O-GlcNAc糖基化修饰的细胞成像及荧光检测新方法研究

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-thiosemicarbazide (FTSC). Based on the previous research progress, our studies will utilize the improved keto sugar substrate synthesis method established by our group, and the strengths of reaction activity of FTSC with ketones under physiological conditions, to establish a simple, efficient and reliable method for imaging O-GlcNAcylation within living cell. Furthermore, we will employ the strengths of β-1,4-galactosyltransferase (GalT) that could transfer UDP-Ac4GalNAc to O-GlcNAc residue, combine with FTSC ligation of ketone, to develop a strategy for detection of O-GlcNAcylation of known protein ex vivo. Our studies will improve and perfect the existing methods for O-GlcNAcylation detection, and facilitate the development of O-GlcNAcylation in proteomics.

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。

蛋白质O-GlcNAc糖基化是细胞内一种常见翻译后修饰，参与调控细胞的生长、分化、凋亡、压力应激等众多生理生化和信号转导活动，而其异常表达则与多种疾病如癌症、老年痴呆症、糖尿病等的发生发展密切相关。由于O-GlcNAc糖基化修饰具有重要的生物学功能和指示意义，对细胞内部进行该种糖基化修饰的动态监测对研究其功能意义重大。而现有的检测方法存在代谢用底物尚未商品化和合成步骤复杂；同时后续的荧光检测需催化剂催化、操作复杂、耗时等缺陷，因而有关O-GlcNAc糖基化修饰的功能机制研究等受到很大限制。通过本研究的开展，建立了生理条件下，荧光试剂与非还原性醛糖、酮糖进行标记的方法，为后续细胞水平O-GlcNAc糖基化修饰的荧光标记和成像研究提供了方法参考；通过建立的化学法合成糖代谢底物类似物，基于寡糖代谢的将带有酮基的底物类似物转移至O-GlcNAc糖链中，建立了O-GlcNAc糖基化修饰的细胞内荧光原位成像的可视化检测方法；为了对其结构进行解析，同时建立了一斧法O-糖链解离同时荧光标记的技术，在此基础上建立了基于稳定同位素O-糖链质谱相对定量分析方法，为研究O-GlcNAc糖基化修饰的结构包括糖基化位点，以及其在疾病发生发展中相关性机制研究提供了有力方法；建立了微量生物糖链的质谱前处理富集纯化方法，为系统性研究生物样品中低丰度表达的糖蛋白的O-GlcNAc糖基化修饰提供了有效方法；初步建立了基于蛋白芯片和化学酶法技术鉴定和筛选O-GlcNAc糖基化修饰的荧光检测方法。在项目的支持下,发表SCI论文5篇;第一作者SCI论文2篇,通讯作者SCI论文1篇;多次参加国内外相关领域专业学术会议,与同行专家进行交流和学习,提交会议论文8篇;培养已毕业研究生5人，在研博士研究生1人，在研硕士研究生4人；项目负责人2014年获评陕西省优秀博士论文奖，2015年以第四参与者获“张树政糖科学优秀青年奖”。