Exploration And Application Of The Structure And Functions Of N-glycosyltransferase

Bacterial surface polysaccharides play an important role in protecting microorganisms from abiotic or biological stress factors,nutrient collection,surface attachment,movement,etc.In addition,bacterial surface polysaccharides can stimulate innate immune responses in many ways.Therefore,bacterial surface polysaccharides have become targets for vaccine design.At present,many methods have been used to synthesize glycoconjugates,among which enzymatic synthesis is a safe,mild and efficient method.Glycoconjugates can be synthesized in vitro through glycosylation catalyzed by glycosyltransferase.The most common type of glycosylation is N-linked glycosylation.Unlike oligosaccharide transferase(OST)which induces glycosylation in the endoplasmic reticulum or bacterial pericellular,HMW1C directly transfers hexose monosaccharides from sugar nucleotides to Asn of specific sequences of receptor proteins(NXS/T;X≠P)in bacterial cytoplasm.We expressed seven enzymes in the HMW1C family(ApNGT,AaNGT,BtNGT,KkNGT,MhNGT,HdNGT,HiNGT)in E.coli.Polypeptides and glycopeptides were isolated by high performance liquid chromatography(HPLC),and the activities of 5 NGTs with high activity were determined in vitro and their glycosylation activities were compared.The structures of NGTs were predicted and modeled by AlphaFold 2.On this basis,the structural informatics analysis of NGTs was carried out.The reasons for the activity differences among NGTs were analyzed from the perspective of structure,and the relationship between the structure and function of NGTs was preliminarily clarified.In order to achieve the goal of synthesis of glycoconjugates better and faster,tool enzymes need to be optimized and modified.The structure and evolution relationship between ApNGT and OGTs of the same family are very similar,but their functions are completely different.Based on the structural informatics analysis,we predicted the location that may affect the recognition of enzymes and substrates,and carried out site directed mutation on ApNGT to obtain 5 mutants with normal expression.In vitro glycosylation experiments were carried out to verify the enzyme activity.It was preliminarily proved that the mutants can use the UDP-GlcNAc glycosylated short peptide DANYTK.On this basis,we propose to use NGTs and capsular polysaccharide serotype 3 synthase to synthesize glycoconjugates.The repeat unit of the capsular polysaccharide serotype 3 is[-3)-β-G1cA-(1,4)-β-Glc-(1-],ApNGT and NGTs of the same family can add the first sugar Glc to Asn at the specific site of peptide substrate Asn-X-Ser/Thr(X≠Pro).CPS3S is a bifunctional enzyme that can alternately add Glc and GlcA.This method provides ideas for the synthesis of glycoconjugates in the future.