Comparison of the C2A Domain of Synaptotagmin-I and Annexin-V As Probes for Detecting Cell Death
Comparison of the C2A Domain of Synaptotagmin-I and Annexin-V As Probes for Detecting Cell Death
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DOI：
10.1021/bc9004415
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发表时间：
2010-05-01
影响因子：
4.7
通讯作者：
Brindle, Kevin M.
中科院分区：
文献类型：
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作者：
Alam, Israt S.;Neves, Andre A.;Brindle, Kevin M.
细胞凋亡的诱导常常伴随着细胞表面磷脂酰丝氨酸 (PS) 的暴露，这已使用放射性核素和 PS 结合蛋白膜联蛋白 V 的荧光标记衍生物进行检测。荧光标记蛋白已在体外广泛用作检测细胞死亡的诊断试剂，放射性核素标记衍生物已进行用于检测治疗后体内肿瘤细胞死亡的临床试验。我们在此表明，突触结合蛋白-I 的 C2A 结构域在通过定点诱变引入的单个半胱氨酸残基上进行了荧光标记，在体外药物处理的小鼠淋巴瘤和人乳腺癌细胞系中，检测到与类似标记的膜联蛋白-V 衍生物相同水平的细胞死亡。然而，C2A 衍生物与活细胞的结合明显减少，因此与膜联蛋白-V 相比，与凋亡和坏死细胞的结合特异性高出 4 倍。 C2A 为开发新一代更具特异性的成像探针以在临床上检测肿瘤细胞死亡提供了潜在途径。
The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell death, and radionuclide-labeled derivatives have undergone clinical trials for detecting tumor cell death in vivo following treatment. We show here that the C2A domain of Synaptotagmin-I, which had been fluorescently labeled at a single cysteine residue introduced by site-directed mutagenesis, detected the same levels of cell death as a similarly labeled Annexin-V derivative, in drug-treated murine lymphoma and human breast cancer cell lines in vitro. However, the C2A derivative showed significantly less binding to viable cells and, as a consequence, up to 4-fold more specific binding to apoptotic and necrotic cells when compared with Annexin-V. C2A offers a potential route for the development of a new generation of more specific imaging probes for the detection of tumor cell death in the clinic.