Kekulandara Dilini N, Samarasinghe Kusal T G, Munkanatta Godage Dhanushka N P, Ahn Young-Hoon
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Org Biomol Chem. 2016 Nov 22;14(46):10886-10893. doi: 10.1039/c6ob02050j.
Protein glutathionylation is one of the major cysteine oxidative modifications in response to reactive oxygen species (ROS). We recently developed a clickable glutathione approach for detecting glutathionylation by using a glutathione synthetase mutant (GS M4) that synthesizes azido-glutathione (γGlu-Cys-azido-Ala) in situ in cells. In order to demonstrate the versatility of clickable glutathione and to increase the chemical tools for detecting glutathionylation, we sought to develop clickable glutathione that uses tetrazine-alkene bioorthogonal chemistry. Here we report two alkene-containing glycine surrogates (allyl-Gly and allyl-Ser) for the biosynthesis of clickable glutathione and their use for detection, enrichment, and identification of glutathionylated proteins. Our results provide chemical tools (allyl-Gly and allyl-Ser for GS M4) for versatile characterization of protein glutathionylation. In addition, we show that the active site of GS can be tuned to introduce a small size chemical tag on glutathione for exploring glutathione function in cells.
蛋白质谷胱甘肽化是细胞对活性氧（ROS）反应时主要的半胱氨酸氧化修饰之一。我们最近开发了一种可点击的谷胱甘肽方法，通过使用一种谷胱甘肽合成酶突变体（GS M4）来检测谷胱甘肽化，该突变体可在细胞内原位合成叠氮基谷胱甘肽（γGlu-Cys-azido-Ala）。为了证明可点击谷胱甘肽的通用性并增加检测谷胱甘肽化的化学工具，我们试图开发利用四嗪-烯烃生物正交化学的可点击谷胱甘肽。在此我们报告了两种用于生物合成可点击谷胱甘肽的含烯丙基甘氨酸替代物（烯丙基甘氨酸和烯丙基丝氨酸）及其用于检测、富集和鉴定谷胱甘肽化蛋白质的用途。我们的结果为蛋白质谷胱甘肽化的通用表征提供了化学工具（用于GS M4的烯丙基甘氨酸和烯丙基丝氨酸）。此外，我们表明可以调节GS的活性位点，在谷胱甘肽上引入一个小尺寸化学标签，以探索谷胱甘肽在细胞中的功能。
