Inactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans
[en] Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.
Research center :
Laboratoire de spectrométrie de masse - MSLab - ULiege Louvain Institute of Biomolecular Science and Technology - UCLouvain
Herman, Xavier; Université Catholique de Louvain - UCL > Louvain Institute of Biomolecular Science and Technology
Far, Johann ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie analytique inorganique
Courtoy, Adeline; Université Catholique de Louvain - UCL > Louvain Institute of Biomolecular Science and Technology
Bouhon, Laurent; Université Catholique de Louvain - UCL > Louvain Institute of Biomolecular Science and Technology
Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique
De Pauw, Edwin ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie analytique inorganique
Chaumont, François; Université Catholique de Louvain - UCL > Louvain Institute of Biomolecular Science and Technology
Navarre, Catherine; Université Catholique de Louvain - UCL > Louvain Institute of Biomolecular Science and Technology
Language :
English
Title :
Inactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans
Service Public de Wallonie DGO6 - WALInnov-Glycocell-1810010 Formation à la Recherche dans l’Industrie et l’Agriculture (FRIA/FNRS) European Union’s Horizon 2020 Research and Innovation Program under grant agreement No. 731077 (EU FT-ICR MS project, INFRAIA-02-2017) European Union and Wallonia program FEDER BIOMED HUB Technology Support (No. 2.2.1/996) European Union’s Horizon 2020 program (EURLipids Interreg Eurogio Meuse-Rhine)
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