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. 2003 Apr;84(4):2382-92.
doi: 10.1016/S0006-3495(03)75044-9.

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Boyan B Bonev  1 , Yuen-Han Lam, Gregor Anderluh, Anthony Watts, Raymond S Norton, Frances Separovic
Affiliations

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Boyan B Bonev et al. Biophys J. 2003 Apr.

Abstract

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.

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Figures

FIGURE 1
FIGURE 1
Phosphorus-31 wideline NMR spectra from hydrated DMPC/SM/EqtII suspensions at 25°C: (a) DMPC; (b) 1000 DMPC: 1 EqtII; (c) 1000 DMPC: 100 SM; (d) 1000 DMPC: 100 SM: 1 EqtII; and (e) 1000 DMPC: 100 SM: 4 EqtII; and at 40°C: (f) DMPC; (g) 1000 DMPC: 1 EqtII; (h) 1000 DMPC: 100 SM; (i) 1000 DMPC: 100 SM: 1 EqtII; and (j) 1000 DMPC: 100 SM: 4 EqtII. The acquisition parameters are described in Materials and Methods.
FIGURE 2
FIGURE 2
Deuterium wideline NMR spectra from hydrated DMPC/SM/EqtII suspensions at 25°C: (a) DMPC; (b) 1000 DMPC: 1 EqtII; (c) 1000 DMPC: 100 SM; (d) 1000 DMPC: 100 SM: 1 EqtII; and (e) 1000 DMPC: 100 SM: 4 EqtII; and at 40°C: (f) DMPC; (g) 1000 DMPC: 1 EqtII; (h) 1000 DMPC: 100 SM; (i) 1000 DMPC: 100 SM: 1 EqtII; and (j) 1000 DMPC: 100 SM: 4 EqtII. The acquisition parameters are described in Materials and Methods.
FIGURE 3
FIGURE 3
Freeze-fracture electron micrographs of hydrated DMPC/SM/EqtII (1000:100:4 mol ratio) suspension (from top to bottom): (a) 46,000× magnification; (b) 130,000× magnification; and (c) another view at 46,000× magnification.
FIGURE 4
FIGURE 4
Phosphorus-31 MAS NMR at 40°C and 12 kHz spinning speed (500 MHz proton frequency) spectra from DMPC/SM mixtures in a MLV suspension: DMPC/SM (10:1 mol ratio) (a), and DMPC/SM /EqtII (1000:100:4 mol ratio) following 20 min benchtop centrifugation to produce a pellet (b), and supernatant (c).
FIGURE 5
FIGURE 5
Phosphorus-31 longitudinal relaxation time, T1, as a function of temperature recorded at 81 MHz from DMPC bilayers (♦), SM bilayers (▪), PC in 1000 DMPC: 100 SM bilayer mixtures (▴), PC in 1000 DMPC: 100 SM bilayers (▴), PC in 1000 DMPC: 4 EqtII bilayer mixtures (◊), PC in 1000 DMPC: 100 SM: 4 EqtII bilayer mixtures (×) and SM in 1000 DMPC: 100 SM: 4 EqtII bilayer mixtures (□). The acquisition parameters are described in Materials and Methods.
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