以谷胱甘肽硫转移酶为受体构建专一识别谷胱甘肽的生物荧光探针及其生物应用

Glutathione (GSH) plays crucial roles in maintaining the appropriate redox status of biological systems. Abnormal levels of GSH were reported to be related with many diseases including neurodegenerative diseases, cancers, AIDS and so on. The development of fluorescent sensors for GSH has attracted a lot of attentions in recent years. Based on the high nucleophilicity of thiol in GSH, the sensors conjugated with GSH, which resulted in fluorescence enhancement or shift in fluorescence emission. However, it is still a challenge to recognize GSH by these small molecule sensors, since other thiol-containing molecules, such as Cysteine (Cys) and Homocysteine (Hcy), can react with sensors in a similar manner. Glutathione S-transferase (GST) is a 26 kDa phase II enzymes that catalyzes the conjugation of GSH to hydrophobic substrate molecules, including a wide range of endogenous and exogenous electrophilic substrates. GST binds GSH with an affinity of 7.1 uM, and displays high selectivity over Cys and Hcy. In this work, we will choose Schistosoma japonicum glutathione S-transferase(sjGST) as a receptor for GSH to construct a selective fluorescent biosensor. A method termed bioorthogonal identification of GSH will be developed, in which a newly developed hydrophobic fluorescent substrate will be firstly bound selectively by a rationally engineered GST, and then react with GSH to release a final fluorescent product with changes in emission intensity or wavelength. Significantly, this biosensor shows high selectivity for GSH with excellent efficiency and sensitivity. The biosensor will be used to detect the abnormal concentration levels of micro scaled GSH in human blood plasma for the diagnosis of HIV infected. By the way of genetically encoded labelling of GST to targetable proteins in various organelles, this biosensor can also be used to map the distribution of GSH in cells.

谷胱甘肽（GSH）是细胞内最主要的含巯基低分子肽，作为解毒剂、抗氧化剂等行使多种生物学功能。GSH的检测可作为多种疾病诊断依据，如神经退行性疾病、癌症、艾滋病等。识别GSH的小分子荧光探针近几年广泛报道，但存在选择性不专一、动态响应窗口窄等缺点。本项目以专一结合GSH的日本血吸虫谷胱甘肽硫转移酶（sjGST）为受体构建生物荧光探针。GST通过催化GSH的巯基与内源或外源性疏水底物偶联而起到解毒作用。本项目通过基因工程方法改造sjGST，与新创造的sjGST荧光底物选择性结合后构建成荧光探针；sjGST选择性结合GSH后催化其巯基与荧光底物发生偶联反应，生成荧光颜色或强度显著变化的产物，从而实现对GSH专一和宽动态响应窗口的荧光检测。将sjGST融合到不同细胞器，研究GSH在细胞内的分布。利用探针检测限低的特点检测血液样本中低浓度的GSH，用于HIV感染诊断。

还原型谷胱甘肽（GSH）是由谷氨酸、半胱氨酸以及甘氨酸组成的一种三肽，参与体内三羧酸循环以及糖代谢，使人体获得高能量。同时GSH能激活多种酶，是一种细胞内重要的代谢物质。另外，GSH还能够参与体内的氧化还原过程，作为解毒剂、抗氧化剂等行使多种生物学功能。GSH的检测可作为多种疾病诊断依据，如神经退行性疾病、癌症、艾滋病等。识别GSH的小分子荧光探针近几年广泛报道，已报道的小分子GSH荧光探针存在选择性不专一、难以响应GSH动态变化、难以检测低浓度GSH、灵敏度易受生物体系复杂环境影响等缺点。本项目以GSH的生物受体谷胱甘肽硫转移酶（GST）为催化剂，将荧光团连接到该酶的专一底物上，构建以GST为受体的专一性识别GSH的生物荧光探针，实现对GSH专一和动态响应的荧光检测。根据酶-底物的专一性识别能力以及探针的高灵敏度，该生物荧光探针不但能够检测血清样本中GSH含量，还能实时检测活细胞内GSH。. 项目执行期间共合成了5个荧光底物。在检测中发现，化合物MPFI-062和 MPFI-074能在sjGST的催化下与GSH专一性作用，化合物中的酶底物上的硝基作为淬灭基团能够淬灭底物荧光，GSH与底物反应后硝基离去，荧光得以显著增强。得益于GST蛋白酶的专一性催化作用，探针对GSH的检测不受体内其他含巯基氨基酸的影响，使其能够在复杂环境中专一响应GSH。基于荧光底物MPFI-062反应后的近红外荧光特性，该探针可用于活细胞内GSH的检测与成像，并且有望应用于活体内GSH的检测。MPFI-074荧光底物能够检测血清内GSH的含量，检测极限能够达到7.9 nM。基于以上研究的相关工作已经发表文章7篇，已经接收的文章3篇，同时申请专利1项。.