The Effects of Cholesterol Oxidation on Erythrocyte Plasma Membranes: A Monolayer Study

Abstract

Cholesterol plays a key role in the molecular and mesoscopic organisation of lipid membranes and it is expected that changes in its molecular structure (e.g., through environmental factors such as oxidative stress) may affect adversely membrane properties and function. In this study, we present evidence that oxidation of cholesterol has significant effects on the mechanical properties, molecular and mesoscopic organisation and lipid-sterol interactions in condensed monolayers composed of the main species found in the inner leaflet of the erythrocyte membrane. Using a combination of experimental methods (static area compressibility, surface dilatational rheology, fluorescence microscopy, and surface sensitive X-ray techniques) and atomistic molecular dynamics simulations, we show that oxidation of cholesterol to 7-ketocholesterol leads to stiffening of the monolayer (under both static and dynamic conditions), significant changes in the monolayer microdomain organisation, disruption in the van der Waals, electrostatic and hydrophobic interactions between the sterol and the other lipid species, and the lipid membrane hydration. Surface sensitive X-ray techniques reveal that, whilst the molecular packing mode is not significantly affected by cholesterol oxidation in these condensed phases, there are subtle changes in membrane thickness and a significant decrease in the coherence length in monolayers containing 7-ketocholesterol.

Keywords: X-ray diffraction; X-ray reflection; erythrocyte membrane; lipid monolayers; molecular dynamics simulations.

Figure 1

(A) Chemical structures of cholesterol and 7-ketocholesterol. (B) The RBC Chol monolayer system. (C) A Voronoi tessellation illustrating the lateral distribution of lipids in a leaflet.

Figure 2

Lipid order parameter ($S_{\mathrm{CD}}$) of the hydrocarbon tails.

Figure 3

(a) Pressure–area isotherms, and (b) static compression moduli, $\epsilon \left(\pi \right)$, for the RBC Chol (purple), RBC KChol (blue) and RBC NChol (green) monolayers on a water subphase. Lettered stars mark points in the isotherms where fluorescence micrographs were recorded.

Figure 4

Rheoviscous properties of RBC IL monolayers determined using stress relaxation experiments (dilatational rheology) for the RBC Chol (purple), RBC KChol (blue) and RBC NChol (green) monolayers, at 30 $\mathrm{m}$$\mathrm{N}$${\mathrm{m}}^{-1}$ and 20 ${}^{\circ }\mathrm{C}$. (a) $\Delta \pi \left(t\right)$ transients (symbols are experimental data, lines are fits), where $\Delta \pi$ is the pressure at a given time, divided by the maximum surface pressure after perturbation $\Delta {\pi }_0(t=0)$. (b) Storage modulus ($G^{\prime }$), loss modulus ($G^{″}$) (upper panel) and the tangent of the phase angle ($tan\varphi =G^{″}/G^{\prime }$, lower panel) extracted via a Fourier transform of $\Delta \pi \left(t\right)$. (c) Cole–Cole plots. (d) Plot of the plateau of the dynamic storage modulus, $G^{\prime }\left(\pi \right)$ (symbols), along with the static elastic modulus, $\epsilon \left(\pi \right)$, from continuous compression experiments (dotted lines). Data extracted from Figure 3. The arrows show the increase in the moduli due to cholesterol oxidation under quasi-static and dynamic deformation.

Figure 5

Fluorescence micrographs of the RBC Chol (a–d), KChol (e–h) and NChol (i–l) monolayers on a water subphase at various surface pressures, corresponding to the points marked in Figure 3 (stars). All monolayers contain 0.02 mol% Rh-DHPE. Each scale bar is 50 $\mathsf{\mu }$$\mathrm{m}$.

Figure 6

PSM-sterol contact probability. Atom label colours: purple is the sterol headgroup; orange the sterol body; green the lipid headgroup; blue the sn-1 tail; and red the sn-2 tail.

Figure 7

Relative hydration of non-hydrogen atoms of cholesterol and 7-ketocholesterol. An atom rendered purple indicates that 20% of the water within 4 Å of cholesterol is also within 4 Å of that atom.

Figure 8

Electron density profiles of the monolayers, derived from the MD simulations.

Figure 9

Surface-sensitive X-ray analysis: (a) Experimental (symbols) X-ray reflectivity curves of RBC Chol (violet), RBC KChol (blue) and RBC NChol (green) at 30 $\mathrm{m}$$\mathrm{N}$${\mathrm{m}}^{-1}$ and best fit curves (lines) (the curves are shifted along the y-axis for clarity); (b) electron density profiles; (c) thicknesses of the monolayer and slabs (lipid headgroup, acyl chains, total); (d) GIXD curves; (e) coherence length (the error bars are given in red). The X-ray experiments were performed using aqueous TRIS buffer at pH = 7.4, 150 $\mathrm{m}$$\mathrm{M}$ NaCl, 50 $\mathrm{m}$$\mathrm{M}$ CaCl₂ subphase at 20 ${}^{\circ }\mathrm{C}$.