Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
Biochemistry. 2012 Apr 24;51(16):3373-82. doi: 10.1021/bi3003132. Epub 2012 Apr 13.
Changes in the cholesterol content of cell membranes affect many physiological and pathological events, including the formation of arterial plaques, the entry of virus into cells, and receptor organization. Measuring the trafficking and distribution of cholesterol is essential to understanding how cells regulate sterol levels in membranes. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target membrane for binding. The specificity of PFO for high levels of cholesterol makes the toxin an attractive tool for studying the distribution and trafficking of cholesterol in cells. However, the use of the native toxin is limited given that binding is triggered only above a determined cholesterol concentration. To this end, we have identified mutations in PFO that altered the threshold for how much cholesterol is required to trigger binding. The cholesterol threshold among different PFO derivatives varied up to 10 mol % sterol, and these variations were not dependent on the lipid composition of the membrane. We characterized the binding of these PFO derivatives on murine macrophage-like cells whose cholesterol content was reduced or augmented. Our findings revealed that engineered PFO derivatives differentially associated with these cells in response to changes in cholesterol levels in the plasma membrane.
细胞膜中胆固醇含量的变化会影响许多生理和病理事件，包括动脉斑块的形成、病毒进入细胞以及受体的组织。测量胆固醇的运输和分布对于了解细胞如何调节膜中固醇水平至关重要。产气荚膜梭菌分泌的细胞溶素 PFO 需要靶细胞膜中的胆固醇才能结合。PFO 对高胆固醇水平的特异性使该毒素成为研究细胞中胆固醇分布和运输的有吸引力的工具。然而，由于只有在确定的胆固醇浓度以上结合才会被触发，因此天然毒素的使用受到限制。为此，我们已经鉴定出 PFO 中的突变，这些突变改变了触发结合所需的胆固醇量的阈值。不同 PFO 衍生物之间的胆固醇阈值差异高达 10 mol %固醇，并且这些变化不依赖于膜的脂质组成。我们对胆固醇含量降低或增加的鼠巨噬样细胞上这些 PFO 衍生物的结合进行了表征。我们的研究结果表明，针对质膜中胆固醇水平的变化，工程化的 PFO 衍生物以不同的方式与这些细胞相关联。
