Human fucosyltransferase IX: Specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization

Abstract

The α3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an α3 linkage onto terminal type II (Galβ4GlcNAc) acceptors, the final step in the biosynthesis of the Lewis x (Le x) epitope, in neurons. In this work, FUT9 cloned from NT2N neurons and overexpressed in HeLa cells (FUT9wt), was found to efficiently fucosylate asialoerythropoietin (asialoEPO), and bovine asialofetuin, but not sialylated EPO. Analysis by HPAEC-PAD and MALDI/TOF-MS revealed predominantly mono-fucosylation by FUT9wt of type II di-, tri- and tetraantennary N-glycans with proximal fucose, with and without N-acetylactosamine repeats from asialoEPO. Minor amounts of difucosylated structures were also found. The results suggested that FUT9 could fucosylate Le x carrier-glycoproteins in neurons. Furthermore, FUT9wt was found to be activated by Mn 2+ and it was capable of synthesizing Le a, although to a lesser extent than Le x and Le y. In vivo, HeLa cells transfected with FUT9wt expressed de novo Le x, as detected by immunofluorescence microscopy. FUT9 was found to be a trans-Golgi and trans-Golgi network (TGN) glycosyltransferase from confocal immunofluorescence co-localization with the markers of the secretory pathway β4-galactosyltransferase ( trans-Golgi and TGN) and TGN-46 (TGN). Deletion of the cytoplasmic domain caused a shift to the cis-Golgi, thus suggesting that information for intra-Golgi localization is contained within the cytoplasmic domain.