Inhibition of Tolaasin Hemolytic Activity by Increase in GD3+-Induced Membrane Rigidity

Abstract

Tolaasin molecule multimerizes and forms a membrane pore, causing brown blotch disease of the cultivated mushrooms. The peptide is a pore-forming bacterial toxin produced by Pseudomonas tolaasii. The pores destroy plasma membrane by interrupting cellular osmotic pressure. Cytotoxicity of tolaasin can be evaluated by measuring hemolytic activity using erythrocytes and blotch formations on the mushroom tissue. Previous studies showed that divalent cations, such as Zn2+, Ni2+, and Cd2+, inhibit tolaasin-induced hemolysis. In this study, Gadolinium ion, Gd3+, known to inhibit the mechanosensitive ion channels, also inhibits the tolaasin activity as a dose-dependent manner. The inhibition of tolaasin-induced hemolysis was observed at 200 μM Gd3+ and completed at 1 mM. Mechanism of Gd3+ action has not been clearly demonstrated; however, it has been known that Gd3+ decreases membrane fluidity. Inhibition of hemolytic activity by Gd3+ was reversible similar to that of Zn2+, but the action mechanisms of these two ions seem to be very different. Although Zn2+ inhibits the ion channel by plugging into the entrance of tolaasin pore, Gd3+ may inhibit the formation of tolaasin channel by changing membrane fluidity. To investigate the effect of Gd3+ on the plasma membrane of erythrocyte, a fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), was inserted into the membrane and the changes in fluorescence intensity were measured. The maximum fluorescence intensity of DPH was measured at 360 nm in excitation and at 456 nm in emission. In the presence of Gd3+, the fluorescence intensity of DPH was increased, suggesting that Gd3+ made the membrane more rigid. Therefore, the Gd3+-mediated increase in membrane rigidity reduces the rate of molecular diffusion in the membrane and may inhibit the multimerization and orientation of tolaasin molecules requiring for membrane pore formation.