Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin.
Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.
Bonev
Boyan B
BB
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
Lam
Yuen-Han
YH
Anderluh
Gregor
G
Watts
Anthony
A
Norton
Raymond S
RS
Separovic
Frances
F
D003064:Cnidarian Venoms / Q000737:chemistry*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008560:Membrane Fluidity; D008854:Microscopy, Electron; D008968:Molecular Conformation; D010539:Permeability; D016062:Porosity; D013109:Sphingomyelins / Q000737:chemistry*; D013329:Structure-Activity Relationship
D003064:Cnidarian Venoms; D008051:Lipid Bilayers; D013109:Sphingomyelins; C012307:equinatoxin; D004134:Dimyristoylphosphatidylcholine