Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation
Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation
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O-GlcNAc 糖基化小分子抑制的无标记电化学生物传感
DOI：
10.1016/j.bios.2017.04.009
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发表时间：
2017
影响因子：
12.6
通讯作者：
Guo Liang-Hong
中科院分区：
文献类型：
--
作者：
Yang Yu;Gu Yuxin;Wan Bin;Ren Xiaomin;Guo Liang-Hong
O-linked n -乙酰氨基葡萄糖（O-GlcNAc）转移酶（OGT）在许多细胞过程和人类疾病（如阿尔茨海默病和II型糖尿病）中调节蛋白质功能起着关键作用，并已成为一个有前景的新靶点。OGT的特异性抑制剂可能是探测o - glcn酰化生物学功能的有价值的工具，但缺乏强大的非辐射检测策略来检测糖基化，阻碍了识别此类化合物的努力。在这里，我们开发了一种新型的无标记电化学生物传感器，用于检测肽o - glcn酰化，该传感器采用蛋白酶保护策略，并以联吡啶锇作为信号报告者介导酪氨酸的电催化氧化。蛋白酶对糖基化肽和未糖基化肽的蛋白水解能力存在很大差异，从而为OGT活性提供了一种感知机制。当o - glcn酰化实现时，糖基化肽不能被蛋白酶K切割，从而导致氧化铟锡（ITO）电极上的高电流响应。然而，当使用小分子成功地抑制o - glcn酰化时，未糖基化的肽可以很容易地被切割并导致低电流信号。肽o - glcn酰化反应是在定义明确的小分子OGT抑制剂存在下进行的。结果表明，该生物传感器可以有效地筛选OGT抑制剂。我们的无标记电化学方法在筛选小分子OGT抑制剂的蛋白质糖基化途径研究中是一个有前途的候选方法。
O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT.