. Ventral midbrain C4 levels were increased (p < 0.001) versus frontal cortex and striatum in sham-perfused rats, whereas after perfusion there were no differences between brain regions. Removal of cerebrovascular contents decreased C4 by 43% in striatum, 52% in frontal cortex, and 69% in ventral midbrain (all p < 0.01 versus sham-perfused means). These results indicate that C4 in the rat brain can be measured quantitatively by ELISA provided that cerebrovascular proteins are removed by perfusion.     

Structural and functional changes in complement protein C4 under UV irradiation

The influence of ultraviolet (UV) light on the structural and functional states of the complement factor C4 was investigated using hemolytic and acid-base titration, PAG electrophoresis, and IR and UV spectrophotometry. UV doses of 75.5 and 755 J/m² initiated C4 activation through changes in the globule structure (increased number of aromatic amino acids and ionogenic groups at the surface). The maximal dose of 2265 J/m² has a destructive effect and decreases its C4 activity in the cascade of hemolytic reactions of the complement system.     

Use of monoclonal antibodies to analyse the expression of a multi-tubulin family

We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti-beta-tubulin monoclonal antibodies, DM1B, DM3B3 and KMX-1 all reacted with the beta 1-tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial beta 2-tubulin isotype - the competence of recognition of this isotype was graded DM1B greater than KMX-1 greater than DM3B3. The anti-alpha-tubulin monoclonal antibody, YOL 1/34 defined the full complement of Physarum alpha-tubulin isotypes, whilst the anti-alpha-tubulin monoclonal antibody, KMP-1 showed a remarkably high degree of isotype specificity. KMP-1 recognises all of the myxamoebal alpha 1-tubulin isotypes but only recognises 3 out of the 4 alpha 1-tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP-1 does not recognise the plasmodial specific alpha 2-tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi-tubulin families.     

Synthesis and regulation of the two human complement C4 genes in stable transfected mouse fibroblasts

The major histocompatibility complex-linked human complement C4 genes are highly homologous in primary structure but give rise to products which differ in complement-activating function. In order to examine the synthesis, function, and regulation of these two genes independently, cloned C4A and C4B genes were transfected into mouse fibroblast L-cells. In the stable transfected cell lines, C4A and C4B are synthesized, undergo a complex series of post-translational modifications, and each functions appropriately in activation of the classical complement pathway. A marked difference in the kinetics of complement component C1-mediated cleavage of the C4A- and C4B-alpha chains was demonstrated in the transfectants and may contribute to the differences in the intrinsic functional activity of the two C4 isotypes. In contrast to the expression of other complement genes which are affected during the hepatic "acute phase response" (factor B, C3), the expression of C4 was not regulated by interleukin-1 or tumor necrosis factor. Interferon-gamma, however, mediated a dose- and time-dependent increase in the expression of the C4 genes. Moreover, interferon had a significantly greater and longer-lasting effect on the synthesis of C4A than that of C4B. Differences in the expression and regulation of these two genes provide insight into the control of complement activation during inflammation.     

Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disease resulting in progressive cognitive decline. Amyloid plaque deposits consisting specifically of β-amyloid peptides that have formed fibrils displaying β-pleated sheet conformation are associated with activated microglia and astrocytes, are colocalized with C1q and other complement activation products, and appear at the time of cognitive decline in AD. Amyloid precursor protein (APP) transgenic mouse models of AD that lack the ability to activate the classical complement pathway display less neuropathology than do the APPQ+/+ mice, consistent with the hypothesis that complement activation and the resultant inflammation may play a role in the pathogenesis of AD. Further investigation of the presence of complement proteins C3 and C4 in the brain of these mice demonstrate that both C3 and C4 deposition increase with age in APPQ+/+ transgenic mice, as expected with the age-dependent increase in fibrillar β-amyloid deposition. In addition, while C4 is predominantly localized on the plaques and/or associated with oligodendrocytes in APPQ+/+ mice, little C4 is detected in APPQ−/− brains consistent with a lack of classical complement pathway activation because of the absence of C1q in these mice. In contrast, plaque and cell associated C3 immunoreactivity is seen in both animal models and, surprisingly, is higher in APPQ−/− than in APPQ+/+ mice, providing evidence for alternative pathway activation. The unexpected increase in C3 levels in the APPQ−/− mice coincident with decreased neuropathology provides support for the hypothesis that complement can mediate protective events as well as detrimental events in this disease. Finally, induced expression of C3 in a subset of astrocytes suggests the existence of differential activation states of these cells.