1. Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III
- Author
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Carolien A. M. Koeleman, Hans Bakker, Irma van Die, Dion E.A. Florack, Dirk Bosch, Geert Stoopen, André M. Deelder, Gerard J. A. Rouwendal, Johannes P. F. G. Helsper, Manfred Wuhrer, and Cornelis H. Hokke
- Subjects
Glycan ,medicine.drug_class ,Electrospray ionization ,Transgene ,Molecular Sequence Data ,Gene Expression ,Genetically modified crops ,transgenic plants ,Monoclonal antibody ,N-Acetylglucosaminyltransferases ,Biochemistry ,Antibodies ,Acetylglucosamine ,beta-1-4 linkage ,N-linked glycosylation ,Polysaccharides ,Tobacco ,medicine ,Humans ,Corporate Staff ,Transgenes ,Concernstaf ,glycoprotein-synthesis ,Gene ,chemistry.chemical_classification ,hen oviduct ,biology ,asparagine-linked oligosaccharides ,Chemistry ,mass-spectrometry ,Plants, Genetically Modified ,carbohydrates (lipids) ,PRI Bioscience ,schistosoma-mansoni ,substrate-specificity ,Carbohydrate Sequence ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,cdna cloning ,biology.protein ,developmental-stage ,Glycoprotein - Abstract
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.
- Published
- 2007
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