Dataset Information

Substrate specificity of FUT8 and chemoenzymatic synthesis of core-fucosylated asymmetric N-glycans.

ABSTRACT: Substrate specificity studies of human FUT8 using 77 structurally-defined N-glycans as acceptors showed a strict requirement towards the ?1,3-mannose branch, but a great promiscuity towards the ?1,6-mannose branch. Accordingly, a chemoenzymatic strategy was developed for the efficient synthesis of core-fucosylated asymmetric N-glycans.

SUBMITTER: Calderon AD

PROVIDER: S-EPMC4852481 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Substrate specificity of FUT8 and chemoenzymatic synthesis of core-fucosylated asymmetric N-glycans.

Calderon Angie D AD Liu Yunpeng Y Li Xu X Wang Xuan X Chen Xi X Li Lei L Wang Peng G PG

Organic & biomolecular chemistry 20160401 17

Substrate specificity studies of human FUT8 using 77 structurally-defined N-glycans as acceptors showed a strict requirement towards the α1,3-mannose branch, but a great promiscuity towards the α1,6-mannose branch. Accordingly, a chemoenzymatic strategy was developed for the efficient synthesis of core-fucosylated asymmetric N-glycans. ...[more]

PMID: 27080952

Similar Datasets

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

Substrate specificity of FUT8 and chemoenzymatic synthesis of core-fucosylated asymmetric N-glycans.

Publications

Substrate specificity of FUT8 and chemoenzymatic synthesis of core-fucosylated asymmetric N-glycans.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets