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

G.J.A. Rouwendal, M. Wuhrer, D.E.A. Florack, C.A.M. Koeleman, A.M. Deelder, H. Bakker, G.M. Stoopen, I. van Die, J.P.F.G. Helsper, C.H. Hokke, H.J. Bosch

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Abstract

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.
Original languageEnglish
Pages (from-to)334-344
JournalGlycobiology
Volume17
Issue number3
DOIs
Publication statusPublished - 2007

Keywords

  • asparagine-linked oligosaccharides
  • glycoprotein-synthesis
  • substrate-specificity
  • cdna cloning
  • hen oviduct
  • developmental-stage
  • schistosoma-mansoni
  • mass-spectrometry
  • transgenic plants
  • beta-1-4 linkage

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