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

我们制备了一种非细胞溶解衍生物的perfringolysin O （θ-毒素），一种胆固醇结合的细胞溶解素，作为胆固醇的探针。已经证明，探针（BCθ；蛋白酶缺口和生物素化的θ-毒素）选择性地与完整细胞的富含胆固醇的膜结构域（脂筏）中的胆固醇结合，并且有助于在细胞表面上可视化富含胆固醇的膜结构域。为了进一步观察活细胞内富含胆固醇的膜结构域，我们建立了一个细胞系统。我们已经证明，BCθ对胆固醇的特异性和高亲和力及其靶向富含胆固醇的膜结构域的特性都可归因于θ-毒素的c端结构域（D4）。基于这一发现，我们构建了D4与增强型绿色荧光蛋白（EGFP）的融合蛋白。首先，融合蛋白EGFP-D4在大肠杆菌中过表达并纯化以进行鉴定。我们证实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片段插入哺乳动物基因表达载体中，并导入小鼠成纤维细胞。我们分离了几种不同水平表达EGFP-D4的稳定细胞系，并在荧光显微镜下发现表达的EGFP-D4与细胞内囊泡结构部分相关。目前正在对囊泡进行表征。

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.