Abstract
Glycosylation is one of the protein post-translational modifications, which plays important roles in normal cell physiology. There are two types of glycosylation: N- and O-linked glycosylation. In mammalian cells, the most common monosaccharides found in the terminus of N- and O-linked oligosaccharides of glycoproteins and glycolipids are sialic acids. Sialic acids have many important functions, including acting as receptors for influenza viruses to invade host cells. Influenza virus vaccines are manufactured in the embryonated chicken eggs, but many problems are involved in this method. Thus, many researches focus on how to produce next generation of influenza vaccines in cultured mammalian cell lines. But, the yields of influenza viruses in cell lines are usually very low compared to those in chicken eggs. In order to decrease the production cost of vaccines produced in cultured mammalian cells, virus yields need to be optimized. According to the infection pathway of influenza viruses, Hatakeyama et al. showed that α2,6-sialyltranseferase overexpressed MDCK cells, which had higher surface sialic acid contents, had higher virus yields when they infected by viruses This result suggested that increase in sialic acid levels may improve the virus yields. In this study, N-acetylmannosamine (ManNAc) and N-acetylglucosamine (GlcNAc) were fed in the culture medium for the purpose of raising sialic acid contents in MDCK and Vero cells. Sialic acid contents increased upon ManNAc supplementation in both MDCK and Vero cells. But, GlcNAc supplement did not affect sialic acid levels in those two cells. These results showed that ManNAc supplementation was a good method for raising surface sialic acid contents in MDCK and Vero cells and also implied that UDP-GlcNAc 2-epimerase regulates rate-limiting step of the pathway, namely biosynthesis of ManNAc, in MDCK and Vero cells. This speculation was supported by the study of Keppler et al. [1] Besides, supplement of ManNAc to CMP-sialic acid synthetase expressed MDCK or Vero cells led to even higher increase in sialic acid level. This result implied that CMP-sialic acid synthetase may also regulate another bottleneck of sialic acid synthesis pathway. The developed method applied in MDCK and Vero cells may have many applications such as production of recombinant proteins or influenza vaccines. Further investigations of these sialylation-increased cells are needed.