Fluorescence Probes for Imaging Basic Carboxypeptidase Activity in Living Cells with High Intracellular Retention
Fluorescence Probes for Imaging Basic Carboxypeptidase Activity in Living Cells with High Intracellular Retention
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用于对具有高细胞内保留的活细胞中的基本羧肽酶活性进行成像的荧光探针
DOI：
10.1021/acs.analchem.0c04793
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发表时间：
2021
影响因子：
7.4
通讯作者：
Urano Yasuteru
中科院分区：
文献类型：
--
作者：
Iwaki Hirohisa;Kamiya Mako;Kawatani Minoru;Kojima Ryosuke;Yamasoba Tatsuya;Urano Yasuteru
碱性羧肽酶（碱性CP）切割肽的C-末端碱性氨基酸，并且它们的活性在某些类型的癌症中上调。因此，检测活细胞中碱性CP的活性不仅对于研究这些酶的生理功能而且对于癌组织的可视化都是重要的。在这里，我们报告了两个荧光素二乙酸酯（FDA）为基础的可激活的荧光探针，命名为5ArgAF-FDA和5LysAF-FDA，其中底物氨基酸精氨酸或赖氨酸通过偶氮甲酰基连接基缀合到苯部分。在七种基本CP之一的CPM的活细胞荧光成像中，5ArgAF-FDA在几分钟内显示出比5LysAF-FDA更大的细胞内荧光增加。通过与碱性CP的抑制剂2-巯基甲基-3-胍基乙基硫代丙酸（MGTA）共孵育，可抑制这种增加。当5ArgAF-FDA应用于具有不同CPM活性的两种乳腺癌细胞系的共培养物时，单个细胞中的荧光增加与CPM的表达水平相关，这表明5ArgAF-FDA由于其高细胞内滞留而具有区分具有不同CPM活性水平的细胞系的能力。我们相信这些探针将是有用的成像与上调的基本CP活动的癌症。
Basic carboxypeptidases (basic CPs) cleave the C-terminal basic amino acid of peptides, and their activity is upregulated in some types of cancers. Therefore, detecting the activity of basic CPs in living cells would be important not only for studying the physiological functions of these enzymes but also for visualization of cancerous tissues. Here, we report two fluorescein diacetate (FDA)-based activatable fluorescence probes, named 5ArgAF-FDA and 5LysAF-FDA, in which the substrate amino acid arginine or lysine is conjugated to the benzene moiety via an azoformyl linker. In live-cell fluorescence imaging of CPM, one of the seven basic CPs, 5ArgAF-FDA showed a larger intracellular fluorescence increase than did 5LysAF-FDA within a few minutes. This increase was inhibited by coincubation with 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), an inhibitor of basic CPs. When 5ArgAF-FDA was applied to a coculture of two breast cancer cell lines with different CPM activities, the fluorescence increase in individual cells was correlated with the expression level of CPM, suggesting that 5ArgAF-FDA has the ability to distinguish cell lines having different levels of CPM activity, owing to its high intracellular retention. We believe these probes will be useful for imaging cancers with upregulated basic CP activity.