Elsevier

Journal of Biotechnology

Volume 257, 10 September 2017, Pages 87-98
Journal of Biotechnology

Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS

Dedicated to Prof. Dr. Alf Pühler on the occasion of his 75th birthday.
https://doi.org/10.1016/j.jbiotec.2017.03.032Get rights and content

Highlights

  • Limited knowledge of molecular productivity enhancing effects of butyrate on CHO.

  • Quick impact of butyrate on histone modification visible by AU-PAGE Western blotting.

  • Straight forward label-free MS-quantification improved view on shifted cellular state.

  • Altered protein level in cell growth, carbohydrate metabolism, protein expression.

  • HDAC inhibition by butyrate also visible in changes of nucleosome modifying factors.

Abstract

Effects of butyrate on CHO producer cells are contradictory, promoting productivity and at the same time repressing proliferation. Though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. As previous proteomic results on this field are mainly based on 2DE-gels, we conducted a label-free MS quantification, based on fast high resolution ESI–MS and a straight forward software solution, to gain insight in shifted cellular processes of CHO cells 25 h after butyrate treatment. 118 proteins or subunits with significantly altered abundances were identified suggesting changes in carbohydrate, protein metabolic and cell cycle processes. Effects of butyrate on the nucleosome assembly as a known direct epigenetic influence on HDAC activity turned out to be unexpectedly fast and persistent, as confirmed by Western blots of histone-H4 acetylation. Contradictory to increased cell specific productivity, most elements of protein metabolism exhibited decreased levels after butyrate treatment. In comparison to published results some overlap of our label free MS data could be observed but also apparently diverging findings, showing the need for complementary omics techniques for a holistic view on cellular processes such as response to butyrate.

Introduction

The global market for biopharmaceuticals has been continuously growing in the last years and is expected to exceed a volume of US$166 billion by 2017 (Shi et al., 2014). Recombinant protein therapeutics are the major category of biopharmaceuticals and about 70% are produced in Chinese Hamster Ovary (CHO) cells (Jayapal et al., 2007, Kim et al., 2012). This popularity of CHO cells is based on easier market approval due to its widespread use in production processes, establishment of powerful gene amplification systems for high specific productivity, the capacity for efficient post-translational modifications (PTMs) and easy adaption to growth in large-scale bioreactors. Numerous strategies have been pursued to further increase cell specific productivity of CHO cells such as optimized feeding strategies, improved media composition, cell line engineering and varying culture conditions, in many cases based on omics approaches (Farrell et al., 2014, Kim et al., 2012, Yee et al., 2008). The productivity promoting effect of butyrate has been widely described and known for more than 20 years (Cherlet and Marc, 2000, Sung et al., 2004). However, sodium butyrate also introduces a detrimental effect on cell growth (Baik and Lee, 2010, Chang et al., 2013, Zhou et al., 2011). Countering this effect by the overexpression of anti-apoptotic proteins has been shown to dramatically increase the protein yield (Kim et al., 2012). Like some other short chain fatty acids, butyrate is known to act as a histone deacetylase (HDAC) inhibitor introducing hyperacetylation of histones (Candido et al., 1978, Davie, 2003, Jiang and Sharfstein, 2008, Riggs et al., 1977). This leads to changes in chromatin structure and accessibility of transcription factors. Different HDAC inhibitors including butyrate have been tested also as potential anti-tumor agents in clinical trials (Kim et al., 2006, Pan et al., 2007, Ververis et al., 2013). Some previous studies investigating the transcriptome or proteome of CHO give a rough overview of butyrate effects on further cellular response after HDAC-inhibition, next to other putative impacts of this short chain fatty acid (Baik et al., 2008, Baik and Lee, 2010, Brinkrolf et al., 2014, Kantardjieff et al., 2010, Klausing et al., 2011, Leon Gatti et al., 2007, Yee et al., 2008, Yee et al., 2009). Recently, we have shown for CHO cells also a rapid and strong impact of butyrate on epigenetic level of DNA-methylation by CpG-microarray approach (Wippermann et al., 2014), and current bisulfite sequencing results underline the complexity of this regulations via DNA modification (Wippermann et al., 2016). However, the complete interaction of metabolic and regulatory elements that leads to increased protein production still remains unclear. Further research on proteomic changes of CHO cells in the presence of butyrate will therefore narrow links between the primary effects of butyrate (HDAC-inhibition, metabolic impact) and following effects leading to increased productivity. Based on such results, cell line engineering should enable the generation of high producer cells without growth-inhibiting effects.

Here we present a gel-free differential proteome analysis of butyrate-treated CHO cells using a straight forward label-free mass spectrometric protein quantification strategy. The broad spectrum of affected protein species offers further insights in the cellular response on butyrate treatment on the level of cell growth, carbohydrate metabolism, protein expression and histone modification. Direct effects of butyrate on nucleosome assembly were confirmed by Western blots of histone-H4 acetylation. So far, most existing proteomic results on this field are based on 2DE-gel technique. Our label free data show the benefit of complementary omic techniques improving our view on the shifted cells state.

Section snippets

Cell culture conditions

The anti-IL-8 antibody producing CHO DP-12 clone #1934 (ATCC CRL-12445) was cultivated in customized, chemically defined, animal component-free medium (Xell, Germany) with addition of 6 mM glutamine, 200 nM methotrexate (MTX) and 0.1 mg/L IGF. Cultivation was carried out in shaking flasks at 37 °C, 5% CO2, 80% humidity and 185 rpm with a culture volume of 50 mL. Cells were inoculated at 5 × 105 cells/mL. Sodium butyrate (Sigma-Aldrich, USA) was added to a final concentration of 2 mM in the cultures 64 h

Results

We investigated the effect of butyrate treatment on CHO DP-12 cells using label-free MS quantification. Six of twelve replicates of CHO cultures were treated with 2 mM sodium butyrate 64 h after inoculation. Samples from the six butyrate-treated and six untreated replicate cultures were taken after further 25 h of growth and analyzed in a label-free MS approach. For detection of effects on histone modification level, Western blots (antibody against histone H4 K5-acetylation) from SDS- and

Discussion

The elucidation of the impact of short-chain fatty acids on the molecular background of producer cells has been object of some omic approaches during the last years. An overview of previous proteomic investigations of butyrate treatment of mammalian production cells has been previously shown (Brinkrolf et al., 2014). So far, these approaches were primarily based on 2DE technique (Baik et al., 2008, Baik and Lee, 2010, van Dyk et al., 2003). In 2DE approaches isoforms, some modifications and

Conclusion

In our label-free MS quantification the sample preparation, MS acquisition and software evaluation were straight forward and no artificial modification of proteins or peptides was necessary. Hence, the presented MS approach has clear advantages compared to classical gel-based but also to most MS-based techniques utilizing labels. The technique is not only time-saving compared to most other “shotgun” quantitative proteomics techniques but also very effective in the detection of differentially

Declaration of competing interest

The authors declare that they have no competing interest.

Authorś contributions

T.N., B.M. and R.H. developed the concept of the study. C.H. and A.S. carried out the cell cultivations. B.M., S.K., W.J. and C.B. performed the MS analysis and data evaluation. N.RdC. conducted the PAGEs and Western blot detections. N.RdC. and R.H. evaluated the Western blot detections. S.P.A. supported the bioinformatics data evaluation. All authors contributed to drafting and reviewing the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Acknowledgments

BM acknowledges the scholarship granted by the CLIB-Graduate Cluster Industrial Biotechnology co-financed by the Ministry of Innovation, Science and Research of North Rhine Westphalia, Germany.

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