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

被引:48
|
作者
Rouwendal, Gerard J. A.
Wuhrer, Manfred
Florack, Dion E. A.
Koeleman, Carolien A. M.
Deelder, Andre M.
Bakker, Hans
Stoopen, Geert M.
van Die, Irma
Helsper, Johannes P. F. G.
Hokke, Cornelis H.
Bosch, Dirk
机构
[1] Univ Wageningen & Res Ctr, Plant Res Int BV, Business Unit Biosci, NL-6708 PB Wageningen, Netherlands
[2] Leiden Univ, Med Ctr, Dept Parasitol, Biomol Mass Spect Unit, NL-2333 ZA Leiden, Netherlands
[3] Free Univ Amsterdam, Dept Mol Cell Biol & Immunol, Glycoimmunol Grp, Med Ctr, NL-1081 BT Amsterdam, Netherlands
[4] Univ Utrecht, Dept Chem, Membrane Enzymol, NL-3584 CH Utrecht, Netherlands
关键词
monoclonal antibody; electrospray ionization mass spectrometry; MALDI; N-glycosylation;
D O I
10.1093/glycob/cwl078
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:334 / 344
页数:11
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