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 in a GnTI knock-out, 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 α1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 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 α1,3-arm 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 erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary 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 α1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8.
Highlights
The asparagine-linked (N-linked) glycosylation is a major posttranslational modification in mammalian systems, which profoundly affects the intrinsic properties and biological functions of proteins [1, 2]
Overexpression of the FUT8 gene in this cell line led to full core fucosylation of EPO-M5, whereas knockdown of this gene resulted in production of EPO glycoforms without core fucosylation, confirming that FUT8 is the sole enzyme responsible for core fucosylation [6]
This led us to hypothesize that in addition to the N-glycan part, the peptide portion of certain glycoprotein may interact with FUT8 to facilitate the catalysis of core fucosylation of the N-glycans that otherwise have only low activity toward FUT8 (Fig. 1B)
Summary
The asparagine-linked (N-linked) glycosylation is a major posttranslational modification in mammalian systems, which profoundly affects the intrinsic properties and biological functions of proteins [1, 2]. In the mammalian N-glycosylation-processing pathway, core fucosylation is preceded by the transfer of an N-acetylglucosamine (GlcNAc), in an ␣1,2-glycosidic linkage, to the 3-arm mannose residue of the oligomannose substrate, Man5GlcNAc2, by N-acetylglucosaminyltransferase I (GnTI) This step was considered a prerequisite for the core fucosylation (Fig. 1A). This led us to hypothesize that in addition to the N-glycan part, the peptide portion of certain glycoprotein (such as EPO) may interact with FUT8 to facilitate the catalysis of core fucosylation of the N-glycans that otherwise have only low activity toward FUT8 (Fig. 1B) To test this hypothesis, we examined different substrates, including the Man5GlcNAc2 glycoforms of EPO and ribonuclease B (EPOM5 and Rb-M5), HIV-1 V3 glycopeptides carrying a Man5GlcNAc2 or a complex type N-glycan (V3-M5 and V3-CT), free M5, and Fmoc-labeled Man5GlcNAc2-Asn (M5Asn-Fmoc). FUT8 was found to fucosylate the Man5GlcNAc2-Asn-Fmoc and even the complex type N-glycan in the context of the V3 peptide, albeit at relatively low efficiency
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