title: Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray.
abstract: O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity.
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Jie
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Suhela
authors.initials: S
authors.lastname: Sharif
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht University, Utrecht, The Netherlands.|PamGene International BV, 's-Hertogenbosch, The Netherlands.
authors.forename: Rob
authors.initials: R
authors.lastname: Ruijtenbeek
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Roland J
authors.initials: RJ
authors.lastname: Pieters
mesh_terms: D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D040081:Protein Array Analysis; D050717:Retinoblastoma-Like Protein p130 / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010455:Peptides; C493837:RBL2 protein, human; D050717:Retinoblastoma-Like Protein p130; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase