Enhanced sialylation of recombinant EPO produced in CHO cells by modulating biosynthesis pathway of sialylation = 시알산 생합성 경로 조절을 통한 CHO 세포 재조합 Erythropoietin 의 시알산 함량 증진

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Sialylation of recombinant therapeutic glycoproteins modulates in vivo half-life associated with therapeutic efficacy. CMP-Neu5Ac, a precursor of sialylation pathway, has known as a limiting factor that increases sialylation of glycoproteins. Here, we initially found that sialylation of recombinant human erythropoietin (rhEPO) was increased by treatment with a potent inhibitor of GSL biosynthesis. The GSL expression level of cells treated with EtDO-P4 was dramatically decreased, while sialic acid content of rhEPO in culture media was significantly increased. We noted that the supplementation of a chemical inhibitor in a large-scale culture is subject to a number of issues including cost and side effect. Thus, we established genetically modified EC2-1H9 CHO cell, which produces the rhEPO having enhanced sialylation by inhibition of GSL synthesis. Ahead of stable CHO cell line construction, we designed small interference RNA targeting Cricetulus griseus UGCG mRNA. These target sequences show no significant homology with any other genes in the genome of mouse, rat and human. In case of EC2-1H9 cells treated with siRNA, the expression level of UGCG and GSLs were significantly decreased, while sialylation of rhEPO in culture media was significantly increased to 50%. These results indicate that the sialylation of glycoproteins is higly related to GSL, and the inhibition of GSL promotes enhanced sialylation of glycoproteins. Furthermore, we established stable CHO cells producing rhEPO under the inhibition of UDP-glucose ceramide glucosyltransferase (UGCG) gene encoding the key enzyme in the initiation of GSL biosynthesis. The expression of gangliosides was inhibited in UGCG repressed cells, whereas sialylation of rhEPO was significantly increased. In addition, a detailed analysis of N-glycosylation showed that di-sialylated N-glycan of rhEPO produced in EC2-1H9-miUGCG1 cells increases by 10.2%, whereas neutral and mono-sialylated N-glycan was reduced compared to rhEPO produced in EC2-1H9 cells. However, we did not observe any change in the sialylation of tri- and tetra-sialylated N-glycans of rhEPO. We found that intracellular CMP-Neu5Ac accumulated by inhibition of GSL biosynthesis, was rapidly consumed in the sialylation of rhEPO inducing dynamic equilibrium. Thus, we were attempted to increase of CMP-Neu5Ac concentration in Golgi by simultaneous overexpression of GNE and CSAT. While the intracellular CMP-Neu5Ac content in EC2-1H9-rCU3 cells was increased about 5.6-fold, the final sialylation was only increased by 44.6% compared to EC2-1H9 control cells. The sialylation of EC2-1H9-rCU3 cells was slightly increased to 9.0% compared to EC2-1H9-miUGCG1 cells, which were induced by inhibition of GSL biosynthesis. In comparison to EC2-1H9-rEKm9 cells, which were overexpressed GNE, CSAT and ST, the sialylation of EC2-1H9-rCU3 cells was only increased to 4.4%.
Kim, Jung Hoeresearcher김정회researcher
한국과학기술원 :생명과학과,
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학위논문(박사) - 한국과학기술원 : 생명과학과, 2017.2,[vii, 94 p. :]


CHO; CMP-Neu5Ac; Erythropoietin; Glycosphingolipid; Sialylation; UGCG; 중국햄스터난소세포; 에리스로포이어틴; 당지질; 시알산

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