Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III.
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.
Rouwendal
Gerard J A
GJ
Business Unit Bioscience, Plant Research International BV, Wageningen University and Research Centre, Droevendaalsesteeg 1 6708 PB Wageningen, The Netherlands. gerard.rouwendal@wur.nl
Wuhrer
Manfred
M
Florack
Dion E A
DE
Koeleman
Carolien A M
CA
Deelder
André M
AM
Bakker
Hans
H
Stoopen
Geert M
GM
van Die
Irma
I
Helsper
Johannes P F G
JP
Hokke
Cornelis H
CH
Bosch
Dirk
D
D000117:Acetylglucosamine / Q000032:analysis / Q000378:metabolism*; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D019076:Transgenes
D000906:Antibodies; D011134:Polysaccharides; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine