Dataset Information

Protein O-fucosylation: structure and function.

ABSTRACT: Fucose is a common terminal modification on protein and lipid glycans. Fucose can also be directly linked to protein via an O-linkage to Serine or Threonine residues located within consensus sequences contained in Epidermal Growth Factor-like (EGF) repeats and Thrombospondin Type 1 Repeats (TSRs). In this context, fucose is added exclusively to properly folded EGF repeats and TSRs by Protein O-fucosyltransferases 1 and 2, respectively. In both cases, the O-linked fucose can also be elongated with other sugars. Here, we describe the biological importance of these O-fucose glycans and molecular mechanisms by which they affect the function of the proteins they modify. O-Fucosylation of EGF repeats modulates the Notch signaling pathway, while O-fucosylation of TSRs is predicted to influence secretion of targets including several extracellular proteases. Recent data show O-fucose glycans mediate their effects by participating in both intermolecular and intramolecular interactions.

SUBMITTER: Holdener BC

PROVIDER: S-EPMC6656592 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Protein O-fucosylation: structure and function.

Holdener Bernadette C BC Haltiwanger Robert S RS

Current opinion in structural biology 20190126

Fucose is a common terminal modification on protein and lipid glycans. Fucose can also be directly linked to protein via an O-linkage to Serine or Threonine residues located within consensus sequences contained in Epidermal Growth Factor-like (EGF) repeats and Thrombospondin Type 1 Repeats (TSRs). In this context, fucose is added exclusively to properly folded EGF repeats and TSRs by Protein O-fucosyltransferases 1 and 2, respectively. In both cases, the O-linked fucose can also be elongated wit ...[more]

PMID: 30690220

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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

Protein O-fucosylation: structure and function.

Publications

Protein O-fucosylation: structure and function.

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