Qi Jieqiong, Wang Ruihong, Zeng Yazhen, Yu Wengong, Gu Yuchao
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.
Prep Biochem Biotechnol. 2017 Aug 9;47(7):699-702. doi: 10.1080/10826068.2017.1303614. Epub 2017 Mar 15.
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.
O-连接的N-乙酰葡糖胺化是一种动态、可逆的翻译后修饰，可调节许多细胞过程。O-连接的N-乙酰葡糖胺转移酶（OGT）是唯一一种将N-乙酰葡糖胺从尿苷二磷酸（UDP）-GlcNAc转移至细胞质和细胞核蛋白特定丝氨酸/苏氨酸残基的酶。OGT活性异常与多种疾病相关，提示OGT可作为一种新型治疗靶点。在本研究中，我们创建了一种基于酶联免疫吸附测定（ELISA）的新方法来检测OGT活性。首先，将酪蛋白激酶II（CKII）（一种著名的OGT底物）包被在ELISA板上。其次，使用抗O-GlcNAc抗体以及辣根过氧化物酶（HRP）标记的二抗检测OGT从UDP-GlcNAc转移至CKII的GlcNAc。最后，使用HRP的底物3,3',5,5'-四甲基联苯胺（TMB）检测反映OGT活性的CKII的O-连接的N-乙酰葡糖胺化水平。基于一系列优化实验，本研究选择RL2抗体用于O-GlcNAc检测，并确定了CKII、OGT和UDP-GlcNAc的浓度。使用市售的OGT抑制剂ST045849验证该系统的功能。总之，本研究展示了一种可用于检测OGT活性和筛选OGT抑制剂的方法。
