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Quantitative characterization of protein N-linked core-fucosylation by an efficient glycan truncation strategy

ABSTRACT: we developed a truncation strategy which efficiently convert intact glycopeptides with hundreds of different glycans into two truncated forms, i.e. a monosaccharide HexNAc and a disaccharide HexNAc+core-Fucose. Further combination with data-independent analysis to form an integrated platform allowed the measurement of site-specific core-fucosylation abundances and the determination of CF occupancy ratio with high reproducibility. Notably, three times of CF sites were identified using this strategy compared to conventional methods based on intact glycopeptide.

ORGANISM(S): 智人（人类）

SUBMITTER: Xin Guo,Mingming Dong

PROVIDER: PXD048491 | JPOST Repository | Tue Jan 06 00:00:00 GMT 2026

REPOSITORIES: jPOST

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Source:

			Action	DRS
	GX_HF_DDA_HS_IGP.raw	Raw
	GX_HF_DDA_HS_IGP_GPSM_DatabaseGlycan.txt	Txt
	GX_HF_DDA_HS_IGP_GPSM_MonosaccharideStepping.txt	Txt
	GX_HF_DDA_HS_IGP_GlycoPeptideQuantificationArea.txt	Txt
	HS_IGP.zip	Other

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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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Quantitative characterization of protein N-linked core-fucosylation by an efficient glycan truncation strategy

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