The role of the Immunoglobulin G1 Fc N-glycan in FcÎ3RIIIa affinity

IgG1 Fc is a remarkable molecule, capable of initiating pro-inflammatory or antiinflammatory responses by binding to Fc receptors. IgG1 Fc has a conserved glycosylation site at N297. The presence of the carbohydrate at N297 is vital for proper Fc function. Intramolecular glycan-polypeptide interactions are thought to impose conformational restrictions on Fc that maintain binding-favorable interactions. Perturbations of these intramolecular interactions may result in disrupted binding of Fc. In this work, we mutate residues at the glycan-polypeptide interface to disrupt the contacts between the glycan and polypeptide. Using MALDI-TOFMS and SPR we demonstrate that Fc mutants display more highly processed glycans and reduced affinity for FcγRIIIa. Furthermore, we mutate residues at the interface of the Cγ2 and Cγ3 domains that do not have highly processed glycans, but still have impaired binding to FcγRIIIa. Finally, we conclude with a mIgG2a Fc protein construct that shows promise for testing Fc functionality in cellular systems.