title: New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro.
abstract: O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3',5,5'-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.
authors.affiliation: a Key Laboratory of Marine Drugs, Chinese Ministry of Education , Ocean University of China , Qingdao , China.|b Key Laboratory of Glycoscience and Glycotechnology of Shandong Province , Ocean University of China , Qingdao , China.|c School of Medicine and Pharmacy , Ocean University of China , Qingdao , China.
authors.forename: Jieqiong
authors.initials: J
authors.lastname: Qi
authors.affiliation: d Outpatient Department , Qingdao Central Hospital , Qingdao , China.
authors.forename: Ruihong
authors.initials: R
authors.lastname: Wang
authors.affiliation: a Key Laboratory of Marine Drugs, Chinese Ministry of Education , Ocean University of China , Qingdao , China.|b Key Laboratory of Glycoscience and Glycotechnology of Shandong Province , Ocean University of China , Qingdao , China.|c School of Medicine and Pharmacy , Ocean University of China , Qingdao , China.
authors.forename: Yazhen
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: a Key Laboratory of Marine Drugs, Chinese Ministry of Education , Ocean University of China , Qingdao , China.|b Key Laboratory of Glycoscience and Glycotechnology of Shandong Province , Ocean University of China , Qingdao , China.|c School of Medicine and Pharmacy , Ocean University of China , Qingdao , China.
authors.forename: Wengong
authors.initials: W
authors.lastname: Yu
authors.affiliation: a Key Laboratory of Marine Drugs, Chinese Ministry of Education , Ocean University of China , Qingdao , China.|b Key Laboratory of Glycoscience and Glycotechnology of Shandong Province , Ocean University of China , Qingdao , China.|c School of Medicine and Pharmacy , Ocean University of China , Qingdao , China.
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Gu
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004800:Enzymes, Immobilized; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D047390:Casein Kinase II; D000117:Acetylglucosamine
keywords: Activity; ELISA; O-GlcNAcylation; OGT