Poster Presentation The 3rd Prato Conference on Pore Forming Proteins 2015

Interaction of fluorescent fusion protein ostreolysin A with artificial lipid membranes is modulated by lysophosphatidylcholine  (#41)

Maja Grundner 1 , Matej Skočaj 2 3 , Jure Derganc 1 , Špela Zemljič Jokhadar 1 , Vesna Hodnik 3 , Peter Maček 2 , Kristina Sepcic 2
  1. Institute of biopysics, Medical faculty, University of Ljubljana, Ljubljana, Slovenia
  2. Institute of cell biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
  3. Department of biology, Biotehnical faculty, University of Ljubljana, Ljubljana, Slovenia

Ostreolysin A (OlyA) is a 15 kDa protein from the aegerolysin family, which binds specifically to membrane domains rich in cholesterol and sphingomyelin [1]. Its interaction with lipid membranes can be abolished by lysophospholipids [2]. Fluorescent fusion protein OlyA-mCherry has recently been produced and shows an interesting potential as a marker for membrane rafts in living or fixed cells [3].

In order to better elucidate the interaction of OlyA-mCherry with lipid membranes, its binding to artificial mono- and bilayers has been assessed in this study. Interaction of OlyA-mCherry and lysophosphatidylcholine with lipid monolayers, lipid droplets, and large and giant unilamellar vesicles has been analysed by using surface plasmon resonance and Langmuir balance.

Surface plasmon resonance experiments using on-chip immobilized OlyA revealed its direct interaction with lysophosphatidylcholine with a KD of 42.5 mM. In low micromolar concentrations, lysophosphatidylcholine was also able to prevent the OlyA-mCherry binding to chip-immobilized lipid bilayers. This suggests the double role of lysophosphatidiylcholine, which can influence the protein binding both via a direct interaction, and by modulating the physical properties of the membrane. The later hypothesis is reinforced also by our experimental finding that lysophosphatidylcholine can decrease the cholesterol availability of sphingomyelin/cholesterol bilayers, making them less susceptible for interaction with the protein.

In contrast to lipid vesicles composed of cholesterol and sphingomyelin, OlyA-mCherry could not bind onto, or penetrate the lipid monolayers of the same lipid composition, indicating that the specific lipid pattern is a prerequisite for its binding. Finally, the specific interaction of OlyA-mCherry with liquid-ordered phase was confirmed by following its binding to phase-segregated giant unilamellar vesicles.

Our combined results suggest that, in addition to physical characteristics and lipid composition of membrane nanodomains, their specific distribution and availability are essential prerequisites for OlyA-mCherry membrane binding.