Expression Of Human FoxA1 And PpGalNAc-T2 And Preliminary Study On O-Glycosylation Of FoxA1 In Vitro

The initiation step of mucin-type O-glycosylation is catalyzed by a large family ofUDP-Gal NAc: polypeptide N-acetylgalactosaminyltransferases(pp Gal NAc-Ts),which transfer a single N-acetylgalactosamine(Gal NAc)monosaccharide to a serine or threonine residue of the polypeptide to form Tn antigen.Then the glycopeptides continue to be catalyzed by a variety of glycosyltransferases to form complex O-linked glycans.Aberrant glycosylation is one of the hall markers of tumor cells.The antigens T and Tn are expressed in most carcinomas and usually absent in healthy tissues.Previous studies in our lab indicated that pp Gal NAc-T4 may modulate the estrogen regulatory network through Fox A1 glycosylation in human breast cancer cells.However,more detailed evidence remains to be known.The confirmation of the O-Gal NAc glycosylation and glycosylation sites of Fox A1 is important elucidate the role of this transcription factor in breast cancer.It was reported that the substrate of pp Gal NAc-T4 is the O-Gal NAc glycosylated protein.In this study,we used pp Gal NAc-T2,which is primary enzyme for the substrate without glycosylation,to catalyze the glycosylation of Fox A1 protein in a cell free system.This research aimed at verifying O-Gal NAc glycosylation and confirming glycosylation sites of Fox A1 to facilitate further investigation of Fox A1 O-Gal NAcosylation.In present study,three truncated parts of Human Fox A1 c DNA(Fox A1-a,b and c)were subcloned into prokaryotic expression vector p ET28a(+).The sequencing results showed that recombinant plasmids p ET28a(+)-Fox A1-a,b and c were subcloned successfully.Fox A1-a,b and c proteins were then recombinant expressed in the E.coli BL21(DE3).The optimized induction conditions was 27?,4h and IPTG concentration of 0.1m M.Then the recombinant proteins were purified by Ni-NTA affinity chromatography.On the other hand,p FLAG-CMV-3-T2 was transfected into 293 T cells using lipofectamine.The supernatant was collected and purified using Anti-FLAG gel to enrich pp Gal NAc-T2.In a cell free reaction system,UDP-Gal NAc was used as glycosyl donor and FAM labelled EA2 peptide was used as receptor substrate.The enzyme activity of pp Gal NAc-T2 was analyzed by high performance liquid chromatography(HPLC).Results showed that catalytic activity of recombinant pp Gal NAc-T2 was good enough to carry out follow-up experiments.Fox A1-a,b and c were then catalyzed by pp Gal NAc-T2 in similar reaction system.O-Gal NAcosylationof Fox A1-c was visualized by VVL Lectin blot and confirmed by mass spectrum analyze.Furthermore,series wild type and mutant peptides derived from Fox A1-c were synthesized and O-Gal NAcosylated by pp Gal NAc-T2 in vitro.Characterization of these glycopeptides revealed that two amino residues of Fox A1,Ser354 and Ser355,were O-Gal NAcosylation sites.Together,these findings suggest that Fox A1 can be O-glycosylated by pp Gal NAc-T2 in vitro.And three O-Gal NAc glycosylation sites are confirmed.