Tabacum Bright Yellow-2 Cells Research Articles

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.

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.

Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells.

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

The proteome complement of Nicotiana tabacum Bright‐Yellow‐2 culture cells

The Nicotiana tabacum Bright-Yellow-2 (BY2) cell line is one of most commonly used plant suspension cell lines and offers interesting properties, such as fast growth, amenability to genetic transformation, and synchronization of cell division. To build a proteome reference map of BY2 cell proteins, we isolated the soluble proteins from N. tabacum BY2 cells at the end of the exponential growth phase and analyzed them by 2-DE and MALDI TOF-TOF. Of the 1422 spots isolated, 795 were identified with a significant score, corresponding to 532 distinct proteins.

Exploring the soluble proteome of TBY-2 cells at the switch towards different cell fates in response to heat shocks

Tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells undergo different fates when exposed for 10 minutes to heat stresses of different severity. A 35 degrees C treatment causes a homeostatic response (HRE) allowing cells to cope with the stress; 55 degrees C triggers processes leading to programmed cell death (PCD), which is complete after 72 h. We have used a proteomic approach to gain insight into the molecular mechanisms defining the fate of TBY-2 cells induced by these two heat stresses. Tandem mass spectrometry (MS/MS) and two-dimensional electrophoresis (2-DE) analysis revealed little overlap of differentially-accumulated proteins: the different severities of heat treatment induced the modulation of specific proteins, some of which are responsible for different cell fates. When the imposed heat shock is beyond a certain threshold, the overall reduced metabolism may be the result of a series of events involving gene expression and oxidative damage that would lead to PCD. Our data suggest that the down-accumulation of several proteins involved in cellular redox homeostasis could provide, until now, an unappreciated contribution to understanding how many partners are involved in promoting the redox impairment leading to PCD. Moreover post-translational modifications seem to play important regulatory roles in the adaptation of TBY-2 cells to different intensities of heat stress.

Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death

An increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H(2)O(2) during time, or directly with known amounts of H(2)O(2). Data presented here indicate that, in TBY-2 cells, a difference in H(2)O(2) level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H(2)O(2)-dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H(2)O(2) generation. Under a threshold value of H(2)O(2) overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD.