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.