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Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase (FUT8) reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc.

Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase (FUT8) reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc.
Author Information (click to view)

Yang Q, Zhang R, Cai H, Wang LX,


Yang Q, Zhang R, Cai H, Wang LX, (click to view)

Yang Q, Zhang R, Cai H, Wang LX,

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The Journal of biological chemistry 2017 07 20() pii 10.1074/jbc.M117.804070

Abstract

The mammalian α1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N-glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the α1,3 mannose arm of N-glycan is essential for FUT8 catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin (EPO) in a GnTI-knockout, FUT8 overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man5GlcNAc2, suggesting that FUT8 can catalyze core fucosylation of N-glycans lacking an α1,3arm GlcNAc in cells. Here, we revisited FUT8 substrate specificity by examining its in vitro activity toward an array of selected N-glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N-glycans lacking an unmasked α1,3arm GlcNAc moiety are not FUT8 substrates. However, Man5GlcNAc2 glycan could be efficiently core fucosylated by FUT8 in an appropriate protein/peptide context, such as with the EPO protein and a V3 polypeptide derived from HIV gp120, and a simple 9-fluorenylmethyl chloroformate protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature biantennary complex type N-glycan also could be core fucosylated by FUT8, albeit at much lower efficiency than the Man5GlcNAc2 peptide. This study represents the first report of in vitro FUT8 catalyzed core fucosylation of N-glycans lacking the α1,3arm GlcNAc moiety. Our results suggested that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8.

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