The PyMOL Molecular Graphics System) using the Fc-glycan from your structure with Protein Data Base (PDB) code 4BYH and a superposition of the atoms of Fc to Fab

The PyMOL Molecular Graphics System) using the Fc-glycan from your structure with Protein Data Base (PDB) code 4BYH and a superposition of the atoms of Fc to Fab. 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. Keywords: core fucosylation, sialylation, core 1,3-fucosyltransferase3-FucTGnTIVhuman 1,3-mannosyl-1,4-core 1,3-fucosyltransferase FAAP24 (FUT11)6-FucTcore 1,6-fucosyltransferaseCH2constant domain of an IgG heavy chainCTScytoplasmic tail, transmembrane domain name and stem regionCxMabcetuximab (Erbitux?)Fabfragment antigen-bindingFcFragment crystallizable region of immunoglobulin GGlcNAcglycosylation mutants lacking plant specific core 1,2- xylose and 1,3-fucose residues Introduction It is well known that immunoglobulins (Ig) circulate as a highly heterogeneously glycosylated combination (microheterogeneity) of an otherwise homogeneous protein backbone, which points to the multiple functions of these proteins. This microheterogeneity may comprise several hundred glycoforms, and it is mainly owed to the presence or absence of sialic acid, galactose, core fucose and bisecting N-acetylglucosamine (GlcNAc). IgG, the simplest Ig isoform, contains a single N-glycosylation site in the constant domain name (Fc), representing a conserved Fructose site in most Ig classes. The Fc-linked carbohydrates are complex-type bi-antennary N-glycans with high levels of core fucosylation (>80%) and a variable quantity of galactose residues. A striking difference of serum IgG Fc glycans to other Igs is the low amount of sialylation, 10% vs 50%.1,2 Many reports have explained variations of IgG Fc glycosylation, especially of the degree of galactosylation, related to diseases and various other physiological changes like age and pregnancy.3 It was found that an absence of sialic acids and low levels of galactosylation might confer important pro-inflammatory properties to IgG.4 Similarly, the absence of core fucose improved the affinity of the Fc to Fc receptors (e.g., FcIII), thereby enhancing antibody-dependent cellular cytotoxicity.5,6 On this basis, glyco-engineered antibodies carrying Fructose afucosylated Fc glycans are currently in clinical development.7,8 Besides the conserved N-glycosylation sites around the Fc portion, additional carbohydrate chains can be linked to the hypervariable regions of Ig (asymmetric antibodies). For instance, up to 25% of IgG molecules isolated from your serum of healthy human subjects have been reported to carry N-glycans on their variable domains.9,10 The amount of asymmetric IgG was found to increase during pregnancy, as well as after the treatment of antibody-producing cells with hormones and cytokines.11,12 Fab-linked glycans from human serum IgG exhibit primarily complex-type bi-antennary N-glycans with high contents of core fucose (80%), bisecting GlcNAc (50%), and sialic acid (80%).9 Depending on their structures and locations, Fructose the Fab glycans may influence IgG effector functions by increasing or decreasing the affinity for the antigen.1 One report suggests that Fab glycosylation could modulate antibody half-life.13 A common obstacle that hampers detailed research on the relationship of Ig glycosylation to functional activities is the incomplete knowledge of how glycan structures are generated and the availability of expression platforms that allow the synthesis of targeted glycoforms. Several expression systems, including mono and multicellular organisms, were developed to address this issue.3,14 Plants appear particularly well suited for the generation of human proteins, including antibodies, with a designed glycosylation profile.14,15 Compelling features are speed and flexibility by which monoclonal antibodies (mAbs) with defined glycosylation profiles can be produced.15 Notably, the system is used for the production of clinical grade ZMAPP, a glycan-engineered antibody cocktail for Ebola treatments.8 Here, we aimed to elucidate factors that contribute to the processing of glycan structures in IgG-based antibodies. We selected cetuximab as a model because it carries 2 glycosylation sites, with one in the Fc and one in the Fab region. A plant-based expression system was used to generate different glycoforms. Glyco-variants terminate with GlcNAc residues, but differ in their core fucosylation (no fucose, 1,3- and 1,6-linkage, referred to as GnGn, GnGnF3 and GnGnF6, respectively). These variants were individually used to co-express, in plants, glycosylation enzymes that catalyze sialylation, branching and bisected structures. Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans by all enzymes. In contrast, Fc glycan processing largely depended on the presence of core fucose. We observed a particularly strong promotion of glycan processing in the presence of plant-specific core 1,3-fucose. Moreover, computer modeling of Fc glycans point to changes in the interactions of Fc carbohydrates upon adding core fucose, corroborating experimental data. Results Generation of cetuximab glycoforms in glycosylation mutant XT/FT synthesizing human.