Sticholysins, Sphingomyelin and Cholesterol: A Closer Look into a Tripartite Interaction

Abstract

Actinoporins are a widely studied group of toxins produced by sea anemones. Their mechanism of action consists of binding to sphingomyelin (SM)-containing membranes. Once attached to the bilayer, they oligomerize and form cation-selective pores. Although SM is usually sufficient to recruit these toxins, the presence of cholesterol in these membranes is also known to greatly facilitate the process. Despite years of research, there are still many unknown details about the actinoporin-membrane interaction. What is the most favorable phase state of the membrane for their action? What happens to SM distribution in the membrane after the toxins bind to the bilayer? What is the exact role of cholesterol and how do these proteins interact with it? To shed some light on these questions we have tested the effect of sticholysins, the actinoporins produced by the Caribbean sea anemone Stichodactyla helianthus, on bilayers of varied compositions. To study the behavior of SMs in the events subsequent to toxin binding, we took advantage of the properties of fluorescent SM analogs labelled with pyrene. By measuring the extent of the excimer to monomer ratio from the emission of pyrene we were able to assess the degree of SM-SM interaction before and after pore formation. Similarly, we used a fluorescent analog of cholesterol, cholestatrienol, to gain further knowledge on the interaction between sticholysins and sterols. To do this, we used a RET approach in which the tryptophan residues of sticholysins acted as donors.