title: Fc gamma receptor glycosylation modulates the binding of IgG glycoforms: a requirement for stable antibody interactions.
abstract: FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.
authors.affiliation: School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College , Pearse St. Dublin 2, Dublin, Ireland.
authors.forename: Jerrard M
authors.initials: JM
authors.lastname: Hayes
authors.forename: Asa
authors.initials: A
authors.lastname: Frostell
authors.forename: Eoin F J
authors.initials: EF
authors.lastname: Cosgrave
authors.forename: Weston B
authors.initials: WB
authors.lastname: Struwe
authors.forename: Oscar
authors.initials: O
authors.lastname: Potter
authors.forename: Gavin P
authors.initials: GP
authors.lastname: Davey
authors.forename: Robert
authors.initials: R
authors.lastname: Karlsson
authors.forename: Cecilia
authors.initials: C
authors.lastname: Anneren
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D000069283:Rituximab; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C489017:FCGR1A protein, human; C499173:FCGR2B protein, human; C496124:FCGR3A protein, human; C428200:FCGR3B protein, human; C080568:Fc gamma receptor IIA; D058851:GPI-Linked Proteins; D007074:Immunoglobulin G; D011134:Polysaccharides; D017452:Receptors, IgG; D011994:Recombinant Proteins; D000069283:Rituximab
keywords: Fcγ receptors; Glycosylation; antibody; glycan analysis; rituximab; surface plasmon resonance