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

Abstract

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.

The advent of plant cells in bioreactors

“…A similar strategy has been developed in plant cells. Efforts have included the inactivation of a fucosyl transferase and a glycosil transferase to obtain human-like glycosylation ( Mercx et al., 2017 ; Smargiasso et al., 2019 ; Herman et al., 2021 ). The presence of endogenous proteases also decreases both quality and quantity of protein production and RNAi strategies have been used to downregulate endogenous proteases with some success ( Mandal et al., 2014 ).…”

Impact of Specific N-Glycan Modifications on the Use of Plant-Produced SARS-CoV-2 Antigens in Serological Assays

“…The inhibitor constitutes one way to circumvent given issues and can be used to avoid false positives with sera from allergic individuals sensitized to CCDs ( Aberer et al, 2017 ). Moreover, it is now possible to produce viral antigens such as RBD in genome edited plants or plant cells that are completely devoid of β1,2-xylose and α1,3-fucose ( Hanania et al, 2017 ; Mercx et al, 2017 ; Jansing et al, 2019 ; Herman et al, 2021 ). Alternatively, RBD and other glycosylated viral antigens can be manufactured in the presence of a pharmacological glycosylation inhibitor such as kifunensine which blocks α-mannose trimming from oligomannosidic N -glycans and thus the formation of processed complex N -glycans ( Xiong et al, 2018 ; Kommineni et al, 2019 ; Shin et al, 2021 ).…”

In vivo deglycosylation of recombinant glycoproteins in tobacco BY‐2 cells

“…Nicotiana tabacum BY-2 cell culture and transformation BY-2 cells were grown as described in Herman et al (2021). D11b cultures (Vasilev et al, 2013) were grown for 10 days in 50 mL of medium without cyclodextrin, in 250 mL Erlenmeyer flasks.…”

“…Simultaneously, to ensure correct identification of glycopeptides, a fraction of some samples (GnTI-KO-IgG2 #25, GnTI-KO-EndoT-IgG2 #9 and RNase B) was deglycosylated according to the procedure described by Smargiasso et al (2019) and Herman et al (2021). These fractions were then submitted to the MELD followed by LC-MS.…”