Sphingomyelin‐Cholesterol Complexes In Plasma Membranes

Abstract

Sphingomyelin(SM) and cholesterol are lipids that are critical for the function of animal cells. Interplay between these two lipids is important for maintaining integrity of the myelin sheath that encases axonal cells and for maintaining functionally distinct pools of cholesterol in the plasma membranes of fibroblast cells. The nature of the interaction between SM and cholesterol has been long debated, with proposals ranging from complexes of the two lipids to no specific interaction between these two lipids.Here, we have developed a sensor to examine the interaction between SM and cholesterol in animal cell membranes. Our sensor is derived from a fungal toxin, ostreolysin A (OlyA). We find that OlyA binds to membranes of CHO‐K1 hamster cells when they contain both SM and cholesterol, but not when levels of either lipid are diminished by treatment with sphingomyelinase (which degrades SM) or cyclodextrin (which depletes cholesterol). In model membranes, OlyA shows no binding to dioleoyl‐phosphatidylcholine – cholesterol membranes even when the cholesterol concentration exceeds 50 mole%. Also, OlyA shows no binding to SM‐containing membranes when epicholesterol (a diasteromer of cholesterol) is present at concentration exceeding 50 mole%. Using X‐ray crystallography, we studied the interaction of OlyA with SM and cholesterol at the atomic level. This structural analysis combined with detailed mutagenesis led us to a single point mutation in OlyA that abolishes its cholesterol specificity while retaining SM specificity. Comparing the X‐ray structures of these two versions of lipid‐bound OlyA combined with ligand docking simulations revealed two distinct binding modes of OlyA to SM. We propose that one of these SM conformations reflects a complex with cholesterol. Studies in live cells show that this pool of SM/cholesterol complexes in plasma membrane is maintained at a constant level across a large range of cholesterol concentrations.Support or Funding InformationThis work was supported by the Welch Foundation (I‐1793) and the American Heart Association (12SDG12040267)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.