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. 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.

Keywords: CHO cells; buthionine sulfoximine; cholesterol; glutathione; proteomics.

Figure 1

Effect of BSO treatment on cell growth, productivity, and GSH content. Gray and blue indicates the feed medium used—control feed medium or low cysteine feed medium, respectively. Circles indicate nontreated bioreactors and triangles indicate BSO‐treated bioreactors. The red dotted lines represent the timing of BSO addition to a medium concentration of 0.5 mM. (a) viable cell concentration (VCC) profile. (b) Product titer in the supernatant over time. (c) Intracellular GSH concentration overtime. (d) mAb specific productivity over time. BSO, buthionine sulfoximine; GSH, glutathione; mAb, monoclonal antibody [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Effect of BSO treatment on glucose, lactate, and alanine uptake/secretion rates. Gray and blue indicates the feed medium used—control feed medium or low cysteine feed medium, respectively. Circles indicate nontreated bioreactors and triangles indicate BSO‐treated bioreactors. The red dotted lines represent the timing of BSO addition to a medium concentration of 0.5 mM. (a) Glucose rate. (b) Lactate rate. (c) Alanine rate. BSO, buthionine sulfoximine [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Heatmap of differentially expressed proteins under BSO treatment in CHO cells. Main cellular function of proteins cluster has been added in the figure. BSO, buthionine sulfoximine; CHO, Chinese hamster ovary; HMG‐CoA, 3‐hydroxy‐3‐methylglutaryl coenzyme A; TCA, tricarboxylic acid [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Glutathione metabolism‐related proteins expression in BSO‐treated cells. The logFC and the adj. p value are represented by the color from green to red. Red indicates a logFC > 0.5 and an adj. p < .05; light red indicates logFC < 0.5 and an adj. p < .05; green indicates a logFC < −0.5 and an adj. p < .05; light green indicates logFC > −0.5 and an adj. p < .05 and gray adj. p > .05. Statistical data have been generated using limma empirical Bayes moderated t test. The BSO‐treated condition (n = 10) have been compared to control conditions (n = 12). BSO, buthionine sulfoximine; GCLc, glutamate–cysteine ligase; GCLm, glutamate–cysteine ligase modifier; GSH, glutathione; GSSG, glutathione disulfide; logFC, log fold change [Color figure can be viewed at wileyonlinelibrary.com]