Construction Of Yeast Cell-based O-GlcNAc Transferase Assay System And Its Application To Screen Novel O-GlcNAcylated Protein

O-GlcNAcylation is a form of post-translational modification wherein single N-acetylglucosamine(GlcNAc)is attached to serine or threonine residues on target proteins.In eukaryotic cell O-GlcNAc addition is mediated by O-GlcNAc transferase(OGT)and hydrolysis by O-GlcNAcase(OGA).O-GlcNAc modification is conserved through eukaryotic cell,however,the budding yeast Saccharomyces cerevisiae notably lacks this protein modification and the genes for the GlcNAc transferase and hydrolase.To study the function of single OGT isoform,OGTs were overexpressed in Saccharomyces cerevisiae individually;the O-GlcNAc modified proteins in sOGT expressing yeast cells were identified,furthermore,homologous of yeast endogenous O-GlcNAc modified proteins were heterogeneously expressed and verified in HEK293 cells.The main conclusions in this study were stated as follow:(1)OGT isoforms differ in N-terminal amino acid sequence,the differences in OGTs caused yeast growth defect were related to their N-terminal region.sOGT caused yeast growth defect depends on its catalytic activity,while that of ncOGT and mOGT were catalytic activity independent.Therefore,sOGT-expressing cells might be an in vivo O-GlcNAc assay system.(2)Mitochondria OGT has an N-termial mitochondrial targeting sequence(MTS),which mediates its targeting to mitochondrial inner membrane.Mitochondria OGT caused yeast growth defect depends on its N-terminal MTS sequence;The expression of MTS sequence caused yeast mitochondrial fusion.Therefore,MTS-expressing yeast cell might be a model for study of mOGT caused mammalian cell apoptosis.(3)O-GlcNAc modified yeast endogenous proteins were enriched by s WGA agarose and indentified by nanoLC-MS/MS in sOGT-expressing yeast cell.There are 468 proteins identified with more than one O-GlcNAc modified peptide,among them 13 yeast proteins possessed human orthologue with the identity higher than 30% in their amino acid.Moreover,the probable O-GlcNAc modification site is conserved between yeast protein and human orthologues.Ubc5,Sup35,Bat2 and Esa1 were verified to be modified by O-GlcNAc in sOGT-expressing cells,which confirmed that the enrichment and analysis method in this study is reliable.(4)The O-GlcNAc modification of human orthologues UBE2D1,HBS1 L,BCAT2 and KAT8 were verified both in sOGT-expressing yeast cells and HEK293 cell.UBE2D1,HBS1 L and BCAT2 were confirmed to be modified by O-GlcNAc.In summary,combine the O-GlcNAc modified protein identification in yeast cell and verification in HEK293 cell together,yeast cell is supposed to be a fairly well model for O-GlcNAc modification study.Yeast cell is expected to be useful for identification of new O-GlcNAcylated proteins.