title: Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG.
abstract: Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies to improve their biotherapeutic potential. One important aspect of this is the modulation of glycosylation as a strategy to improve efficacy. Here, we describe mutations of Y407 in the CH3 domain of IgG1 and IgG4 that significantly increase sialylation, galactosylation, and branching of the N-linked glycans in the CH2 domain. These mutations also promote the formation of monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass spectrometry was used to probe conformational changes in IgG1-Y407E, revealing, as expected, a more exposed CH3-CH3 dimerization interface. Additionally, allosteric structural effects in the CH2 domain and in the CH2-CH3 interface were identified, providing a possible explanation for the dramatic change in glycosylation. Thus, the mutation of Y407 in the CH3 domain remarkably affects both antibody conformation and glycosylation, which not only alters our understanding of antibody structure, but also reveals possibilities for obtaining recombinant IgG with glycosylation tailored for clinical applications.
authors: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors: Rebecca J
authors: RJ
authors: Rose
authors: Patrick H C
authors: PH
authors: van Berkel
authors: Ewald T J
authors: ET
authors: van den Bremer
authors: Aran F
authors: AF
authors: Labrijn
authors: Tom
authors: T
authors: Vink
authors: Janine
authors: J
authors: Schuurman
authors: Albert J R
authors: AJ
authors: Heck
authors: Paul W H I
authors: PW
authors: Parren
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D019158:N-Acetylneuraminic Acid; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D019158:N-Acetylneuraminic Acid
keywords: N-linked glycosylation; antibody structure; hydrogen-deuterium exchange; native mass spectrometry; sialic acid