Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

Abstract

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. The study of industrial CHO cell lines have demonstrated a close link between GSH metabolism and clone productivity. However, a deep investigation is still required to understand this correlation and highlights new potential targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions in order 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). Cysteine supply modulation have revealed a correlation between intracellular GSH and product titer over time. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and non-treated cells has highlighted a slow down of the TCA cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the glutathione related proteome has been observed with a up-regulation of the regulatory subunit of glutamate cysteine ligase and a down-regulation of a specific glutathione transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global down-regulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it can be the missing part of the jigsaw to finally elucidate the link between GSH synthesis and productivity.