On the importance of the phosphocholine methyl groups for sphingomyelin/cholesterol interactions in membranes: a study with ceramide phosphoethanolamine

Abstract

In this study, we have examined how the headgroup size and properties affect the membrane properties of sphingomyelin and interactions with cholesterol. We prepared N-palmitoyl ceramide phosphoethanolamine (PCPE) and compared its membrane behavior with D-erythro-N-palmitoyl-sphingomyelin (PSM), both in monolayers and bilayers. The pure PCPE monolayer did not show a phase transition at 22 degrees C (in contrast to PSM), but displayed a much higher inverse isothermal compressibility as compared to the PSM monolayer, indicating stronger intermolecular interactions between PCPEs than between PSMs. At 37 degrees C the PCPE monolayer was more expanded (than at 22 degrees C) and displayed a rather poorly defined phase transition. When cholesterol was comixed into the monolayer, a condensing effect of cholesterol on the lateral packing of the lipids in the monolayer could be observed. The phase transition from an ordered to a disordered state in bilayer membranes was determined by diphenylhexatriene steady-state anisotropy. Whereas the PSM bilayer became disordered at 41 degrees C, the PCPE bilayer main transition occurred around 64 degrees C. The diphenylhexatriene steady-state anisotropy values were similar in both PCPE and PSM bilayers before and after the phase transition, suggesting that the order in the hydrophobic core in both bilayer types was rather similar. The emission from Laurdan was blue shifted in PCPE bilayers in the gel phase when compared to the emission spectra from PSM bilayers, and the blue-shifted component in PCPE bilayers was retained also after the phase transition, suggesting that Laurdan molecules sensed a more hydrophobic environment at the PCPE interface compared to the PSM interface both below and above the bilayer melting temperature. Whereas PSM was able to form sterol-enriched domains in dominantly fluid bilayers (as determined from cholestatrienol dequenching experiments), PCPE failed to form such domains, suggesting that the size and/or properties of the headgroup was important for stabilizing sphingolipid/sterol interaction. In conclusion, our study has highlighted how the headgroup in sphingomyelin affect its membrane properties and interactions with cholesterol.

FIGURE 1

Force-area isoterms of binary mixtures of PCPE and cholesterol. (A) The surface pressure versus mean molecular area isotherms for PCPE at 22°C and 37°C. (B) The surface pressure versus mean molecular area isotherms for mixtures containing 0 (long-dashed line), 25 (dash-dot-dash line), 50 (short-dashed line), 75 (dotted line), or 100 (solid line) mol % cholesterol in PCPE at 37°C. The monolayers were compressed on water at a speed not exceeding 10 Ų/molecule/min.

FIGURE 2

Inverse isothermal compressibility coefficient of phospholipid monolayers plotted against the surface pressure. Monolayers of PSM (—▴—), DMPC (–□–), DMPE (·⋯▾⋯·), and PCPE (—··○··—)were compressed on water at 22°C at a speed of 10 mm/min to a given target pressure. The barrier was programmed to oscillate for 2 min with an area change set to 0.5% and the frequency at 40 mHz. The curves are representative curves of two separate experiments.

FIGURE 3

Steady-state anisotropy (r_ss) of DPH in bilayer membranes. The bilayers were prepared from pure PSM and from a mixture 33 mol % cholesterol in PSM (panel A), or from pure PCPE and from a mixture 33 mol % cholesterol in PCPE bilayers (panel B). The anisotropy of DPH is drawn as a function of temperature.

FIGURE 4

Emission spectra of Laurdan in bilayer membranes. The emission spectra of Laurdan in pure PSM and PCPE bilayers are shown in panel A both 5°C below and above the T_m. Panel B shows Laurdan emission in PSM and PCPE membranes containing 33 mol % cholesterol both 5°C below and above the T_m. All emission spectra in the graph were background subtracted, corrected, and normalized. The solid line is PSM below the transition, and the dotted line is PSM above the transition. The dashed line is PCPE below the transition, and the dash-dot-dash line is PCPE above the transition.

FIGURE 5

GP_ex values of Laurdan in PCPE and PSM bilayers as a function of temperature. Laurdan emission spectra in PCPE vesicles were recorded stepwise by 5°C between 35°C and 85°C, in PSM vesicles between 10°C and 65°C. Vesicles contained 1 mol % Laurdan, and the GP_ex value was calculated as described above.

FIGURE 6

Quenching of CTL emission by 12SLPC in phospholipids bilayers as a function of temperature. Sample F (quenched) consisted of POPC/12SLPC/phospholipids/cholesterol/CTL (35:30:30:4:1 molar ratio); 12SLPC was replaced by POPC in sample F_o (unquenched). The phospholipids used were PSM, PCPE, or PSM/PCPE 1:1 molar ratio. The temperature was increased by 5°C/min and the F/F_o ratio was calculated.