DETECTING CHANGES IN CHOLESTEROL ACTIVITY (ACCESSIBILITY) AT THE MEMBRANE SURFACE USING PERFRINGOLYSIN O MUTANTS
Cholesterol is an essential component of mammalian cell membranes and it is important to regulate the structure and function of lipid bilayers. Changes in cholesterol levels are involved in many physiological and pathological events such as the formation of arterial plaques, viral entry into cells, sperm capacitation, and receptor organization. Determination of cholesterol trafficking and distribution is essential for understanding how cells regulate cholesterol activity. A cholesterol probe capable of distinguishing changes in cholesterol chemical activity within membranes would facilitate investigations in this area. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target cell membrane for binding. The specificity of PFO for high levels of active cholesterol makes the toxin a potential tool for the detection of cholesterol distribution and trafficking. In an effort to adapt PFO into a molecular probe capable of sensing changes in membrane cholesterol activity, we have taken a non-lytic derivative of PFO and introduced several point mutations in the membrane-interacting domain 4 loops. These mutations altered the threshold of cholesterol concentration required in the membrane to trigger binding . The cholesterol-dependent binding of each PFO derivative was tested on model membranes containing different amounts of cholesterol. Three PFO derivatives were selected to test their binding to macrophage plasma membranes. These three derivatives showed differential binding to cell membranes treated with ?-methyl-cyclodextrin/cholesterol mixtures. Our data showed that the produced PFO derivatives differentially bind to model and biological membranes containing different cholesterol activity (or accessibility).