Enhancing the sialylation of recombinant EPO produced in CHO cells via the inhibition of glycosphingolipid biosynthesis
| Autor: | Seung-Yeol Park, Jung Hoe Kim, Makoto Ito, Chan Yeong Kwak, Chung Geun Lee, Nozomu Okino |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Pyrrolidines Monosaccharide Transport Proteins lcsh:Medicine Context (language use) CHO Cells Glycosphingolipids Article Cell Line law.invention Propanolamines 03 medical and health sciences chemistry.chemical_compound Cricetulus Biosynthesis law Cricetinae Animals lcsh:Science Erythropoietin chemistry.chemical_classification Multidisciplinary 030102 biochemistry & molecular biology Chemistry Chinese hamster ovary cell lcsh:R Glycosphingolipid Recombinant Proteins Cell biology carbohydrates (lipids) 030104 developmental biology Cell culture cardiovascular system Recombinant DNA lcsh:Q lipids (amino acids peptides and proteins) Glycoprotein Intracellular |
| Zdroj: | Scientific Reports, Vol 7, Iss 1, Pp 1-9 (2017) Scientific Reports |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-017-13609-4 |
| Popis: | Sialylation regulates the in vivo half-life of recombinant therapeutic glycoproteins, affecting their therapeutic efficacy. Levels of the precursor molecule cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) are considered a limiting factor in the sialylation of glycoproteins. Here, we show that by reducing the amount of intracellular CMP-Neu5Ac consumed for glycosphingolipid (GSL) biosynthesis, we can increase the sialylation of recombinant human erythropoietin (rhEPO) produced in CHO cells. Initially, we found that treating CHO cells with a potent inhibitor of GSL biosynthesis increases the sialylation of the rhEPO they produce. Then, we established a stable CHO cell line that produces rhEPO in the context of repression of the key GSL biosynthetic enzyme UDP-glucose ceramide glucosyltransferase (UGCG). These UGCG-depleted cells show reduced levels of gangliosides and significantly elevated levels of rhEPO sialylation. Upon further analysis of the resulting N-glycosylation pattern, we discovered that the enhanced rhEPO sialylation could be attributed to a decrease in neutral and mono-sialylated N-glycans and an increase in di-sialylated N-glycans. Our results suggest that the therapeutic efficacy of rhEPO produced in CHO cells can be improved by shunting intracellular CMP-Neu5Ac away from GSL biosynthesis and toward glycoprotein sialylation. |
| Databáze: | OpenAIRE |
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