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Gene Expression in NS0 cells under ammonium stress

ABSTRACT: Ammonium is a waste product that inhibits cell growth, recombinant protein production, and protein glycosylation in mammalian cell culture. Recent studies have demonstrated that ammonium adversely affects glycosylation-related gene expression in Chinese hamster ovary (CHO) cells. However, since the CHO cell line species has not been fully sequenced, a glycosylation transcriptome analysis is not possible in this cell line. Therefore, to further understand the effects of ammonium on glycosylation-related gene expression, NS0 cells, a mouse myeloma cell line, were cultured under elevated ammonium. NS0 cells are similar to CHO cells, in that the NS0 cells are anchorage-independent and commonly used to commercially produce recombinant proteins. Additionally, DNA microarrays containing all known mouse glycosylation-related genes were available to be used to examine gene expression. NS0 cells were cultured under normal (control), elevated ammonium, elevated salt, and elevated ammonium with proline. It was observed that the control and treatments culture growth rates were not significantly different; however, the final cell densities were significantly different. The DNA microarray data was analyzed using a Welch ANOVA test with a Benjamini and Hochberg false discovery rate correction for the multiple comparisons of the glycosylation-genes. No significant difference in gene expression levels between the four conditions examined were observed. The results of this study demonstrated that NS0 cells, at the gene expression level, are insensitive to ammonium. Thus, the decreased glycosylation observed in NS0 cell cultures at elevated ammonium is likely due to changes in synthesis and degradation enzyme activity. In contrast, CHO cells have decreased glycosylation levels due to decreased sialytransferase gene expression and not increased degradation enzyme activity. Therefore, even though NS0 and CHO cells are both commonly used recombinant hosts for glycoprotein synthesis, it appears that NS0 and CHO cells had different control mechanisms respect to glycosylation-related gene expression under elevated ammonium.

ORGANISM(S): Mus musculus

PROVIDER: GSE17495 | GEO | 2009/08/05

SECONDARY ACCESSION(S): PRJNA118931

REPOSITORIES: GEO

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Project description:Ammonium is a waste product that inhibits cell growth, recombinant protein production, and protein glycosylation in mammalian cell culture. Recent studies have demonstrated that ammonium adversely affects glycosylation-related gene expression in Chinese hamster ovary (CHO) cells. However, since the CHO cell line species has not been fully sequenced, a glycosylation transcriptome analysis is not possible in this cell line. Therefore, to further understand the effects of ammonium on glycosylation-related gene expression, NS0 cells, a mouse myeloma cell line, were cultured under elevated ammonium. NS0 cells are similar to CHO cells, in that the NS0 cells are anchorage-independent and commonly used to commercially produce recombinant proteins. Additionally, DNA microarrays containing all known mouse glycosylation-related genes were available to be used to examine gene expression. NS0 cells were cultured under normal (control), elevated ammonium, elevated salt, and elevated ammonium with proline. It was observed that the control and treatments culture growth rates were not significantly different; however, the final cell densities were significantly different. The DNA microarray data was analyzed using a Welch ANOVA test with a Benjamini and Hochberg false discovery rate correction for the multiple comparisons of the glycosylation-genes. No significant difference in gene expression levels between the four conditions examined were observed. The results of this study demonstrated that NS0 cells, at the gene expression level, are insensitive to ammonium. Thus, the decreased glycosylation observed in NS0 cell cultures at elevated ammonium is likely due to changes in synthesis and degradation enzyme activity. In contrast, CHO cells have decreased glycosylation levels due to decreased sialytransferase gene expression and not increased degradation enzyme activity. Therefore, even though NS0 and CHO cells are both commonly used recombinant hosts for glycoprotein synthesis, it appears that NS0 and CHO cells had different control mechanisms respect to glycosylation-related gene expression under elevated ammonium. NS0 cells (ECACC#85110503), originally from the European Collection of Cell Culture, were donated to Clemson University by Merck, Inc. NS0 cells are a mouse myeloma cell line with lymphoblast morphology, non-secreting clone, and cholesterol auxotroph. NS0 cells cultured under four conditions were examined: Control (C), Ammonium-Stressed (A), Salt-Stressed (S), and Ammonium-Stressed with Proline added to mitigate the negative effects of ammonium (P). Triplicates of each condition were used. The cultures were be monitored during the normal batch growth phase. To identify genes sensitive to ammonium in growing cultures, the 90-h time point was selected for RNA isolation and gene expression analysis. Other culture parameters that were monitored include: Cell density, viability, and glucose.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.

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Gene Expression in NS0 cells under ammonium stress

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