Establishment of a cellular system for visualizing intracellular lipid rafts using a new cholesterol-binding probe

使用新型胆固醇结合探针建立细胞内脂筏可视化细胞系统

• 批准号: 14580660
• 负责人: HAYASHI Masami
• 金额: $ 2.3万
• 依托单位: Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580660/
• 关键词: CHOLESTEROL; FIBROBLAST; LIPID RAFTS; PERERINGOLYSIN O; VISUALIZATION; GREEN FLUORESCENT PROTEIN; MICRODOMAIN; ラフト; 線維芽細胞; 脂質マイクロドメイン; 脂質ミクロドメイン; MDCK細胞

We prepared a non-cytolytic derivative of perfringolysin O (θ-toxin), a cholesterol-binding cytolysin, as a probe for cholesterol. It is already shown that the probe (BCθ; protease-nicked and biotinylated θ-toxin) binds selectively to cholesterol in cholesterol-rich membrane domains (lipid rafts) of intact cells, and is useful for visualizing cholesterol-rich membrane domains on the cell surface. To visualize further intracellular cholesterol-rich membrane domains of living cells, we established a cellular system. We have shown that the specificity and high affinity of BCθ for cholesterol and its property of targeting to cholesterol-rich membrane domains can all be ascribed to the C-terminal domain (D4) of θ-toxin. Based on this finding, we constructed a fused protein of D4 and enhanced green fluorescent protein (EGFP). First, the fused protein, EGFP-D4,was overexpressed in E. coli and purified for characterization. We confirmed that EGFP-D4 is a fluorescent protein targeting to cholesterol-rich membrane domains. Then, an EGFP-D4 DNA fragment was inserted into a vector for mammalian gene expression and introduced into mouse fibroblasts. We isolated several stable cell lines expressing EGFP-D4 at variable levels and showed that the expressed EGFP-D4 was partially associated with intracellular vesicular structures by fluorescence microscopy. Characterization of the vesicles is now under way.

我们制备了产气荚膜梭菌溶素O（θ-毒素）的非溶细胞衍生物，作为胆固醇的探针。已经表明，探针（BCθ;蛋白酶切口和生物素化的θ-毒素）选择性地结合完整细胞的富含胆固醇的膜结构域（脂筏）中的胆固醇，并且可用于可视化细胞表面上的富含胆固醇的膜结构域。为了进一步可视化活细胞的细胞内富含胆固醇的膜结构域，我们建立了细胞系统。BCθ对胆固醇的特异性和高亲和力以及其靶向富含胆固醇的膜结构域的特性都可以归因于θ毒素的C-末端结构域（D4）。在此基础上，我们构建了D4与增强型绿色荧光蛋白（EGFP）的融合蛋白。首先，在大肠杆菌中过量表达融合蛋白EGFP-D4。大肠杆菌并纯化以进行表征。我们证实了EGFP-D4是一种靶向富含胆固醇的膜结构域的荧光蛋白。然后，将EGFP-D4 DNA片段插入用于哺乳动物基因表达的载体中并导入小鼠成纤维细胞。我们分离了几个稳定的细胞株表达EGFP-D4在不同的水平，并表明所表达的EGFP-D4部分与细胞内的囊泡结构的荧光显微镜。目前正在对囊泡进行表征。

项目成果

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Ohno-Iwashita, Y.等人：“Perfringolysin O，一种胆固醇结合溶细胞素，作为脂筏探针”厌氧菌。

Heijnen, H.F.G.et al.: "Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains"J.Thrombosis and Haemostasis. 1(6). 1161-1173 (2003)

Heijnen, H.F.G. 等人：“血小板丝状伪足中筏的浓度与这些域中 c-Src 和 CD63 的募集相关”J. 血栓形成和止血。

Terashita, A. et al.: "Lipid binding activity of a neuron-specific protein NAP-22 studied in vivo and in vitro"J. Neurosci. Res.. 70(2). 172-179 (2002)

Terashita, A. 等人：“体内和体外研究的神经元特异性蛋白 NAP-22 的脂质结合活性”J.

Reid, P.C.et al.: "A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain"J.Lipid Res.. 45(3). 582-591 (2004)

Reid, P.C. 等人：“一种新型胆固醇染色揭示了 Niemann-Pick 型 C1 小鼠大脑中早期神经元胆固醇的积累”J.Lipid Res. 45(3)。

Aoki, T.et al.: "Sequestration of cross-linked membrane molecules to caveolae in two different pathways"Acta Histochemica et Cytochemica. 36(2). 165-171 (2003)

Aoki，T.等人：“交联膜分子以两种不同途径螯合到小窝”《组织化学与细胞化学学报》。