Abstract
Introduction
Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.
Objectives
The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.
Methods
To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.
Results
Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.
Conclusion
Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.
Similar content being viewed by others
References
Agrawal, N., Dasaradhi, P. V., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA interference: Biology, mechanism, and applications. Microbiology Molecular Biology Review, 67, 657–685.
Akashi, H., & Gojobori, T. (2002). Metabolic efficiency and amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis. Proceedings of the National Academy of Sciences, 99, 3695–3700.
Altamirano, C., Berrios, J., Vergara, M., & Becerra, S. (2013). Advances in improving mammalian cells metabolism for recombinant protein production. Electronic Journal of Biotechnology 16(3), 10
Andersen, K. B., & Von Meyenburg, K. (1977). Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli. The Journal of Biological Chemistry, 252, 4151–4156.
Atkinson, D. E. (1968). Energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry, 7, 4030–4034.
Begley, P., Francis-Mcintyre, S., Dunn, W. B., Broadhurst, D. I., Halsall, A., Tseng, A., et al. (2009). Development and performance of a gas chromatography–time-of-flight mass spectrometry analysis for large-scale nontargeted metabolomic studies of human serum. Analytical Chemistry, 81, 7038–7046.
Beinert, H., Kennedy, M. C., & Stout, C. D. (1996). Aconitase as iron–sulfur protein, enzyme, and iron-regulatory protein. Chemical Reviews, 96, 2335–2374.
Bordag, N., Janakiraman, V., Nachtigall, J., González Maldonado, S., Bethan, B., Laine, J.-P., et al. (2016). Fast filtration of bacterial or mammalian suspension cell cultures for optimal metabolomics results. PLoS ONE, 11, e0159389.
Breusch, F. L. (1937). Citric acid in tissue metabolism. Journal Physiological Chemistry, 250, 262–280.
Brezinsky, S. C. G., Chiang, G. G., Szilvasi, A., Mohan, S., Shapiro, R. I., Maclean, A., et al. (2003). A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. Journal of Immunological Methods, 277, 141–155.
Burdon, R. H., Alliangana, D., & Gill, V. (1995). Hydrogen peroxide and the proliferation of BHK-21 cells. Free Radical Research, 23, 471–486.
Cairns, R. A., Harris, I. S., & Mak, T. W. (2011). Regulation of cancer cell metabolism. Nature Reviews Cancer, 11, 85–95.
Cantu, D., Fulton, R. E., Drechsel, D. A., & Patel, M. (2011). Mitochondrial aconitase knockdown attenuates paraquat-induced dopaminergic cell death via decreased cellular metabolism and release of iron and H2O2. Journal of Neurochemistry, 118, 79–92.
Cantu, D., Schaack, J., & Patel, M. (2009). Oxidative inactivation of mitochondrial aconitase results in iron and H2O2-mediated neurotoxicity in rat primary mesencephalic cultures. PLoS ONE, 4, e7095.
Chan, C. Y., Carmack, C. S., Long, D. D., Maliyekkel, A., Shao, Y., Roninson, I. B., & Ding, Y. (2009). A structural interpretation of the effect of GC-content on efficiency of RNA interference. BMC Bioinformatics, 10, S33–S33.
Chevaillier, P. (1993). Pest sequences in nuclear proteins. International Journal of Biochemistry, 25, 479–482.
Clay, H. B., Parl, A. K., Mitchell, S. L., Singh, L., Bell, L. N., & Murdock, D. G. (2016). Altering the mitochondrial fatty acid synthesis (mtFASII) pathway modulates cellular metabolic states and bioactive lipid profiles as revealed by metabolomic profiling. PLoS ONE, 11, e0151171.
Cooper, G. M. (2000). Metabolic energy. In The cell: A molecular approach (2nd ed.). Sunderland, MA: Sinauer Associates.
Costello, L. C., & Franklin, R. B. (2013). A review of the important central role of altered citrate metabolism during the process of stem cell differentiation. Journal of Regenerative Medicine & Tissue Engineering, 2, 1.
Da Veiga Moreira, J., Peres, S., Steyaert, J.-M., Bigan, E., Paulevé, L., Nogueira, M. L. et al. (2015). Cell cycle progression is regulated by intertwined redox oscillators. Theoretical Biology and Medical Modelling, 12, 10.
Deberardinis, R. J., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S., & Thompson, C. B. (2007). Beyond aerobic glycolysis: Transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proceedings of the National Academy of Sciences of the United States of America, 104, 19345–19350.
Dietmair, S., Hodson, M. P., Quek, L. E., Timmins, N. E., Chrysanthopoulos, P., Jacob, S. S., et al. (2012a). Metabolite profiling of CHO cells with different growth characteristics. Biotechnology and bioengineering, 109, 1404–1414.
Dietmair, S., Hodson, M. P., Quek, L.-E., Timmins, N. E., Gray, P., & Nielsen, L. K. (2012b). A multi-omics analysis of recombinant protein production in Hek293 Cells. PLoS ONE, 7, e43394.
Ding, Y., Xu, L., Jovanovic, B. D., Helenowski, I. B., Kelly, D. L., Catalona, W. J., Yang, X. J., Pins, M., & Bergan, R. C. (2007). The methodology used to measure differential gene expression affects the outcome. Journal of Biomolecular Techniques, 18, 321–330.
Dunn, W. B., Broadhurst, D., Begley, P., Zelena, E., Francis-Mcintyre, S., Anderson, N., et al. (2011). Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nature Protocols, 6, 1060–1083.
Dykxhoorn, D. M., Novina, C. D., & Sharp, P. A. (2003). Killing the messenger: Short RNAs that silence gene expression. Nature Reviews Molecular Cell Biology, 4, 457–467.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.
Fiszer-Kierzkowska, A., Vydra, N., Wysocka-Wycisk, A., Kronekova, Z., Jarząb, M., Lisowska, K. M., et al. (2011). Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells. BMC Molecular Biology, 12, 27.
Gardner, P. R., Raineri, I., Epstein, L. B., & White, C. W. (1995). Superoxide radical and iron modulate aconitase activity in mammalian cells. Journal of Biological Chemistry, 270, 13399–13405.
Goh, D. L., Patel, A., Thomas, G. H., Salomons, G. S., Schor, D. S., Jakobs, C., et al. (2002). Characterization of the human gene encoding alpha-aminoadipate aminotransferase (AADAT). Molecular Genetics and Metabolism, 76, 172–180.
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T., & Weber, K. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. Journal of Cell Science, 114, 4557–4565.
Hardie, D. G. (2007). AMP-activated/SNF1 protein kinases: Conserved guardians of cellular energy. Nature Reviews Molecular Cell Biology, 8, 774–785.
Hardie, D. G., & Hawley, S. A. (2001). AMP-activated protein kinase: The energy charge hypothesis revisited. BioEssays, 23, 1112–1119.
Jacobsen, L. B., Calvin, S. A., & Lobenhofer, E. K. (2009). Transcriptional effects of transfection: The potential for misinterpretation of gene expression data generated from transiently transfected cells. BioTechniques, 47, 617.
Jitrapakdee, S., Maurice, M. S., Rayment, I., Cleland, W. W., Wallace, J. C., & Attwood, P. V. (2008). Structure, Mechanism and Regulation of Pyruvate Carboxylase. The Biochemical Journal, 413, 369–387.
Kanehisa, M., & Goto, S. (2000). KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Research, 28, 27–30.
Kaul, S., Sharma, S. S., & Mehta, I. K. (2008). Free radical scavenging potential of L-proline: Evidence from in vitro assays. Amino Acids, 34, 315–320.
Krebs, H. A., & Holzach, O. (1952). The conversion of citrate into cis-aconitate and isocitrate in the presence of aconitase. Biochemical Journal, 52, 527–528.
Lai, T., Yang, Y., & Ng, S. K. (2013). Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals, 6, 579–603.
León, Z., García-Cañaveras, J. C., Donato, M. T., & Lahoz, A. (2013). Mammalian cell metabolomics: Experimental design and sample preparation. Electrophoresis, 34, 2762–2775.
Li, F., Vijayasankaran, N., Shen, A., Kiss, R., & Amanullah, A. (2010). Cell culture processes for monoclonal antibody production. mAbs, 2, 466–477.
Liou, G.-Y., & Storz, P. (2010). Reactive oxygen species in cancer. Free Radical Research, 44, 479–496.
Locasale, J. W., & Cantley, L. C. (2011). Metabolic flux and the regulation of mammalian cell growth. Cell Metabolism, 14, 443–451.
Luo, J., Vijayasankaran, N., Autsen, J., Santuray, R., Hudson, T., Amanullah, A., et al. (2012). Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process. Biotechnology and Bioengineering, 109, 146–156.
Martius, C. (1937). The metabolism of citric acid. Journal Physiological Chemistry, 247, 104–110.
Mason, M., Sweeney, B., Cain, K., Stephens, P., & Sharfstein, S. (2014). Reduced culture temperature differentially affects expression and biophysical properties of monoclonal antibody variants. Antibodies, 3, 253.
Mckenna, T. (2009). Oxidative stress on mammalian cell cultures during recombinant protein expression (Doctoral dissertation, Linköping University Electronic Press).
Mulukutla, B. C., Khan, S., Lange, A., & Hu, W.-S. (2010). Glucose metabolism in mammalian cell culture: New insights for tweaking vintage pathways. Trends in Biotechnology, 28, 476–484.
Murakami, K., & Yoshino, M. (1997). Inactivation of aconitase in yeast exposed to oxidative stress. IUBMB Life, 41, 481–486.
O’donnell-Tormey, J., Nathan, C. F., Lanks, K., Deboer, C. J., & De La Harpe, J. (1987). Secretion of pyruvate. An antioxidant defense of mammalian cells. The Journal of Experimental Medicine, 165, 500–514.
Oka, S., Hsu, C. P., & Sadoshima, J. (2012). Regulation of cell survival and death by pyridine nucleotides. Circulation Research, 111, 611–627.
Ott, M., Gogvadze, V., Orrenius, S., & Zhivotovsky, B. (2007). Mitochondria, oxidative stress and cell death. Apoptosis, 12, 913–922.
Owen, O. E., Kalhan, S. C., & Hanson, R. W. (2002). The key role of anaplerosis and cataplerosis for citric acid cycle function. Journal of Biological Chemistry, 277, 30409–30412.
Ozturk, S., & Hu, W. S. (2005). Cell culture technology for pharmaceutical and cell-based therapies. CRC Press, Boca Raton.
Phang, J. M., Donald, S. P., Pandhare, J., & Liu, Y. (2008). The metabolism of proline, a stress substrate, modulates carcinogenic pathways. Amino Acids, 35, 681–690.
Quek, L. E., Dietmair, S., Kromer, J. O., & Nielsen, L. K. (2010). Metabolic flux analysis in mammalian cell culture. Metabolic Engineering, 12, 161–171.
Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., & Khvorova, A. (2004). Rational siRNA design for RNA interference. Nature Biotechnology, 22, 326–330.
Rice-Evans, C. A., & Burdon, R. H. (1994). Free radical damage and its control. Elsevier, Amsterdam.
Rohatgi, N., Nielsen, T. K., Bjørn, S. P., Axelsson, I., Paglia, G., Voldborg, B. G., et al. (2014). Biochemical characterization of human gluconokinase and the proposed metabolic impact of gluconic acid as determined by constraint based metabolic network analysis. PLoS ONE, 9, e98760.
Sellick, C. A., Hansen, R., Maqsood, A. R., Dunn, W. B., Stephens, G. M., Goodacre, R., et al. (2009). Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells. Analytical Chemistry, 81, 174–183.
Sellick, C. A., Hansen, R., Stephens, G. M., Goodacre, R., & Dickson, A. J. (2011). Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nature Protocols, 6, 1241–1249.
Seth, G., Hossler, P., Yee, J. C., & Hu, W. S. (2006). Engineering cells for cell culture bioprocessing–physiological fundamentals. Advances in Biochemical Engineering Biotechnology, 101, 119–164.
Tavender, T. J., & Bulleid, N. J. (2010). Peroxiredoxin IV protects cells from oxidative stress by removing H2O2 produced during disulphide formation. Journal of Cell Science, 123, 2672–2679.
Templeton, N., Dean, J., Reddy, P., & Young, J. D. (2013). Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed-batch CHO cell culture. Biotechnology and Bioengineering, 110, 2013–2024.
Vander Heiden, M. G., Cantley, L. C., & Thompson, C. B. (2009). Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science, 324, 1029–1033.
Vásquez-Vivar, J., Kalyanaraman, B., & Kennedy, M. C. (2000). Mitochondrial aconitase is a source of hydroxyl radical: An electron spin resonance investigation. Journal of Biological Chemistry, 275, 14064–14069.
Villadsen, J., Nielsen, J., & Lidén, G. (2011). Chemicals from metabolic pathways. In Bioreaction engineering principles. New York: Springer.
Wedge, D. C., Allwood, J. W., Dunn, W., Vaughan, A. A., Simpson, K., Brown, M., et al. (2011). Is serum or plasma more appropriate for intersubject comparisons in metabolomic studies? An assessment in patients with small-cell lung cancer. Analytical Chemistry, 83, 6689–6697.
Wieland, O. H. (1983). The mammalian pyruvate dehydrogenase complex: Structure and regulation. In Reviews of physiology, biochemistry and pharmacology. Berlin: Springer.
Winterbourn, C. C. (1995). Toxicity of iron and hydrogen peroxide: The Fenton reaction. Toxicology Letters, 82–83, 969–974.
Wu, G., Bazer, F. W., Burghardt, R. C., Johnson, G. A., Kim, S. W., Knabe, D. A., et al. (2011). Proline and hydroxyproline metabolism: Implications for animal and human nutrition. Amino Acids, 40, 1053–1063.
Yan, L.-J., Levine, R. L., & Sohal, R. S. (1997). Oxidative damage during aging targets mitochondrial aconitase. Proceedings of the National Academy of Sciences, 94, 11168–11172.
Yang, R.-Z., Park, S., Reagan, W. J., Goldstein, R., Zhong, S., Lawton, M., et al. (2009). Alanine aminotransferase isoenzymes: Molecular cloning and quantitative analysis of tissue expression in rats and serum elevation in liver toxicity. Hepatology, 49, 598–607.
Young, J. D. (2013). Metabolic flux rewiring in mammalian cell cultures. Current Opinion in Biotechnology, 24, 1108–1115.
Yuan, H.-X., Xiong, Y., & Guan, K.-L. (2013). Nutrient sensing, metabolism, and cell growth control. Molecular Cell, 49, 379–387.
Acknowledgements
The authors thank Dr Sam Heywood (UCB) for his critical review of this manuscript. We would also like to thank Profs Alan Dickson and Pedro Mendes (University of Manchester) for their insight and expertise that assisted this research.
Author information
Authors and Affiliations
Contributions
Conceived and designed experiments: ND DH RG DM. Performed the experiments: ND. GC-MS analysis: DT ND. Data analyses: ND DH RG DT DM. Wrote and commented in the paper: ND DH RG DT DM.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human and animal participants
This article does not contain any studies with human participants or animals performed by any of the authors.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Dhami, N., Trivedi, D.K., Goodacre, R. et al. Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells. Metabolomics 14, 136 (2018). https://doi.org/10.1007/s11306-018-1430-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11306-018-1430-0