Exploration And Application Of The Enzymatic Character Of N-glycosyltraferases

Protein glycosylation is one of the most prominent post-translational protein modifications.Recently,production of recombinant proteins using E.coli in industrial applications to obtain glycosylated therapeutic proteins has progressed steadily.Chemical method is the traditional method for producing glycoprotein conjugates.The use of chemical methods adding sugar chains to carrier proteins has many drawbacks such as many steps,high cost,uncertain glycosylation sites,the number of sugar chains is difficult to control.Biological synthesis can effectively avoid these drawbacks.Recently,a cytoplasmic soluble N-glycosyltransferase(NGT)has been discovered in bacteria.It transfers nucleotide-activated donors to asparagine side chains in the conserved N-(X(?)P)-S/T sequon.The number of NGT-like N-glycosyltransferase studied is limited.And NGT-mediated glycoprotein conjugate vaccines have not been reported.Based on the great value of NGT,understanding the enzymatic properties of NGT is important in the field of glycobiology.We chose Streptococcus pneumoniae type 3,which is often found in Streptococcus pneumoniae vaccine.On the basis of understanding the enzymatic properties of NGT,we will establish a synthase-dependent glycoprotein vaccine production platform and provide a novel glycoprotein conjugate vaccine synthesis production method.In the second chapter of this thesis,we investigates the enzymatic properties of NGT from four different sources.Here,we expressed and purified four different N-glycosyltransferases derived from diverse bacteria,including Actinobacillus pleuropneumoniae(ApNGT),Aggregatibacter aphrophilus(AaNGT),Kingella kingae(KkNGT)and Bibersteinia trehalosi(BtNGT)and measured their catalytic activities with four synthesized peptides via in vitro glycosylation assays.We then analyzed and compared the glycosylation efficiencies of these four NGT isoforms,which showed distinct substrate selectivities.We sought to probe peptide specificities among various NGT isoforms,which could broaden the application of NGT-catalyzed N-glycosylation of a variety of therapeutic proteins.In the third chapter of this thesis,the biological method achieves glycosylation modification of carrier protein by NGT.Based on the experimental results of the first part,NGT isoforms have different peptide substrate preferences,we identifies a N-glycosyltransferase to recognize substrate protein or modifies the substrate protein sequence to achieve the recognition of N-glycosyltransferase.NGT and the carrier protein are co-expressed in E.coli to achieve glycosylation modification of the carrier protein.In the fourth chapter of this thesis,we will establish a S.pneumoniae type 3 polysaccharide protein vaccine platform.We have obtained the genes for S.pneumoniae type 3 synthase Cps3s.On the basis of the previous step,we have completed the NGT-mediated glycosylation of the carrier protein.The glycosylated carrier protein is a glycosyl acceptor,and the glycosyl donor is UDP-Glc,UDP-GlcUA,and further utilizes Cps3S synthetase to produce a polysaccharide conjugate sugar chain.Because of Cps3S synthase is a transmembrane protein,purification of Cps3S synthase by heterologous expression in E.coli have been blocked.