Fixation of the first component of complement by immune complexes: effect of reduction and fragmentation of antibody.
ABSTRACT: The integrity of both covalent and non-covalent bonds between the heavy chains of rabbit immunoglobulin gamma antibody is essential for maximal efficiency of binding of the first component of complement to immune complexes.
Project description: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.
Project description:The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.
Project description: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.
Project description:A tryptic fragment (A) of Mr 25000 was prepared from bovine secretory component. The fragment binds polymeric immunoglobulin, although 9 times less effectively than secretory component on a molar basis. The fragment has four buried half-cystine residues and two exposed half-cystine residues. It gives rise to two fragments of Mr 11000-13000 on prolonged digestion with trypsin, and these do not bind polymeric immunoglobulin. It is proposed that fragment A consists of two immunoglobulin-like domains. Bovine secretory component was found to have 9-11 buried half-cystine residues and four exposed half-cystine residues. Reduction and alkylation of the exposed residues decreases the binding of polymeric immunoglobulin by 3-fold. Initial tryptic cleavage of bovine secretory component gives a fragment (Q) disulphide-bridged to a further fragment (T). Fragment Q is similar in size to a three-domain immunoglobulin fragment, and fragment T is similar in size to a two-domain immunoglobulin fragment. The two-domain fragment A is derived from fragment Q by further tryptic cleavage. The results are compatible with the proposal by Mostov, Friedlander & Blobel [(1984) Nature (London) 308, 37-43] that secretory component consists of multiple immunoglobulin-like domains. The results also indicate that optimal binding of polymeric immunoglobulin involves several domains stabilized by an exposed disulphide bridge.
Project description:Protein-A-Fc-fragment complexes were observed in sedimentation-velocity experiments by ultracentrifugation. The interaction was studied by protein-fluorescence-quenching titrations of the Fc fragment with protein A, allowing the dissociation constant to be determined under a variety of conditions. The first component of the complement pathway, C1, is activated by complexes of protein A with rabbit IgG (immunoglobulin G), and the structural basis for this interaction was studied by using n.m.r. (nuclear magnetic resonance). The four Fc-fragment binding sites on protein A were shown to contain aromatic amino acids, and to be connected by mobile hydrophilic regions. Neither n.m.r. nor proton-relaxation-enhancement studies show evidence of a large conformational change of the Fc fragment on binding protein A, and this suggests that the cross-linking of the Fc fragments may be primarily responsible for the activation of component C1. This is supported by the inability of a univalent tryptic fragment of protein A to activate complement fixation by rabbit IgG.
Project description:Preformed immune aggregates, containing antigen and either IgG (immunoglobulin G) or F(ab')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement. Both the IgG and F(ab')2 immune aggregates bound C3b, the activated form of the complement component C3, in a similar manner, 2-3% of the C3 available in the serum being bound to the aggregates as C3b, and the rest remaining in the fluid phase as inactive C3b or uncleaved C3. It was found that the C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes could be demonstrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b-antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, which suggested that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent mol.wts. of 360000 and 580000, corresponding to either one to two C3b molecules respectively bound to one molecule of antibody. On reduction of the C3b-IgG complexes it was found that the beta-chain, but not the alpha'-chain, of C3b was released along with all the light chain of IgG but only about half or less of the heavy chain of IgG. These results indicate that, during activation of the alternative pathway of complement by immune aggregates containing IgG antibody, the alpha'-chain of C3b may become covalently bound at one or two sites in the Fd portion of the heavy chain of IgG.
Project description:Thiol compounds have been investigated as inhibitors of the covalent binding reaction of human complement protein C4 using Sepharose-C1s as a combined activating and binding surface. o- and p-substituted aminothiophenols are equally effective inhibitors, whereas the m-substituted compound is a less potent inhibitor. The anti-hypertensive drug captopril is also shown to inhibit the covalent binding reaction. A comparison of the effects of these compounds on the covalent binding reaction of isolated C4A and C4B has been made. Results suggest that a Pro-to-Leu substitution in C4B is likely to account for the differences in inhibitory potency of C4B compared with C4A observed with the aromatic inhibitors.
Project description:The relative lability of the interchain disulphide bonds of mouse G(2a)-myeloma protein 5563 was studied as a function of 2-mercaptoethanol concentration. Analysis of partial-reduction mixtures by polyacrylamide-gel electrophoresis and microdensitometry showed that the disulphide bonds between light and heavy chains are much more susceptible to reduction than the bonds between heavy chains. At a low concentration of 2-mercaptoethanol (10mm) the major dissociable products of mouse immunoglobulin G are heavy-chain dimers and free light chains. These findings contrast with the reported behaviour of rabbit immunoglobulin G, for which the lability of inter-heavy-chain bonds was found to exceed that of the bonds linking light and heavy chains (Hong & Nisonoff, 1965); the relative stability of rabbit immunoglobulin G interchain bonds was confirmed in the present study. Examination of human immunoglobulin G and an immunoglobulin G (gamma2) of guinea pig showed that at least in the majority of molecules, as with mouse immunoglobulin G, the disulphide bonds between light and heavy chains are more susceptible to reduction than the inter-heavy-chain bonds.
Project description:The complement protein C3, when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, 'nascent' C3b, which is known to bind covalently to certain surfaces. The characteristics of the covalent binding reaction have been studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 microseconds for the reactive intermediate has been calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of oxygen and nitrogen nucleophiles. Activation of C3 in the presence of radioactive forms of four such nucleophiles, phenylhydrazine, methylamine, glycerol and glucosamine results in apparent covalent incorporation of the nucleophile into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of the binding of C3 to Sepharose-trypsin. The radioactive nucleophiles may be considered as 'active-site' labels for C3.
Project description:The precipitin reaction is enhanced in the presence of polysaccharides (Hellsing, 1966). This reaction has now been studied in detail with labelled antigen ((125)I-labelled human serum albumin) and antibody ((131)I-labelled rabbit anti-albumin immunoglobulin G). The relative proportions of antigen and antibody in the precipitates are unchanged by the addition of dextran in spite of the increased precipitation. The ratio of antibody to antigen in the soluble immune complexes decreases with increasing polysaccharide concentration. This can be interpreted as a decrease in the aggregate size of the complexes. At the same time the amount of free antigen in the solution increases. The results are consistent with a decrease in solubility, primarily of the large immune aggregates, together with a shift in the equilibrium between small and large complexes. The effect is in accord with a steric-exclusion phenomenon.