Identification Of O-GalNAc And O-GlcNAc Modification Sites Of Human FOXA1

Glycosylation is one of the major protein post-translational modifications.More than50% proteins of the cells are glycosylated.Glycosylation of protein plays an important regulatory role in many biological processes such as cellular immunity,signal transduction,protein translation regulation and protein degradation.Glycosylations include Nglycosylation,O-glycosylation and GPI-anchored glycosylation.O-glycosylation occurs on the hydroxyl oxygen of the serine or threonine residue of protein.O-linked N-acetylgalactosamine,O-GalNAc modification and O-linked N-acetylglucosamine,O-GlcNAc modification are two types of important O-linked glycosylation.O-GalNAcylation initiates glycosylation under the catalysis of a family of GalNAc transferases(ppGalNAc-Ts)containing more than 20 isozymes,and continues to extend under the catalysis of other specific glycosyltransferases to form Branched oxygen connects complex glycan structures.O-GlcNAcylation is a dynamic and reversible post-translational modification occurring on serine or threonine residue of protein.O-GlcNAc transferase(OGT)catalyzes the transfer of an GlcNAc moiety from the donor substrate UDP-GlcNAc onto serine/threonine residues of nuclear and cytoplasmic proteins.The substrates of these two O-linked glycosylation types are widely distributed and have different functions,and participate in the regulation of the development of almost all important physiological processes and major diseases.Our previous studies showed that the forkhead box protein A1(FOXA1),an important transcription factor maybe potentially O-GalNAcylated and O-GlcNAcylated,which affect the physiological function of FOXA1.But the glycosylation site is unknown.In this study,the O-GalNAcylation and O-GlcNAcylation sites of FOXA1 were identified by Mass Spectrometry through establishing O-GalNAcylation and O-GlcNAcylation reaction systems in vitro.Firstly,secreted recombinant ppGalNAc-T2 proteins with verified ppGalNAc-T2 enzyme activity were purified from 293 T cell culture by affinity chromatography.Further,the recombinant human FOXA1 proteins expressed and purified from E.coli were incubated with the purified ppGalNAc-T2 enzyme and UDP-GalNAc as the substrate donor in vitro.By specific lectin VVL blot analysis,FOXA1 was validated to be O-GalNAcylated byppGalNAc-T2 in vitro.ESI-ETD-MS/MS analysis of this O-GalNAcylated FOXA1 showed that S355 is the O-GalNAcylation site of FOXA1 protein by ppGalNAc-T2 in vitro.Next,the pCMVPuro-FOXA1-HA was transfected into the 293 T cell to obtain the HA-FOXA1 recombinant protein.The target protein FOXA1 was enriched by HA-label immunoprecipitation,and it was verified that FOXA1 can be O-GlcNAcylated in cells.The human OGT and the hisdine(His)-tagged FOXA1 recombinant protein were co-expressed in E.coli BL21(DE3)to establish an O-GlcNAcylation reaction system in prokaryotic cells.O-GlcNAcylated FOXA1 was obtained by Affinity purification,and the results of ESI-ETD-MS/MS show that the O-GlcNAcylation sites of FOXA1 is S298,S301,T432,S441 and S443.In summary,FOXA1 protein can be O-GalNAcylated by glycosylation reaction system in vitro.It was identified by mass spectrometry that S355 of FOXA1 is an O-GalNAcylation site by ppGalNAc-T2 in vitro.S298,S301,T432,S441 and S443 of FOXA1 are O-GlcNAcylation sites.The identification of glycosylation sites will provide experimental basis for the study of FOXA1 protein glycosylation function,and also provide feasible experimental systems and protocols for the identification of glycosylation and modification sites of other proteins in vitro.