| Carbohydrates (also known as Glycans) constitute one of the major classes of bio-macromolecules in living organisms, they not only are essential components of cells but also play important roles in a variety of vital biological processes. Carbohydrates are of diverse structural complexities, they exist as oligosaccharides, polysaccharides and glycoconjugates (including glycoprotein, glycolipid, lipopolysaccharides). Specific changes in glycan profiles have been associated with certain disease states such as cancer and inflammation, illustrating the potential of using glycans in clinical diagnosis and perhaps as targets to develop therapeutics.The biosynthesis of glycans and glycoconjugates is catalyzed by glycosyltransferases (GT), which directly determined the structure of the glycans. Corresponding to the diverse structural complexities of glycans, GT constitute one of the largest enzyme families in nature. Statistically, around 1-2% of the genes in the sequenced genomes are putative GT, whereas only a small portion of which were characterized. GT catalyze the transfer of glycan moieties from glycan donor to acceptor to form a specific glycosidic bond. Most of the GT utilize sugar nucleotides (such as UDP-Gal, GDP-Fuc) as donors, which are called Leloir type GT. Others could utilize lipid (such as dolichol) linked sugar as donor, and called non-Leloir type GT. The acceptor of GT could be monosaccharides, oligosaccharides, polysaccharides, polypeptides, proteins, lipids, small molecules and even DNA.The application of glycans in modern medical science relies on large scale synthesis of such molecules. Enzymatic synthesis is the major approach for the synthesis, and Leloir type GT are mainly used. However, there are two unsolved problems:1) Few GT can be used for large scale glycan synthesis; 2) Sugar nucleotides are both expensive and hard to obtain.O-Polysaccharides (OPS) is one of the major bacterial polysaccharides, constituted by O-repeating units (up to 100 copies). O-PS covalently linked to core oligosaccharide and lipid-A to form complete LPS. O-PS exhibit vast structural complexities, for example, there are 181 O-PS with distinct structures in E. coli and 90 in Samonella. Based on the structural complexities, bacteria are divided into different O-serotypes, such as E. coli 086 and 0157. Increasing evidences show that O-PS play essential roles in bacteria-host interactions, and involved in bacterial infection. Thus, investigations on O-PS biosynthesis pathway will facilitate the development of pharmaceutics targeting on such bacteria.Glycosylation is one of the most common posttranslational modifications of proteins in eukaryotes. A recent analysis on the genome/protein databases revealed that more than half of all proteins in nature would be glycoproteins. Glycosylation modules protein's structure and function in biological systems in many ways. For example, the oligosaccharide components in glycoproteins are implicated to play roles in many intercellular recognition processes such as cell adhesion, tumor metastasis, pathogen infections, and immune responses. On the other hand, glycosylation can influence protein's biosynthesis, folding, antigenicity, immunogenicity, serum half-life. Such properties have been utilized in development of glycoprotein pharmaceutics. Thus, investigations on details of protein glycosylation will facilitate the development of glycoprotein pharmaceutics.We targeted on these glycobiology problems in this dissertation. Firstly, We characterized a bifunctional UDP-GlcNAc/Glc 4-Epimerase from E. coli O-PS biosynthesis gene cluster, provided biochemical details that facilitate the application in oligosaccharide synthesis. Secondly, we developed a high-throughput method for indentifying GT activities, provided a platform for rapid and efficient GT identification. Thirdly, we in vitro reconstituted the O-PS biosynthesis pathway, provided a model for detailed mechanism investigation toward this pathway. Lastly, we have overexpressed the first bacterial oligosaccharyltransferase, Pg1B, which makes the investigation related to N-glycosylation more easier.UDP-Gal and UDP-GalNAc are important sugar donors in the synthesis of a variety of human related antigens. However, they are hard to prepare and significantly expensive, one way to solve this problem is the addition of UDP-GlcNAc/Glc 4-epimerase into the reactions. A gne gene in E. coli O86:B7 O-PS gene cluster were thought to encode such an enzyme. To confirm this annotation, overexpression, purification and biochemical characterization of Gne was performed. By using capillary electrophoresis, we showed that Gne can catalyze the interconversion of both UDP-GlcNAc/GalNAc and UDP-Glc/Gal. The Km values of Gne for UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc are 370, 295,323, and 373μM, respectively. The comparison of the kinetic parameters of Gne from E. coli 086:B7 to those of other characterized UDP-GlcNAc/Glc 4-Epimerases indicated that it is a bifunctional UDP-GlcNAc/Glc 4-Epimerase. Moreover, the calculated kcat/Km values for UDP-GalNAc and UDP-Gal are approximately two to four times higher than those for UDP-GlcNAc and UDP-Glc, suggesting that Gne is slightly more efficient for the epimerization of UDP-GalNAc and UDP-Gal. One mutation (S306Y) retained the activity for non-acetylated substrate, but totally abolished the activity for N-acetylated substrate, indicating that residue S306 plays an important role in the determination of substrate specificity.There are nearly 40,000 putative GT genes among the sequenced genomes. However, due to the difficulty in characterizing such enzymes, only a very small portion of them were used in oligosaccharide synthesis. Herein, we developed a high-throughput GT indentification method, including mainly three steps:1) Rapid GT cloning:LIC were used to clone 97 GT. LIC does not need to design specific restriction sites for each GT, it uses less DNA and less time-consuming; 2) In vitro protein expression:Expressway Cell-Free E. coli Protein Expression System were used to express 97 GT, the expression was finished within 4 hours, and 77 of them were detected in western blot; 3) MALDI-MS Label Free Detection:we prepared a glyco-array with 25 acceptors and 7 donors. All the enzymes were analyzed with each donor and acceptor, the number of total reactions was 34300. The bio-chips were rinsed and applied to MALDI-TOF/MS directly. We found 9 new GT using this method.Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared GalNAc-PP-Und, the E. coli 086 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway. Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, Pg1B, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous Pg1B proteins. Pg1B was overexpressed in E. coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified Pg1B was verified using a chemically synthesized sugar donor:GalNAc-PP-Und and a synthesized peptide acceptor. The result confirms that Pg1B is solely responsible for the oligosaccharyltransferase activity and complements the finding that Pg1B exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of Pg1B using the PhoA/LacZ fusion method. The topological model shows that Pg1B possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common Ncyt-CPeri topology with 11 transmembrane segments for the STT3 family proteins.In summary, through detailed investigation on GT, we established an O-PS in vitro biosynthesis model, enriched the basic knowledge of bacterial polysaccharide biosynthesis; developed and high-throughput enzyme assay method, provided a platform for rapid GT identification; in addition, we obtained large quantities of Pg1B protein, facilitated further investigation on the mechanisms of N-glycosylation.
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