Dataset Information

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 (?1,3- and ?1,6-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 ?1,3-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.

SUBMITTER: Castilho A

PROVIDER: S-EPMC4622071 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation.

Castilho Alexandra A Gruber Clemens C Thader Andreas A Oostenbrink Chris C Pechlaner Maria M Steinkellner Herta H Altmann Friedrich F

mAbs 20150101 5

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 Gl ...[more]

PMID: 26067753

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Project description:FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

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Dataset Information

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation.

Publications

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation.

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