ASSEMBLY OF FLAMMUTOXIN, A CYTOLYTIC PROTEIN FROM THE EDIBLE MUSHROOMFLAMMULINA VELUTIPES, INTO A PORE-FORMING RING-SHAPED OLIGOMER ON THETARGET-CELL

Flammutoxin has been previously isolated as a cardiotoxic and cytolyti c polypeptide of 22 or 32 kDa from the fruiting bodies of the edible m ushroom Flammulina velutipes. In the present study, we purified flammu toxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non -electrolytes with hydrodynamic diameters of > 5.0 nm, although it cau sed leakage of potassium ions and swelling of the cells under the same conditions, (3) Experiments including solubilization of cell-bound to xin with 2% (w/v) SDS at 20 degrees C and subsequent Western immunoblo ts showed that flammutoxin formed a band corresponding to 180 kDa unde r the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a r ing-shaped structure with outer and inner diameters of 10 and 5 nm, re spectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 degrees C and isolated by a sucrose-gradient u ltracentrifugation. These data indicated that flammutoxin assembles in to a ring-shaped oligomer possessing a hydrophilic pore of 4-5 nm on t arget cells.