title: Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation.
abstract: We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF(3), and GnGnF(6) CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.
authors.affiliation: a Department of Applied Genetics and Cell Biology ; University of Natural Resources and Life Sciences ; Vienna , Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.forename: Clemens
authors.initials: C
authors.lastname: Gruber
authors.forename: Andreas
authors.initials: A
authors.lastname: Thader
authors.forename: Chris
authors.initials: C
authors.lastname: Oostenbrink
authors.forename: Maria
authors.initials: M
authors.lastname: Pechlaner
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000068818:Cetuximab / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005643:Fucose / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; D005643:Fucose; D000068818:Cetuximab
keywords: IgG; Nicotiana benthamiana; bisected glycans; cetuximab; core fucosylation; glycan modelling; sialylation