A BIOTINYLATED PERFRINGOLYSIN-O DERIVATIVE - A NEW PROBE FOR DETECTION OF CELL-SURFACE CHOLESTEROL

theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostr idium perfringens. To detect cell surface cholesterol, we prepared a t heta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as th eta-toxin without cytolytic activity. Human erythrocytes, V79 cells an d human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by e ither flow cytometry or laser confocal microscopy. The fluorescence in tensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither tryp an blue nor propidium iodide, indicating that BC theta stained just th e outer surface of the plasma membrane of vital cells. Treatment of th e cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensit y of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold. suggesting higher cholesterol contents in the inner layer of thp plasma membrane. When cells were cultured wi th cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVE C. This indicates that cell surface cholesterol may be provided in dif ferent ways with these two cell lines. These results suggest that BC t heta can be a useful probe for visualizing cell surface cholesterol an d for evaluating the effects of cellular events on the topology and di stribution of cholesterol. (C) 1997 Elsevier Science B.V.