title: Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.
abstract: The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.
authors.affiliation: Department of Science Education-Chemistry Major, Daegu University , Gyeongbuk 712-714, South Korea.
authors.forename: Eun J
authors.initials: EJ
authors.lastname: Kim
authors.forename: Lara K
authors.initials: LK
authors.lastname: Abramowitz
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Bond
authors.forename: Dona C
authors.initials: DC
authors.lastname: Love
authors.forename: Dong W
authors.initials: DW
authors.lastname: Kang
authors.forename: Hans F
authors.initials: HF
authors.lastname: Leucke
authors.forename: Dae W
authors.initials: DW
authors.lastname: Kang
authors.forename: Jong-Seog
authors.initials: JS
authors.lastname: Ahn
authors.forename: John A
authors.initials: JA
authors.lastname: Hanover
mesh_terms: D004305:Dose-Response Relationship, Drug; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D015394:Molecular Structure; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009532:Nickel / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; D009532:Nickel; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D001619:beta-N-Acetylhexosaminidases