Relevance of lipid phase coexistence and membrane fluidity on membrane insertion and permeabilizing activity of Sticholysins, two pore-forming toxins

LOHANS PEDRERA PUENTES; URIS ROS QUINCOCES; RAFAEL SÁNCHEZ PUPO; MARÍA LAURA FANANI; BRUNO MAGGIO; CARLOS ÁLVAREZ VALCÁRCEL

Tucuman

Congreso; XLI Reunion Annual Sociedad Argentina de Biofísica; 2012

Sticholysin I and II (Sts) are pore-forming proteins produced by the sea anemone Stichodactyla helianthus belonging to the actinoporin family (1). Those toxins binds oligomerize and finally destabilize natural and artificial membranes through the formation of oligomeric pores. Binding and pore-formation of actinoporins depend on the physico-chemical properties of the membrane. The effect of fluidity and coexistence of lipid microdomains has been barely examined for these proteins. We performed a systematic study of the effect of sphingomyelin (SM) content in phosphatidylcholine (PC)/SM mixed monolayers and liposomes on both toxins binding and permeabilizing activity. Our studies demonstrated that the binding and pore inducing capability of both proteins are maximal against mixed membranes containing gel/fluid phase. We studied how variations in fatty acid chain length and unsaturation of PC modify binding and activity of Sts on model PC:SM systems. Sts´ activity increased with decreasing acyl chain length or saturation of PC-fatty acids. Also, their association to membrane occurred more effectively when including PCs with shorter or unsaturated fatty acyl chains. Since those PCs turn liposomal membranes less tightly packed, this can explain the higher propensity of toxin molecules to bind and oligomerize under these conditions. To get insights into the role of lipid microdomains on the action of Sts we performed studies in SM, PC and sterol systems that promote or not lateral domain formation. The coexistence of gel/fluid phases in PC:SM mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in PC:SM:sterol mixtures favor toxin-membrane association and oligomerization. This could expand the disordered phase, a more suitable environment than lipid rafts domains for N-terminal insertion and pore formation by Sts. Altogether these results stress the relevance of membrane fluidity and domain formation for St’s activity and consequently pore-formation.