Phase transition in sphingomyelin/cholesterol vesicles observed with EPR coincides with the increased activity of a cytolytic protein ostreolysin

K. Rebolj1, B. Bakrač1, T. Koklič2, K. Sepčić1, P. Maček1, M. Šentjurc2

1Department of Biology, Biotechnical faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia; 2J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

Ostreolysin (Oly) is a cytolytic 15 kDa protein from the edible oyster mushroom (Pleurotus ostreatus). Preliminary results have shown the specific interaction of Oly with liquid-ordered membrane domains, Lo¹. In this work sonicated vesicles composed of varying proportions of cholesterol (Chol) and sphingomyelin (SM) were used to mimic membrane Lo domains. The domain structure of spin labelled vesicles was studied using two different spin probes (5-doxyl derivative of palmitic acid methyl ester and spin-labelled cholesterol). The major change in lateral domain structure at 25°C was observed between 30 and 40 mol % of Chol vs. SM. This is in accordance with previous studies of the same mixtures with DPH fluorescence quenching², showing the appearance of Lo phase above 30% Chol. The results also coincide with ostreolysin binding and permeabilization of the same vesicles, both effects appearing above 30% Chol. EPR measurements of ostreolysin binding showed no effect on membrane arrangement, suggesting that Oly does not induce the formation of liquid-ordered domains in membranes. We conclude that EPR is a suitable technique for studying the heterogeneous domain structure of lipid membranes. The observed phase transitions are comparable to those observed with other techniques. Moreover, EPR with GHOST condensation routine enables the determination of different modes of spin label motion which originate from nanoscale lateral membrane regions, which are not commonly detected with other techniques.
¹Sepčić K., Berne S., Rebolj K., Batista U., Plemenitaš A., Šentjurc M., Maček P. (2004) Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains. FEBS Lett. 575(1-3): 81-5.
²de Almeida R.F.M., Fedorov A., Prieto M. (2003) Sphingomyelin/Phosphatidylcholin/Cholesterol Phase Diagram: Boundaries and Composition of Lipid Rafts. Biophy. J. 85, 2406-2416.

Email: katja.rebolj@bf.uni-lj.si

Address: Department of Biology, Biotechnical faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia