Global Identification Of O-GlcNAc Transferase (OGT) Interactors And Substrates Based On A Human Proteome Microarray

As one of the most important protein post-translational modifications (PTMs),glycosylation plays critical roles in a variety of biological processes. Recent studiesshow that O-GlcNAcylation is highly related to many challenging diseases. Since thediscovery of O-GlcNAcylation, there are>1,000proteins that could be O-GlcNAcylatedhave been identified.But there is only one gene for coding OGT and are three transcriptisoforms, namely ncOGT, mOGT, sOGT. The fundamental question here is that how a singleOGT could specifically modify>1,000protein substrates? It is said that OGT may be mostlyregulated by its interactions with a myriad of binding partners to form various substratespecifc holoenzyme complexes, each with unique protein specificity. However, in humancell there are only more than30OGT interacting proteins have been identified，and it is notenough for fully elucidating the substrate specificity and function of OGT, besides all theproteins were interacted with ncOGT. O-GlcNAcylation is similar to proteinphosphorylation, but in human cells, comparied with phosphorylated proteins,O-GlcNAcylated proteins is fewer, thus more O-GlcNAcylated substrates of OGT alsoneed to be discovered.Proteome microarrays, provides a versatile platform for characterization of hundreds ofthousands of proteins in a highly parallel and high-throughput manner. Based on a humanmicroarray, we can globally screening the human OGT interacting proteins andO-GlcNAcylated substrates. In the study,Twenty-nine OGT interactors were successfullyidentified. Bioinformatics analysis like PANTHER classification system showed that theseinteracting proteins play a variety of roles in a wide range of cellular functions and highlyenriched in intra-Golgi vesicle-mediated transport and vitamin biosynthetic process. Thensix of the candidates choosed to validate by Co-immunoprecipitation in293T cells andKinetic bingding parameters. At last two proteins, PSAT1and HAAO were interacted withncOGT and the two proteins were also O-GlcNAcylated. Besides,we also analysed the enzyme activity of ncOGT and sOGT by a peptidemicroarray, the results showed that sOGT can O-GlcNAcylated its substrates. Thenwe try to improve the reaction condition and finally confirmed a optimization choice.In our future study,we can globally identifying substrates proteins by a humanmicroarray,combined the two part results,it may serve as start point for furtherfunctional analysis.