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Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fc? receptor IIIa.

ABSTRACT: Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fc? receptor IIIa (Fc?RIIIa), depending on N-glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N-glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble form of Fc?RIIIa (sFc?RIIIa) in complex with IgG1-Fc. Our simulation highlights increased conformational fluctuation of the N-glycan at Asn162 of sFc?RIIIa upon fucosylation of IgG1-Fc, consistent with crystallographic data giving no interpretable electron density for this N-glycan, except for the innermost part. The fucose residue disrupts optimum intermolecular carbohydrate-carbohydrate interactions, rendering this sFc?RIIIa glycan distal from the Fc glycan. Moreover, our simulation demonstrates that core fucosylation of IgG1-Fc affects conformational dynamics and rearrangements of surrounding amino acid residues, typified by Tyr296 of IgG1-Fc, which was more extensively involved in the interaction with sFc?RIIIa without Fc core fucosylation. Our findings offer a structural foundation for designing and developing therapeutic antibodies with improved ADCC activity.

SUBMITTER: Sakae Y

PROVIDER: S-EPMC5653758 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa.

Sakae Yoshitake Y Satoh Tadashi T Yagi Hirokazu H Yanaka Saeko S Yamaguchi Takumi T Isoda Yuya Y Iida Shigeru S Okamoto Yuko Y Kato Koichi K

Scientific reports 20171023 1

Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fcγ receptor IIIa (FcγRIIIa), depending on N-glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N-glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble for ...[more]

PMID: 29062024

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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.

Dataset Information

Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fc? receptor IIIa.

Publications

Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa.

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