Engineering Intracellular CMP-Sialic Acid Metabolism into Insect Cells and Methods to Enhance Its Generation

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• 作者：Karthik Viswanathan ; Someet Narang ; Stephan Hinderlich ; Yuan C. Lee ; and Michael J. Betenbaugh
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 24, 2005
• 年：2005
• 卷：44
• 期：20
• 页码：7526 - 7534
• 全文大小：276K
• 年卷期：v.44,no.20(May 24, 2005)
• ISSN：1520-4995

文摘

Previous studies have reported that insect cell lines lack the capacity to generate endogenouslythe nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, thebiosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstratedfor the first time in insect cells by expressing the mammalian genes required for the multistep conversionof endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase(EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressedin insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levelssynthesized were found to be lower than those achieved with ManNAc supplementation due to feedbackinhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site forfeedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAcfeeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increasedthe levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAcsupplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels ofUDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance thelevels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation ofcomplex glycoproteins in insect cells and other cell culture systems.