Human fucosyltransferase IX: specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization.
The alpha3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an alpha3 linkage onto terminal type II (Galbeta4GlcNAc) acceptors, the final step in the biosynthesis of the Lewisx (Lex) 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 Lex carrier-glycoproteins in neurons. Furthermore, FUT9wt was found to be activated by Mn2+ and it was capable of synthesizing Lea, although to a lesser extent than Lex and Ley. In vivo, HeLa cells transfected with FUT9wt expressed de novo Lex, 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 beta4-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.
Brito
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Instituto de Tecnologia Química e Biológica, Avenida da República, 2780-157 Oeiras, Portugal.
Kandzia
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Graça
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Conradt
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Costa
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D000595:Amino Acid Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D016256:Lewis X Antigen / Q000096:biosynthesis; D009154:Mutation / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D006023:Glycoproteins; D016256:Lewis X Antigen; D011134:Polysaccharides; C519500:FUT9 protein, human; D005647:Fucosyltransferases