Exploration Of The Enzymatic Property Of A Novel N-Glycosyltraferases And Modification Of N-glycan On Proteins

Sugars could be devided into three types including monosaccharide,oligosaccharide and polysacchride.Saccharides and glycoconjugates are very essential in biogenic activities and play important roles in various life process.Polysaccharides existed on the surface of bacteria can be devided into three types including LPS,CPS,EPS among which LPS is a particular component of the outer membrane of Gram-negative bacteria which is composed of Lipid A,core polysaccharide,and a distal polysaccharide(O-antigen).O-antigen which located on the surface of bacteria is composed of repeated saccharides unit with identitical structure.O-antigen is closely related to bacterial virulence and plays essential roles in bacterial signal recognition,adhesion and immune evasion.According to the different length of saccharides chain of O-anigen,it can be classified into three types,long type(19-25 repeated saccharide units),intermediate type(10-18 repeated saccharide unit),short type(<10 repeated saccharide unit).O-antigen could induce host specific immunoreaction under certain circumstances and the saccharides chain composition of the O-antigen are closely associated with the antigenicity of the germs.As baceria N-glycosylation pathway becomes better understood,our lab have prepared sugar-protein conjugateing vaccine using E.coli 0157:H7 O-antigen and MBP through the method of bacterial fermentation.The purified protein was veried through Western blotand MALDI-TOF-MS.Sugar-protein conjugateing vaccine accquired through biological method is more homogeneous and can be more essily prepared than chemical method.Rescent studies have found a new glycosyltransferase,HMW1C,which is a N-glycosyltransferase existing in cytoplasm that can use the free UDP-Glc or UDP-Gal and transfer them to the asparagine residue of the polypeptide directly.It is clearly different from the Pg1B family glycosyltransferase and is therefore a new N-glycosyltransferase.Due to the crystal structure of HMW1C have not been acquired,the most understood glycosyltransferase is ApNGT whose sequence is highly homologous to it.ApNGT is derived from Actinobacillus pleuropneumoniae.In the second chapter,this thesis presents the studies of two glycosyltransferase,AaNGT and BtNGT,which is derived from Aggregatibacter aphrophilus and Bibersteinia trehalosi respectively.Their enzymatic properties were thorouthly studied.Some specific UDP were chosed for the study of the substrate specificity of the glycosyltransferase AaNGT,BtNGT and ApNGT.In the third chapter of this thesis,the building and verification of glycoprotein modification platform were described.E.coli W3110 was modified causing gene deletion of wbbl gene in O-antigen gene cluster.Therefore O-antigen only carries a GlcNAc unit.After waal gene of O-antigen ligase was knocked out,PglB could transfer GlcNAc of free lipid carrier in periplasmic space to MBP generating MBP-GlcNAc.Therefore,glycans on the glycoprotein could be modified by various glycosyltransferase in vitro using this platform which would have a far-reaching influence on the study of glycoprotein.In the forth chapter,this thesis presents the studies about the modification of glycans on the glycoprotein through wzz gene.Three wzz gene were choosed:E.coli 086:B7 wzz(short type),E.coli0157:H7 wzz(intermediate type)?E.coliO127 wzz(long type).Three wzz genes of different source were inserted in the plasmid pBAD24-mbp-pglb using the method of ccdB toxalbumin inverse screening technology.Then the expression strain,E.coli W3110 Awaal pYES1L-0157rfb pBAD24-wzz-mbp-pglb,was successfully constructed.Finally,the recombinant glycoprotein was expressed and glycans on the glycoprotein were further examined.