Enhancing the sialylation of recombinant EPO produced in CHO cells via the inhibition of glycosphingolipid biosynthesis

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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.
Publisher
NATURE PUBLISHING GROUP
Issue Date
2017-10
Language
English
Article Type
Article
Keywords

HAMSTER OVARY CELLS; GROWTH-FACTOR RECEPTOR; SIALIC-ACID CONTENT; GLUCOSYLCERAMIDE SYNTHASE; INTERFERON-GAMMA; INSECT CELLS; GLYCOSYLATION; ERYTHROPOIETIN; PROTEIN; EXPRESSION

Citation

SCIENTIFIC REPORTS, v.7

ISSN
2045-2322
DOI
10.1038/s41598-017-13609-4
URI
http://hdl.handle.net/10203/226836
Appears in Collection
BS-Journal Papers(저널논문)
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