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Glycobiology Advance Access published online on December 19, 2006
Glycobiology, doi:10.1093/glycob/cwl078
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III

Gerard J.A. Rouwendal1,*, Manfred Wuhrer3, Dion E.A. Florack1, Carolien A. M. Koeleman3, André M. Deelder3, Hans Bakker1,2, Geert M. Stoopen1, Irma van Die4, Johannes P.F.G. Helsper1, Cornelis H. Hokke3 and Dirk Bosch1,5

1 Business Unit Bioscience, Plant Research International B.V., Wageningen University and Research Centre, Droevendaalsesteeg 1 6708 PB Wageningen, The Netherlands
3 Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
4 Glycoimmunology Group, Department of Molecular Cell Biology and Immunology, VU Medical Center, van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands
5 Membrane Enzymology, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands

* To whom correspondence should be addressed: Tel: 31 (0)317 477144 Fax: 31 (0)317 418094 E-mail: gerard.rouwendal{at}wur.nl

Received on October 20, 2006; revised on December 1, 2006; accepted on December 5, 2006

In this study we show that introduction of the human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide (2-AB), profiling was performed using normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reverse-phase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal GlcNAc residues in contrast to wild-type plants where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.

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

2 Current adress: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover, Germany


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