O-GlcNAc糖基化修饰的细胞成像及荧光检测新方法研究

项目名称： O-GlcNAc糖基化修饰的细胞成像及荧光检测新方法研究

项目编号： No.31300678

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 张英

作者单位： 西北大学

项目金额： 20万元

中文摘要： O-GlcNAc糖基化修饰是一种常见的蛋白质翻译后修饰方式，其在细胞内的异常表达与多种疾病的发生发展密切相关。因此对O-GlcNAc糖基化修饰水平进行动态检测，可作为研究疾病发生发展及诊断治疗的参考依据。由于细胞内O-GlcNAc糖基化修饰蛋白的定位及种类有多样性、表达丰度低，高灵敏度和特异性的检测手段相对缺乏。现有的荧光检测方法存在检测用底物尚未商品化及不易合成、荧光检测操作复杂等缺点，阻碍了O-GlcNAc糖基化修饰深入研究。本项目将利用本实验室建立的FTSC荧光成像技术及改进的底物合成方法，发展一套操作简单、高效可靠的检测方法，应用于O-GlcNAc糖基化修饰的活细胞荧光成像；同时建立基于蛋白芯片技术，体外大规模筛查蛋白O-GlcNAc糖基化修饰的荧光检测新方法。本研究将改进和完善O-GlcNAc糖基化修饰荧光检测方法，对进一步推动O-GlcNAc糖基化修饰蛋白质组学研究有重要意义。

中文关键词： O-糖基化；O-GlcNAc糖基化；荧光素-5-氨基硫脲；成像；荧光检测

英文摘要： O-GlcNAcylation, is one of the most common and important posttranslational modification way of protein. And its abnormal expression within cells is associated with variety of deseases such as Alzheimer's disease, diabetes, tumor occurence, et al. Thus, the detection of dynamic level of O-GlcNAcylation, could be used as an effective information for investigating of disease occurrence,development, diagnosis and treatment. Due to the fact that the location and structure complexity, and microscale expression of O-GlcNAcylation glycoprotein within cells, very few sensitive and specific methods can be used for O-GlcNAcylation detecction. And the existing methods used for detecting O-GlcNAcylation suffer from some drawbacks including commercial unavailability and tedious synthesis steps of keto sugar substrates, additional catalysts for labelling reaction and tedious procedure during fluorescence detection. Hence, the detection of O-GlcNAcylation has been hindered in some extent. In our previous work, we have established an innovative synthesis route for the rapid synthesis of the unnatural substrate, 2-keto Ac4GlcNAc or 2-keto Ac4GalNAc, to provide us with basic material used for O-GlcNAcylation detection. Besides that, we also have developed a fluorescent imaging profile technology using fluorecent tag, fluorescein-5

英文关键词： O-glycosylation；O-GlcNAcylation；fluorescein-5-thiosemicarbazide；imaging；fluorescent detection