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.
- asparagine-linked oligosaccharides
- cdna cloning
- hen oviduct
- transgenic plants
- beta-1-4 linkage