Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells

Wang, Qiong; Wang, Tiexin; Yang, Shuang; Sha, Sha; Wu, Wells W.; Chen, Yiqun; Paul, Jackson T.; Shen, Rong-Fong; Cipollo, John F.; Betenbaugh, Michael J.

doi:10.1016/j.ymben.2020.06.007

Cited by 10 publications

(8 citation statements)

References 30 publications

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“…The overexpression of B3GNT2 and/or B4GALT1 is responsible for the elongation of glycans. Previously, overexpression of B4GALT1 was found to result in the elongation of glycans on CHO cells [31]. From the flow cytometry analysis, B4GALT1 appeared to be the limiting factor for the elongation of glycans in MDCK cells, while the elongation was limited by B3GNT2 in hCK cells.…”

Section: Discussionmentioning

confidence: 90%

“…Recently, we and others have shown that poly-LacNAc containing N -glycans are critical for the binding of contemporary H3N2 viruses [8, 12]. Therefore, we used the glycosyltransferases B3GNT2 and B4GALT1 to increase the biosynthesis of LacNAc repeating units to produce extended N -glycans [12, 25, 26, 31]. We hypothesized that the overexpression of B3GNT2 and/or B4GALT1 in MDCK and hCK cells would produce appropriate glycan receptors for recent H3N2 (subclade 3C.2a) IAVs.…”

Section: Resultsmentioning

confidence: 99%

“…Changes in sugar nucleotide levels have been observed in cells after overexpression of B3GNT2 and B4GALT1 [31], which may be a limiting factor in the elongation of glycans. Therefore, the concentrations of sugar nucleotides in the cell lysates of MDCK WT, hCK WT, and hCK-B3GNT2 cells were measured by mass spectrometry (Fig.…”

Section: Sugar Nucleotides Are Not a Limiting Factor In The Biosynthe...mentioning

confidence: 99%

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Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

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Bestebroer

et al. 2022

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Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

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Section: Discussionmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Sugar Nucleotides Are Not a Limiting Factor In The Biosynthe...mentioning

confidence: 99%

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Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

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Bestebroer

et al. 2022

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“…However, the PTM pattern from CHO cells is not identical to human PTMs (Dumont et al, 2016) and the incompatibility with some types of proteins negatively affects drug efficacy, potency, or stability (Kuriakose et al 2016;Goh and Ng 2017). Therefore, besides further development of the CHO cell line to meet the high-quality PTM requirements and increased yields (Datta et al 2013;Koffas et al, 2018;Liang et al, 2020;Tejwani et al, 2018;Fouladiha et al, 2020;Wang et al, 2020), a lot of focus on improving hosts for biopharmaceutical production is on cell factories derived from human cells with the natural ability of generating human PTMs, such as the human embryonic kidney 293 (HEK293) cells (Almo and Love 2014;Malm et al, 2020;Tegel et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

et al. 2022

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“…However, the PTM pattern of CHO cells is not identical to human PTMs (Dumont et al, 2016) and the incompatibility with some types of proteins negatively affects drug efficacy, potency or stability (Goh and Ng, 2017;Kuriakose et al, 2016). Therefore, besides further development of the CHO cell-line to meet the high-quality PTM requirements and increased yields (Datta et al, 2013;Fouladiha et al, 2020;Koffas et al, 2018;Liang et al, 2020;Tejwani et al, 2018;Wang et al, 2020), a lot of focus on improving hosts for biopharmaceutical production is on cell factories derived from human cells with the natural ability of generating human PTMs, such as the Human Embryonic Kidney 293 HEK293cells (Almo and Love, 2014;Malm et al, 2020;Tegel et al, 2020).…”

mentioning

confidence: 99%

Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

Saghaleyni

Malm

Zrimec

et al. 2020

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SummaryHigher eukaryotic cell lines like HEK293 are the preferred hosts for production of therapeutic proteins requiring human post translational processing. However, recombinant protein production can result in severe stress on the cellular machinery, resulting in limited titre and product quality. To investigate the cellular and metabolic characteristics associated with these limitations, we compared erythropoietin (secretory) and GFP (non-secretory) protein producer HEK293 cell-lines using transcriptomics analysis. Despite the high demand for ATP in all protein producer clones, a significantly higher capacity for ATP production was observed with erythropoietin producers as evidenced by the enrichment of upregulated genes in the oxidative phosphorylation pathway. In addition, ribosomal genes exhibited specific patterns of expression depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased erythropoietin secretion, we detected higher ER stress, including upregulation of the ATF6B gene. Our results are significant in recognizing key pathways for recombinant protein production and identifying potential target genes for further development of secretory power in mammalian cell factories.In BriefAlthough the protein secretion process has been widely studied, the complexity of it leaves many questions with regards to defining bottlenecks for successful protein secretion to be answered. By investigating the transcriptomic profiles of different HEK293 clones with varying translational rates producing either the secreted protein erythropoietin or the intracellular GFP, we reveal that high ATP production and improved capacity of specific post-translational pathways are key factors associated with boosting erythropoietin production.HighlightsTranscriptomics analysis of a panel of HEK293 stable cell lines expressing GFP or erythropoietin (EPO) at varying translational ratesExpression of mitochondrial ribosomal genes is positively correlated with EPO secretionExpression of different cytosolic ribosomal genes are correlated with productivity in a recombinant-protein specific mannerHigh EPO producing clones have significant upregulation of ATF6B, potentially enabling a beneficial ER stress response to cope with high protein secretionGraphical Abstract

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Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells

Cited by 10 publications

References 30 publications

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

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