Research paper Human fucosyltransferase IX: Specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization C. Brito a , S. Kandzia b , T. Grac ¸a b , H.S. Conradt b , J. Costa a,c, * a Instituto de Tecnologia Quı ´mica e Biolo ´gica, Avenida da Repu ´ blica, 2780-157 Oeiras, Portugal b GlycoThera GmbH, Inhoffenstrasse 7, D-38124 Braunschweig, Germany c Instituto de Biologia Experimental e Tecnolo ´ gica, Apartado 12, 2781-901 Oeiras, Portugal Received 7 December 2007; accepted 5 March 2008 Available online 19 March 2008 Abstract The a3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an a3 linkage onto terminal type II (Galb4GlcNAc) 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 2þ 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 ex- pressed 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 b4-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. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Fucosyltransferase IX; Lewis x ; N-Glycans; Erythropoietin; Golgi localization 1. Introduction Glycosyltransferases (GTs) mediate the transfer of mono- saccharide residues from nucleotide sugar donors to glyco- conjugates along their transport in the secretory pathway. a3-Fucosyltransferases (a3-FUTs) catalyse the transfer of fucose in an a3 linkage, onto terminal Galb4GlcNAc motifs in the oligosaccharide chains, the final step in the biosynthesis of the Lewis x (Le x ) epitope. FUT3, FUT4, FUT5, FUT6, FUT7 and FUT9 constitute the human a3/4-FUT family (reviewed in [1]), presenting the type II membrane topology characteristics of Golgi resident GTs: a short N-terminal cytosolic tail, a single transmembrane domain, a flexible region adjacent to the trans- membrane domain called stem and a large C-terminal globular catalytic domain, facing the lumen of the Golgi [2]. FUT10 and FUT11 are possible a3/4-FUTs based on their homology with Abbreviations: AF, asialofetuin; AP-1, adaptor protein 1; BFA, brefeldin A; CNS, central nervous system; dHex, deoxyhexose; EPO, erythropoietin; FUT9, fucosyltransferase IX; FUT, fucosyltransferase; GT, glycosyltransfer- ase; Hex, hexose; HexNAc, N-acetylhexosamine; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection; LacNAc, N- acetylactosamine; Le a , Lewis a ; Le x , Lewis x ; Le y , Lewis y ; MALDI/TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MTOC, microtubule organizing centre; sLacNAc, sialyl-N-acetylactosamine; sLe x , sialyl-Lewis x ; TFA, trifluoroacetic acid; TGN, trans-Golgi network; TX-100, Triton X-100. * Corresponding author. Laboratory of Glycobiology, Instituto de Tecnologia Quı ´mica e Biolo ´gica, Avenida da Repu ´blica, Apartado 127, 2781-901 Oeiras, Portugal. Tel.: þ351 214 469 437; fax: þ351 214 411 277. E-mail address: jcosta@itqb.unl.pt (J. Costa). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.03.002 Available online at www.sciencedirect.com Biochimie 90 (2008) 1279e1290 www.elsevier.com/locate/biochi