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Glycobiology Advance Access originally published online on May 3, 2006
Glycobiology 2006 16(8):748-756; doi:10.1093/glycob/cwj119
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

Max Crispin1–3, David J. Harvey2, Veronica T. Chang5, Chao Yu5, A. Radu Aricescu4, E. Yvonne Jones4, Simon J. Davis5, Raymond A. Dwek2 and Pauline M. Rudd2

3 Division of Structural Biology and 4 Cancer Research UK Receptor Structure Research Group, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford OX3 7BN, UK; 5 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK

1 To whom correspondence should be addressed; e-mail: max{at}strubi.ox.ac.uk

2 Present address: Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Received on March 6, 2006; revised on April 20, 2006; accepted on April 20, 2006

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the {alpha}-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of {alpha}-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal {alpha}1–2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi {alpha}-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.

Key words: electrospray ionization mass spectrometry / fucosyltransferase / matrix-assisted laser desorption / ionization (MALDI) / N-linked glycosylation


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