Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.
Valentine ChevallierErwin M SchoofLaetitia MalphettesMikael Rørdam AndersenChristopher Thomas WorkmanPublished in: Biotechnology and bioengineering (2020)
Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH-related proteome has been observed with an upregulation of the regulatory subunit of glutamate-cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.
Keyphrases
- fluorescent probe
- induced apoptosis
- living cells
- cell cycle arrest
- signaling pathway
- cell proliferation
- climate change
- endoplasmic reticulum stress
- heavy metals
- wastewater treatment
- randomized controlled trial
- cell death
- cell therapy
- ms ms
- oxidative stress
- low density lipoprotein
- risk assessment
- single cell
- poor prognosis
- mesenchymal stem cells
- study protocol
- combination therapy