Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex

J Biol Chem. 2004 Jun 25;279(26):26975-82. doi: 10.1074/jbc.M402676200. Epub 2004 Apr 14.


Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cholesterol / chemistry
  • Dogs
  • Erythrocyte Membrane / drug effects
  • Erythrocyte Membrane / metabolism
  • Erythrocytes / drug effects
  • Erythrocytes / metabolism
  • Hemoglobins / metabolism
  • Hemolysin Proteins / chemistry*
  • Hemolysin Proteins / isolation & purification
  • Hemolysin Proteins / metabolism*
  • Hemolysis / drug effects
  • Horses
  • Humans
  • Liposomes / chemistry
  • Liposomes / metabolism
  • Liposomes / pharmacology
  • Mycotoxins / chemistry*
  • Mycotoxins / isolation & purification
  • Mycotoxins / metabolism*
  • Phospholipids / chemistry
  • Pleurotus / chemistry*
  • Potassium / chemistry
  • Potassium / metabolism
  • Rabbits
  • Sheep
  • Sphingomyelins / metabolism
  • Sphingomyelins / pharmacology*


  • Hemoglobins
  • Hemolysin Proteins
  • Liposomes
  • Mycotoxins
  • Phospholipids
  • Sphingomyelins
  • Cholesterol
  • Potassium