Visualization of Phagosomal Hydrogen Peroxide Production by a Novel Fluorescent Probe That Is Localized via SNAP-tag Labeling
Visualization of Phagosomal Hydrogen Peroxide Production by a Novel Fluorescent Probe That Is Localized via SNAP-tag Labeling
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DOI：
10.1021/ac501041w
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发表时间：
2014-06-17
影响因子：
7.4
通讯作者：
Sumimoto, Hideki
中科院分区：
文献类型：
--
作者：
Abo, Masahiro;Minakami, Reiko;Sumimoto, Hideki
过氧化氢（H2 O2）是活性氧（ROS）的一种，在机体防御和细胞信号转导等方面发挥着重要的生理作用。在摄入入侵微生物期间，专职吞噬细胞如巨噬细胞将H2 O2特异性地释放到吞噬体中，以将毒性ROS导向被吞噬的微生物。虽然H2 O2被认为在生命系统中发挥离散的影响，这取决于它的生产，积累和消费的位置，有探测这种氧代谢产物的技术在亚细胞分辨率的高分子特异性的限制。在这里，我们描述了一个O-6-苄基鸟嘌呤衍生物的5-（4-硝基苯甲酰基）-羰基荧光素（NBzF-BG），一种新的H2 O2特异性荧光探针的发展; NBzF-BG共价和选择性地与SNAP标签蛋白结合，导致形成的荧光团-蛋白质结合物（SNAP-NBzF）。SNAP-NBzF与H2 O2快速反应，从而显示出9倍的荧光增强。当SNAP-标签在HEK 293 T细胞和RAW264.7巨噬细胞中表达为与血小板衍生生长因子受体（PDGFR）的跨膜结构域C端融合的蛋白质时，标签存在于质膜的外部; NBzF-BG与细胞表面SNAP-标签的缀合使得能够检测外源添加的H2 O2。我们还证明了H2 O2的分子成像，这是内源性产生的巨噬细胞的吞噬体ingglutaminIgG包被的乳胶珠。因此，NBzF-BG与SNAP标签技术相结合，应该是一种有用的工具，以测量活细胞中H2 O2的局部生产。
Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O-6-benzylguanine derivative of 5-(4-nitrobenzoyI)-carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence. When SNAP-tag is expressed in HEK293T cells and RAW264.7 macrophages as a protein C-terminally fused to the transmembrane domain of platelet-derived growth factor receptor (PDGFR), the tag is presented on the outside of the plasma membrane; conjugation of NBzF-BG with the cell surface SNAP-tag enables detection of H2O2 added exogenously. We also demonstrate molecular imaging of H2O2 that is endogenously produced in phagosomes of macrophages ingesting IgG-coated latex beads. Thus, NBzF-BG, combined with the SNAP-tag technology, should be useful as a tool to measure local production of H2O2 in living cells.