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Glycobiology Advance Access published online on May 3, 2006
Glycobiology, 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
Received March 6, 2006
Revised April 20, 2006
Accepted April 20, 2006

Article

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

Max Crispin 1 *, David J. Harvey 2, Veronica T. Chang 3, Chao Yu 3, A. Radu Aricescu 4, E. Yvonne Jones 4, Simon J. Davis 3, Raymond A. Dwek 2, and Pauline M. Rudd 2

1 Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford OX3 7BN, UK
2 Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
3 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
4 Cancer Research UK Receptor Structure Research Group, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford OX3 7BN, UK

* To whom correspondence should be addressed.
Max Crispin, E-mail: max{at}strubi.ox.ac.uk


  Abstract

A mammalian 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 mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S or Chinese hamster ovary cells (CHO) Lec3.2.8.1 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, 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.

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