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

我们制备了一种与胆固醇结合的细胞溶血素θ-毒素的非溶细胞性衍生物，作为胆固醇的探针。已经证明，该探针(BCθ；蛋白酶刻痕和生物素标记的θ毒素)选择性地与完整细胞的富含胆固醇的膜域(脂筏)中的胆固醇结合，并有助于显示细胞表面的富含胆固醇的膜域。为了进一步显示活细胞内富含胆固醇的膜域，我们建立了一个细胞系统。我们发现BC-θ对胆固醇的特异性和高亲和力以及其靶向高胆固醇膜结构域的特性都可以归因于θ-毒素的C-末端结构域(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，在大肠杆菌中高效表达融合蛋白EGFP-D4，并对其进行纯化鉴定。我们证实了EGFP-D4是一种针对富含胆固醇膜结构域的荧光蛋白。然后，将EGFP-D4DNA片段插入哺乳动物基因表达载体，并导入小鼠成纤维细胞。我们分离了几个稳定表达不同水平的EGFP-D4的细胞系，荧光显微镜下显示表达的EGFP-D4部分与细胞内的囊泡结构有关。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.