Glycobiology Advance Access originally published online on December 19, 2006
Glycobiology 2007 17(3):334-344; doi:10.1093/glycob/cwl078
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Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III
3 Business Unit Bioscience, Plant Research International B.V., Wageningen University and Research Centre, Droevendaalsesteeg 1 6708 PB Wageningen, The Netherlands
4 Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
5 Glycoimmunology Group, Department of Molecular Cell Biology and Immunology, VU Medical Center, van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands
6 Membrane Enzymology, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
1 To whom correspondence should be addressed; 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 human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants 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, profiling was performed using normal-phase high-performance liquid chromatography 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 N-acetylglucosamine (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 / MALDI / N-glycosylation
2 Present address: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover, Germany.