AUTHOR=Herman Xavier , Far Johann , Courtoy Adeline , Bouhon Laurent , Quinton Loïc , De Pauw Edwin , Chaumont François , Navarre Catherine 

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

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.634023

DOI=10.3389/fpls.2021.634023

ISSN=1664-462X

ABSTRACT=<p><italic>Nicotiana tabacum</italic> 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 <italic>N</italic>-glycans. However, <italic>N</italic>-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 <italic>N</italic>-glycans was obtained using CRISPR/Cas9 edition of two <italic>N-acetylglucosaminyltransferase I (GnTI)</italic> genes, whose activity is a prerequisite for the formation of all complex <italic>N</italic>-glycans. The suppression of complex <italic>N</italic>-glycans in the <italic>GnTI</italic>-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 <italic>GnTI</italic> and α<italic>1,3-fucosyltransferase</italic> (<italic>FucT</italic>) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the <italic>GnTI/FucT</italic>-KO cell lines. Absence of complex <italic>N</italic>-glycans on secreted glycoproteins of <italic>GnTI</italic>-KO and <italic>GnTI/FucT</italic>-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose <italic>N</italic>-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of <italic>N</italic>-glycan homogeneity and the high-mannose <italic>N</italic>-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.</p>