| O-GlcNAc glycosylation and recently discovered O-GlcNAc6P glycosylation with a phosphate group on the 6-hydroxyl group,are hot topics in the field of protein glycosylation.Chemoenzymatic glycan labeling is one of the most effective methods for O-GlcNAc studies.Capitalizing the unique specificity of a galactosyltransferase BGalT-Y289L,galactose derivatives can be added onto the GlcNAc residue.The chemical reaction handle on the galactose derivatives enables further labeling with fluorophores for detection or biotin for affinity enrichment.BGalT-Y289L,aβ1-4-galactosyltransferase(GalT)mutant originating from bovine,has to be recombinantly expressed in E.coli as inclusion bodies and refolded in vitro to obtain active enzyme.The failure to achieve soluble expression is a major obstacle to its application.Other GalTs including a β1-4 galactosyltransferase from Helicobacter pylori(Hp1,4GalT)may also be explored for chemoenzymatic glycan labeling.One aim of this thesis is to express soluble Hp1,4GalT and BGalT-Y289L in E.coli and screening their activity for O-GlcNAc6P extension.In order to screen GalT that can recognize GlcNAc-6P,we synthesized GlcNAc-6P and its derivative 6P-GlcNAc-S-pNP in Chapter 2.We cloned the GlcNAc kinase gene NagK and obtained active NagK.GlcNAc-SH was phosphorylated at C-6 by NagK to obtain 6P-GlcNAc-SH,then p-nitrophenol was added to 6P-GlcNAc-SH via sulfhydryl alkylation to get the target compound 6P-GlcNAc-S-pNP.In Chapter 3,the optimized BGalT-Y289L and Hp1-4GalT coding sequences were synthesized and inserted into a serial of expression vectors.For Hp1,4GalT,we fused the target gene with maltose binding protein and obtained soluble active enzyme.For BGalT-Y289L,soluble expression was achived by fusion with the propeptide of lipase from Staphylococcus hyicus.Although the soluble expressed Hp1,4GalT and BGalT-Y289L were unable to recognize 6P-GlcNAc-S-pNP as the acceptor,they were active with GlcNAc as the acceptor thus presented as useful tools for O-GlcNAc labeling and glycan synthesis.Chapter 4 of this thesis is about enzymatic synthesis of nucleotide sugars.Nucleotide sugars are essential glycosyl donors for Leloir-glycosyltransferases.The accessibility of nucleotide sugars is indispensable for the synthesis,modification and application of oligosaccharides,glycoproteins,glycolipids and sugar-modified small molecules.L-Arabinose exists ubiquitously in plant and some microorganisms,and is the second abundant pentose in plant.The synthesis of L-arabinose-containing glycan is catalyzed by arabinosyltransferase with UDP-L-arabinose(UDP-Ara)as the donor.A chemoenzymatic synthesis route has already been established,in which UDP-Ara was synthesized by Arabidopsis thaliana UDP-sugar pyrophosphorylase(AtUSP)with chemically synthesized L-arabinose 1-phosphate(Ara-1-P)as the substrate.However,there were two Ara-1-P isomers from chemical synthesis,β-Ara-1-P and α-Ara-1-P,and AtUSP only uses β-Ara-1-P.Thus,this method has several disadvantages including laborious chemical synthesis involving multiple steps,low yield with enzymatic synthesis,and time-consuming.In order to simplify the in vitro synthesis of UDP-Ara,we screened out an arabinokinase from Paludisphaera borealis(PbAraK).Recombinant PbAraK was obtained by soluble expression in E.coli.The substrate specificity and optimal reaction conditions were investigated.PbAraK can recognize L-arabinose and L-ribose to synthesize corresponding sugar 1-phsophastes.Metal ions were needed in the reaction.The optimal reaction temperature is 45℃ and the optimal pH is 7.5-8.0.Then,an one-pot-three-enzyme synthesis system for UDP-Ara was established with PbAraK,AtUSP(EC 2.7.7.64)and inorganic pyrophosphatase(EC 3.6.1.1).PbAraK-catalyzed synthesis of Ara-1-P possess merits including absolute stereoselectivity,ease operation,environmental-friendly and low cost.The established UDP-Ara synthesis method is easily to scale-up and has potential value in glycan synthesis. |
PDF Full Text Request