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

ABSTRACT: 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.

SUBMITTER: Kwak CY

PROVIDER: S-EPMC5638827 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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

Kwak Chan-Yeong CY Park Seung-Yeol SY Lee Chung-Geun CG Okino Nozomu N Ito Makoto M Kim Jung Hoe JH

Scientific reports 20171012 1

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 ce ...[more]

PMID: 29026192

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Project description:Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C6-ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias.

Dataset Information

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

Publications

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

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