| UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases play a key role in the first step of O-linked glycanprotein oligosaccharide biosynthesis by transferring an N-acetyl galactosamine to the hydroxyl group of a serine or threonine residue of peptide or protein,forming GalNAc ?1-O-Ser/Thr,also named Tn antigen.The formation of Tn antigen is closely related to the occurrence and metastasis of tumor.It is not usually found on healthy cell surfaces,but can be found in 70-90%cancer.It is therefore of great importance to synthesize Tn antigen and study its biological function.Our study was focused on the biosynthesis of Tn antigen from low-cost glycosyl donor UDP-GlcNAc by advanced enzyme engineering.Enzymatic synthesis for Tn antigen is a more preferable method than chemical method,due to the mild reaction condition,less pollution and high purity of product.1.The enzymatic activity detection of ppGalNAc-Ts from Homo Sapiens In this study,we subcloned ppGalNAc-T2 and ppGalNAc-T8 genes from Homo sapiens based on recombinant plasmids(pPICZ?B-HSGT)in Pichiapastoris which stored in our lab,ligated the gene fragment into pET30 vector.And the genes were expressted in E.coli,their enzymatic activity after purification were detected with the glycosyl donor UDP-GalNAc and artificial synthetic acceptor EA2-peptide and EA2-W-peptide.The results showed the conversion of ppGalNAc-T2 was 44.45%and 16.82%,ppGalNAc-T8 is 5.58%and 0.22%,respectively.Then ppGalNAc-T2 was chosen for being applied in biosynthesis of Tn antigen.2.The biosynthesis of Tn antigen on peptide The pathway for Tn antigen biosynthesis on mucin5AC core polypeptide was desighed in this paper in order to simulate the nature form of Tn antigen and study its biological functiona.Due to the high cost of the glycosyl donor(UDP-GalNAc)of Tn antigen,UDP-GalNAc would be obtained from low cost UDP-GalNAc with UDP-glucose-4-epimerase from Crassostrea gigas,firstly,and mucin5AC core polypeptide aslo from Homo sapiens would be as the glycosyl acceptor to synthesize Tn antigen Therefore Tn antigen on mucin5AC core polypeptide was synthesized with ppGalNAc-T2 and CGIUGE4 which UDP-GlcNAc as the glycosyl donor,mucin5AC core polypeptide as the glycosyl acceptor In addition,the inducible vectors are widely used in prokaryotic expression vectors for recombinant protein in vitro,the prokaryotic expression vectors were modified directly with constitutive regulatory sequence for simulating the expression by constitutively and overcoming the disadvantage of inducible vectors.Then two genes in the synthetic pathway of Tn antigen and fusion mucin5AC was applied into the constructed vector of prokaryotic expression vectors without any induction,respectively.One-pot reaction with ppGalNAc-T2,CGIUGE4 and fusion MUC5AC successfully obtained Tn antigen.The details of results are as follows:(1)The construction of prokaryotic expression vectors and the expression of green fluorescent protein gene into the constructed vectorsThe regulatory sequences of six resistance genes(Blasticidin,Tetracycline,Chloram-phenicol,Streptomycin,Kanamycin,Hygromycin)were analyzed in bioinformation database,then was grouped with different digestion sites in tandem,forming a unique constitutive regulatory sequence(CRS),which can promote the expression of insert target genes.And CRS after DNA synthesis was introduced into commercial expression vector pETDuet-1/pACYCDuet-1/CDFDuet-1/RSFDuet-1 to obtain the constitutive vectors(pET-CRS/pACYC-CRS/CDF-CRS/RSF-CRS)and to get the initiator element for expression of insert target genes.The green fluorescent protein genes were cloned into the modified vectors with different digestion sites,the results indicated the efficient expression vectors without any induction suitable for prokaryotic expression system were constructed.Therefore ppGalNAc-T2 was applied into CDF-CRS vector,CGIUGE4 was applied into pACYC-CRS vector for Tn antigen biosynthesis.(2)The design of Fusion MUC5AC and the biosynthesis of Tn antigenThe amino acid sequence encoded by MUC5AC gene from Homo sapiens was analyzed by bioinformatics to find the core peptide sequence of mucin5AC protein domain which contain multiple repeat S/T sequence,therefore fusion MUC5AC gene fragment was designed and then ligated to the RSFDuet-1 vector,forming constitutive recombinant vector,moreover was expressed in E.coli without induction successfully.The recombinant ppGalNAc-T2 and CGIUGE4 were put into one-pot reaction obtain Tn antigen with UDP-GlcNAc as the glycosyl donor and fusion mucin5AC as the glycosyl acceptor.The results indicated no more than 10 Tn antigen were detected both in the reaction with UDP-GlcNAc as glycosyl dondor and the positive control with UDP-GalNAc as glycosyl dondor,and we obtain the Tn antigen from low cost UDP-GlcNAc by our methold and our modified constitutive vector. |
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