Viswanathan Karthik, Narang Someet, Hinderlich Stephan, Lee Yuan C, Betenbaugh Michael J
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 2005 May 24;44(20):7526-34. doi: 10.1021/bi047477y.
Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of 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 coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.
先前的研究报道，昆虫细胞系缺乏内源性生成糖缀合物唾液酸化所需的核苷酸糖CMP - Neu5Ac的能力。在本研究中，通过表达将内源性UDP - GlcNAc多步转化为CMP - Neu5Ac所需的哺乳动物基因，首次在昆虫细胞中证明了完全从内源性代谢物合成这种活化形式的唾液酸。使用杆状病毒表达载体在昆虫细胞中共表达UDP - GlcNAc - 2 - 表异构酶/甘露糖胺激酶（EK）、唾液酸9 - 磷酸合酶（SAS）和CMP - 唾液酸合成酶（CSAS）的基因，但由于CMP - Neu5Ac对EK酶的反馈抑制，发现合成的CMP - Neu5Ac和前体Neu5Ac水平低于补充甘露糖胺时的水平。当使用反馈调节位点已发生突变的类唾液酸尿症突变型EK基因时，CMP - Neu5Ac的合成水平比野生型EK高出4倍以上，比喂食甘露糖胺时高出2.5倍。向培养基中添加UDP - GlcNAc的前体N - 乙酰葡糖胺（GlcNAc），可使CMP - Neu5Ac水平进一步提高，比补充甘露糖胺时高出7.5倍，在较高水平的UDP - GlcNAc下，在Neu5Ac转化为CMP - Neu5Ac过程中形成了一个瓶颈。本研究提供了一种有用的生化策略，用于合成和提高唾液酸化供体分子CMP - Neu5Ac的水平，CMP - Neu5Ac是昆虫细胞和其他细胞培养系统中生成复杂糖蛋白的关键限制性底物。
