Quantitative analysis of the interaction between immune complex and C1q complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of C1q to immune precipitates.
A novel method was developed for the analysis of the interaction of large multivalent ligands with surfaces (matrices) to analyse the binding of complement subcomponent C1q to immune precipitates. Our new evaluation method provides quantitative data characteristic of the C1q-immune-complex interaction and of the structure of the immune complex as well. To reveal the functional role of domain-domain interactions in the Fc part of IgG the binding of C1q to different anti-ovalbumin IgG-ovalbumin immune complexes was studied. Immune-complex precipitates composed of rabbit IgG in which the non-covalent or covalent bonds between the heavy chains had been eliminated were used. Non-covalent bonds were abolished by splitting off the CH3 domains, i.e. by using Facb fragments, and the covalent contact was broken by reduction and alkylation of the single inter-heavy-chain disulphide bond. The quantitative analysis of the binding curves provides a dissociation constant (K) of 200 nM for the interaction between C1q and immune precipitate formed from native IgG. Surprisingly, for immune precipitates composed of Facb fragments or IgG in which the inter-heavy-chain disulphide bond had been selectively reduced and alkylated, stronger binding (K = 30 nM) was observed. In this case, however, changes in the structure of the immune-complex matrix were also detected. These structural changes may account for the strengthening of the C1q-immune-complex interaction, which can be strongly influenced by the flexibility and the binding-site pattern of the immune-complex precipitates. These results suggest that domain-domain interactions in the Fc part of IgG affect the segmental mobility of IgG molecules and the spatial arrangement of the immune-complex matrix rather than the affinity of individual C1q-binding sites on IgG.
D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology*; D011232:Chemical Precipitation; D003166:Complement Activating Enzymes / Q000276:immunology*; D003172:Complement C1 / Q000276:immunology*; D015922:Complement C1q; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000276:immunology*; D010047:Ovalbumin / Q000276:immunology; D011817:Rabbits; D014461:Ultracentrifugation
D000936:Antigen-Antibody Complex; D003172:Complement C1; D007074:Immunoglobulin G; D015922:Complement C1q; D010047:Ovalbumin; D003166:Complement Activating Enzymes