Discussion

In this study, in order to characterize oxidative stress response in CHO cells, we have modulated the GSH synthesis through two approaches: by the reduction of cysteine supply and by the inhibition of GSH biosynthesis. Reducing cysteine concentration in the feed by 50% did not appear to influence cell growth, but led to a decrease of 24% in titer and of 50% in intracellular glutathione. However, inhibition of GCL activity by BSO led to significant depletion of GSH as well as a reduced cell growth and titer. In addition to classical physiological characterization and metabolite profiling, proteomics was performed at different stages of the culture. Interestingly, no significant differences in protein expression were observed in the reduced cysteine feed condition, whereas 63 proteins displayed different expression levels in the BSO-treated conditions suggesting an adaptation to oxidative stress.
In a recent study, it was shown that a decrease of cysteine supply led to a depletion of glutathione (Ali et al., 2019). As a consequence, the oxidative stress generated led to cell death, titer decrease and differential expression of proteins. In our study, the decrease of cysteine supply did not lead to intracellular GSH depletion. Therefore, we did not observe cell death and significant protein differential expression (adjusted p value<0.05, LogFC threshold : 0.5), while specific productivity was substantially reduced. Indeed, a clear correlation over time between specific productivity and GSH intracellular concentration was observed. It can thus be suggested that in response to a reduced cysteine supply and as a consequence a reduce GSH availability, the cell metabolism initially decreases the recombinant protein production to reduce ROS production. The regulation of this phenomena can be due to a differential expression of non-detected host cell proteins such as transcription factors. It could also be due to other regulatory mechanisms such as protein phosphorylations, which are not detected in this type of proteomic analysis.
Partial inhibition of GCL using BSO has been suggested as a selection system to enrich for a cell population with higher productivity (Feary et al., 2017). In our case, specific productivity is not increased by the BSO treatment, because the concentration used appeared to fully inhibit the GCL enzyme. However, the increase in cell diameter and the overexpression of GCLc and GCLm were consistent with previous observations (Feary et al., 2017). Interestingly, in the present study, GCLm was more overexpressed than GCLc in BSO-treated cells. In the control condition, GCLm expression decreases over time following the intracellular GSH increase. However, a constitutive expression of GCLc was observed (Figure S3). Despite a likely higher level of GCLc protein in CHO cells, it looks as though dynamic expression of GCLm is the most important parameter in the regulation of de novo synthesis of GSH during the process. In a previous study in CHO cells, it has been demonstrated that up-regulation of GCLm by cell engineering increase GSH content, but surprisingly not the GCL activity (Orellana et al., 2017). In the present study, in the presence of BSO, CHO cells try to compensate for GCL inhibition by producing more GCLm. However, this response was not able to restore normal intracellular GSH levels in our experimental conditions. As cystine cannot be used for GSH synthesis, it is possible that the potential secretion observed from day 8 happened to avoid accumulation of this amino acid in the cell. Indeed, cysteine/cystine accumulation can potentially influence the intracellular redox potential.
In addition to the attempt to up-regulate GSH production when inhibited by BSO, cells recycled GSH through the overexpression ofS -formylglutathione and GSH reductase. Likewise, GSH catabolism through the gamma-glutamyl cyclotransferase was down-regulated. Interestingly, the GST Mu enzymes 1, 5 and 6 were down-regulated. However other GSTs from other families (omega, alpha and pi) have been detected and were not interpreted as down-regulated. This can be due to the difference of substrate selectivity. For example, GSTs Mu are in general more efficient for nucleophilic aromatic substitution and less selective than GSTs Alpha (Eaton et al., 1999; Salinas et al., 1999). GSTs Alpha is the only family able to reduce hydroperoxides. They are also involved in lipid peroxidation by product detoxification such as Acrolein and 4-hydroxy-2-nonenal (Stevens et al., 2008; Yang et al., 2016). Moreover, some GSTs can have additional activities to conjugation such as GST Pi 1 which can bind to c-Jun N-terminal kinase (JNK) and GST Mu 1 which can bind to apoptosis signal-regulating kinase 1 (ASK1), and modulate apoptosis signaling pathways (Allocati et al., 2018; Armstrong, 2010).
Beside GSH metabolism, other responses to oxidative stress were observed. The main one was the overexpression of the heme oxygenase 1 already observed on day 6 and amplified on day 10. Increase of the free heme detoxification is usually observed under oxidative stress (Gozzelino et al., 2010). However, we also observed an up-regulation of the transferrin receptor protein 1 and the 5-aminolevulinate (ALA) synthase. The first is involved in iron transport and the second is the rate-limiting enzyme in heme synthesis. However, there is an inconsistency with the regulation of heme biosynthesis described in literature as ALA synthase is usually down-regulated when the heme oxygenase 1 is up-regulated (Ajioka et al., 2006; Fujii et al., 2004).
The heme oxygenase 1 gene expression is regulated by the Nuclear factor E2-related factor 2 (Nrf2). This factor is retained in the cytoplasm through a complex with Keap1 under normal conditions. Under oxidative stress, it is translocated to the nucleus and binds to the antioxidant response element (ARE). The overexpression of Gclm and heme oxygenase 1 in the BSO-treated cells suggests an activation of the Keap1-Nrf2 pathway. Moreover Sequestosome 1, also called p62, is also overexpressed (Supplementary material 1). This proteins is known to compete with Nrf2 for the interaction with Keap1 leading to a stabilization of free Nrf2 (Wei et al., 2019). Others proteins related to the antioxidant defense (catalase, superoxide dismutase [Mn], thioredoxin 1, glutathione reductase ) and NADPH regeneration through the oxPPP pathway (Glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconate dehydrogenase) have been measured as differentially expressed in the BSO-treated cells, but with a lower logFC magnitude (Tonelli et al., 2018) (Supplementary Table 1). This observation can support the hypothesis of an Nrf2 activation. One of the limitations with this explanation is that GSTs Mu genes have also been described as Nrf2 target and are significantly down-regulated.
Another response to oxidative stress is the down-regulation of intracellular ROS production. The main source of ROS within the cell is the respiratory chain (Turrens, 2003). In this context, mitochondria proteins should be the main targets of activity reduction. The general down-regulation of proteins involved in the oxidative phosphorylation and the TCA cycle observed in BSO conditions confirm this hypothesis. Acyl-CoA synthetase family member 2, involved in the activation of fatty acid is also down-regulated (LogFC =-0.79, adj. p. value=1.11x1010). This observation suggested a decrease of Acetyl CoA supply to the TCA through the beta oxidation pathway. This hypothesis was supported by the down-regulation of other enzymes involved in the beta oxidation process such as the Carnitine O-palmitoyltransferase 2 or the Acetyl-CoA acetyltransferase (Supplementary table 1). Consequently, carbon fluxes through the TCA cycle were reduced and pyruvate accumulated in the cells. Indeed, glycolysis enzymes were not down-regulated and the glucose uptake was constant. Other enzymes involved in pyruvate production such as the malic enzyme [NAD] were down-regulated. Therefore, pyruvate surplus was converted to lactate and alanine which are produced from day 6 in BSO-treated cells.
Another down-regulated process in response to glutathione depletion was lipid metabolism and especially the cholesterol de novo synthesis pathway. Cholesterol plays a major role in membrane fluidity regulation. Moreover, cholesterol regulation may also play a role on protein secretion reduction as it is an essential building block of secretion vesicles (Wang et al., 2000). Recently, it has been shown that the increase of cholesterol synthesis with the up-regulation of a micro RNA can increase the productivity of CHO cell lines by increasing their secretion capacity (Loh et al., 2017). It is then possible that the increase of productivity and glutathione content observed over time during the process are also link to cholesterol regulation.
One hypothesis that can be proposed to explain the down-regulation of cholesterol synthesis under glutathione depletion is the accumulation of oxysterols in the ER. The expression of enzymes involved in cholesterol synthesis is regulated by a common transcription factor SREBP2. SERBPs are retained in the ER membrane by forming a complex with the SERBP cleavage-activating protein (SCAP) and the insulin-induced gene protein (Insig). The retention of the complex is controlled by cholesterol and by oxysterol concentrations (Howe et al., 2016). As oxysterol is a byproduct of cholesterol biosynthesis, it is a signal for cholesterol overproduction for the cell. Oxysterols can be enzymatically derived, especially by the cytochrome P450 reductase, or direct products of cholesterol autoxidation (Olkkonen et al., 2012). Hence, it could be hypothesized that the BSO treatment led to an increase of oxysterols in the ER (Micheletta et al., 2006).
Another possible explanation is that the reduction of the cholesterol synthesis could be an attempt to decrease the use of NADPH. Indeed, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase use two NADPH molecules to reduce HMG-CoA to mevalonate (Burg et al., 2011). Moreover, NADPH electrons are transferred by the cytochrome P450 reductase to squalene monooxygenase and lanosterol demethylase during cholesterol synthesis (Porter, 2015). In total, four NADPH molecules are used to produce one cholesterol molecule from acetyl-CoA, which is stoichiometrically a lot. Decreasing this activity may also help to maintain NADPH/NADP+ redox homeostasis and indirectly counteract oxidative stress.
Another hypothesis is the down-regulation of cholesterol to favorize GSH import in the mitochondria. Indeed, cholesterol has been reported as a mitochondrial GSH transport regulator (Ribas et al., 2016). Accumulation of cholesterol in the mitochondria membrane has been shown to impair the activity of some membrane proteins such as the 2-oxoglutarate carrier which exports 2-oxoglutarate in the cytosol in exchange of the import of GSH in the mitochondria. Moreover, it has been shown that accumulation of mitochondrial cholesterol can damage the respiratory chain complexes assembly (Solsona-Vilarrasa et al., 2019). Under GSH depletion, the cells potentially tried to stabilize the mitochondria membrane and favorize GSH import in the mitochondria matrix by lowering cholesterol.
We showed that reducing GSH intracellular content by half led to a decrease productivity of heterologous protein production despite a modest number of changes in the host cell protein expression profile. However, GSH depletion resulted in an adaptation of GSH metabolism and triggered an oxidative stress response. In addition, cells died and recombinant protein was completely stopped. Thanks to these extreme conditions, this study have lighted up that the modulation GSH thanks to BSO also impacted lipid biosynthesis, especially cholesterol which play a role in protein secretion. Thus, in order to finally figure out how glutathione metabolism is linked to productivity , further work should include a control of cholesterol metabolism.