Effect of reduction and alkylation on structure and function of rabbit IgG antibody—II. Effects on classical pathway C3 convertase formation

One of the basic structural features of human IgG1 is the arrangement of the disulfide bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgG1 molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometry (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disulfide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, disulfide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CH1 domains had similar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgG1kappa and IgG1lambda was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disulfide bonds was smaller in IgG1kappa than in IgG1lambda. The intra chain disulfide bonds in the Fab region of IgG1kappa were also less susceptible than disulfide bonds in the Fab region of IgG1lambda.

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Ranking the Susceptibility of Disulfide Bonds in Human IgG1 Antibodies by Reduction, Differential Alkylation, and LC−MS Analysis

Liu

et al. 2010

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Quantitative analysis of the interaction between immune complex and Clq complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of Clq to immune precipitates

Vonderviszt¹,

Török²,

Lakatos³

et al. 1987

Biochemical Journal

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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.

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Effect of reduction and alkylation on structure and function of rabbit IgG antibody—II. Effects on classical pathway C3 convertase formation

Cited by 2 publications

References 25 publications

Ranking the Susceptibility of Disulfide Bonds in Human IgG1 Antibodies by Reduction, Differential Alkylation, and LC−MS Analysis

Ranking the Susceptibility of Disulfide Bonds in Human IgG1 Antibodies by Reduction, Differential Alkylation, and LC−MS Analysis

Quantitative analysis of the interaction between immune complex and Clq complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of Clq to immune precipitates

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