Abstract
Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin gamma1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcgammaRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcgammaRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.
Highlights
Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function
The impact of posttranslational modifications (PTMs)1 on protein structure is more understood for some modifications, it is less defined for other PTMs and in many cases is protein-dependent
Bear in mind that in this IgG1 there was essentially no change in receptor binding activity upon oxidation, but there was enhanced receptor binding upon hypergalactosylation (Fig. 1). These results suggest two potential scenarios that are consistent with the receptor binding data. (a) The glycans, the terminal galactose, are themselves involved in the interaction with Fc␥RIIIa, or (b) Changes in the glycans cause conformational changes, which force the Fc portion into a conformation more amenable to Fc␥RIIIa binding (a theory proposed by Krapp et al [38])
Summary
Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. Because afucosylated-hypergalactosylated IgG1 has a significant increase in affinity for Fc␥RIIIa and much higher activation (Fig. 1B), close to 50ϫ more than the native form, a Kd (20 nM) that was 50ϫ lower than that for the native IgG1 complex was used to approximate the relative proportions of bound and free species in the exchange experiment.
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