Enzymatic Synthesis Of Sialylated Oligosaccharides And Functional Indentification Of Bacterial Sialyltransferase WfaN

Sialic acid, as a common monosaccharide, is widely present in mammal cells. Recently it has also been found in bacteria especially in human pathogen bacteria. In pathogen bacteria sialic acid is always a component of lipopolysaccharide or capsular polysaccharide. Sialylated glycoconjugate in bacteria plays an important role in virulence, so enzymes for the synthesis of these structures have already become an intriguing target in clinical treatment and scientific research.Sialylated oligosaccharides or sialylated glycoconjugates are ubiquitous in mammal. Sialylated sugar chains can not only participate in stabilization of protein structure and extension of half-life period of glycoproteins, and be an important component of gangliosides participant in brain’s development, but also play a vital role in lots of biological processes. Moreover, they mediate cell-cell recognition, cell adhesion and signal transduction. Also, some sialylated oligosaccharides are key structural components in some-antigenic determinant involved in the development and metastasis of tumor. These sialylated antigens have become significant molecular markes in cancer diagnosis and staging, and will probably be applied to tumor immunotherapy. Thus, synthesis of sialylated oligosaccharides in vitro is particularly important.Sialyltransferases (STs) are indispensable for biological synthesis of sialylated oligosaccharides. Compared with sialyltransferases from mammal, those from bacteria are easily over-expressed in the soluble form by prokaryotes expression system such as Escherichia coli BL21(DE3). Interestingly, some bacteria STs show broad substrate specificity, using active N-acetylneuraminic acid derivatives as their sugar donors, which riches the variety of sialosides.Enzymatic synthesis of sialylated oligosaccharides can be carried out in one-pot reaction system in which CMP-Neu5Ac synthase and sialyltransferase sequentially catalyze the synthetic reaction. This synthetic strategy facilitates the purification process and shortens the synthetic time. In Chapter2of this thesis, we enzymatically synthesized Neu5Acα2,3Gal and Neu5Acα2,61actose. Pd2,6ST from Photobacterium damsela is responsible for the formation of α2,6linkage,and PmST1from Pasteurella multocidafor the formation of α2,3linkage. Activated CMP-Neu5Ac can be synthesized by NmCSS from Neisseria meningitides. All of these three enzymes have been previously reported. This thesis adopted one-pot synthetic strategy to synthesize these two sialosides. Neu5Acα2,3Gal and Neu5Acα2,61actose can be used in many research fields. Pd2,6ST is the first α2,6sialyltransferase cloned from bacteria and expressed in E. coli. This enzyme demonstrates flexible donor and acceptor substrate specificities. Off all the bacterial ST reported so far, PmST1display the most relaxed substrate speicificity. The second chapter focuses on the synthesis and purification of sialosides in large scale, with the method of one-pot system, utilizing Pd2,6ST and NmCSS or PmST1expressed by the common prokaryotic expression E. coli BL21(DE3).Sialic acid reported in P. damsel, P. multocida and Haemophilus, and vertebrates is usually attached to galactose or GaINAc via α2,3or α2,6linkage, or to Sia via a2,8linkage; however, sialic acid in O-antigens of both E.coli024and E.coli056is linked to glucose via α2,3bond. The putative enzymes involved in transferring of sialic aicd towards glucose are enzymes WfaN (in E. coli024) and WfaR (in E. coli056). The known acceptors for ST include galactose, and its amino-derived carbohydrate, and sialic acid; whereas there is no research reported on ST whose acceptor is glucose.The third chapter of this thesis is to clone wfaN gene from E. coli024O-antigen biosynthetic gene cluster, and to futher express and purify the recombinant WfaN sialyltransferase. We also identified the catalytic activity of the enzyme and its biological functions based on in vitro reactions and in vivo gene-knockout (in chapter4). The studies on this sialyltransferase will rich our knowledge of the structures, mechanisms, and functions of glycotransferases. In addition, the ST obtained by this thesis may be used to produce specified sugar structure, and then potentially research on the structure and function of sugar chain, with latent application to medication and clinical diagnosis.