Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

Abstract

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38 degrees C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20 degrees -50 degrees C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

Fig. 1.

Domain morphology in ternary mixtures of EPC/ESM/sterol (45/45/10 mol%). (A) cholesterol (B) 7-dehydrocholesterol. Domain separation was revealed using the fluorescent lipid Texas red-DPPE (1 mol%). Observation temperatures are reported as indicated. Bar = 20 µm. EPC, egg yolk L-α-phosphatidylcholine; ESM, egg yolk sphingomyelin.

Fig. 2.

Domain morphology in ternary mixtures of EPC/ESM/sterol (40/40/20 mol%) as a function of temperature: (A) cholesterol and (B) 7-dehydrocholesterol probed by fluorescent lipid Texas red-DPPE (1 mol%). Bar 20 μm. EPC, egg yolk L-α-phosphatidylcholine; ESM, egg yolk sphingomyelin.

Fig. 3.

Domain morphology in ternary mixtures of EPC/ESM/sterol (34/33/33 mol%). (A) cholesterol and (B) 7-dehydrocholesterol. Domain separation was revealed using the fluorescent lipid Texas red-DPPE (1 mol%). Observation temperatures are reported as indicated. Bar = 20 µm. EPC, egg yolk L-α-phosphatidylcholine; ESM, egg yolk sphingomyelin.

Fig. 4.

SAXS showing the first and second order of the lamellar Bragg reflections of an aqueous liposomal dispersion of ESM/sterol mixtures (A). Lipid proportions are given as mol%. Samples were firstly equilibrated at 20°C and diffractograms were subsequently recorded at 1°C interval during heating to 50°C (rate +2°C/min shown by arrows). (B) Plots of the temperature dependence of the distinct components identified by peak fitting of diffractograms shown in 4A. Variations of the d-spacings are shown as a function of the temperature for binary lipid mixtures. Filled circles = cholesterol; open circles = 7-dehydrocholesterol. CHOL, cholesterol; 7-DHC, 7-dehydrocholesterol; ESM, egg yolk sphingomyelin; L_d, liquid-disordered phase; L_o, liquid-ordered phase; SAXS, small-angle X-ray scattering.

Fig. 5.

Plots of first-order lamellar reflections (filled circles) recorded at 30°C for binary mixtures of (A) ESM codispersed with 40 mol% cholesterol and (B) 7-DHC. Peaks are fitted with Gaussian/Lorentzian curves (open circles) and separated into designated peak 1 (long d-spacing, liquid-ordered component) and peak 2 (liquid-disordered component). 7-DHC, 7-dehydrocholesterol; ESM, egg yolk sphingomyelin.

Fig. 6.

Transversal electron density profiles of the two structures deconvolved from the aqueous dispersion of binary mixtures of ESM/cholesterol (solid line) and ESM/dehydrocholesterol (dashed line). Concentration are in mol% for recording at 20° and 50°C as indicated. Peaks 1 and 2, as indicated in Fig. 5, correspond to the lamellar liquid-ordered and liquid-disordered components separated from the raw diffractograms (Fig. 4A) by peak fitting (Fig. 5). ESM, egg yolk sphingomyelin.

Fig. 7.

Deconvolution of WAXS intensity profiles recorded at 30°C for the aqueous dispersion of (A) ESM (no sterol added). Binary mixtures ESM/cholesterol (B) 90/10 mol% and (C) 80/20 mol% are compared with ESM/7-DHC at (D) 90/10 mol% and (E) 80/20 mol%. 7-DHC, 7-dehydrocholesterol; ESM, egg yolk sphingomyelin; WAXS, wide-angle X-ray scattering.

Fig. 8.

Order parameters and fractions of the 16 NS spin-labeled probe in the distinct environments distinguished by spectral simulation in ESM/sterol binary mixtures. Proportions are given as mol%: phase assignments are based on ESR and X-ray observations (NA, no assignment). CHOL, cholesterol; 7-DHC, 7-dehydrocholesterol; EPC, egg yolk L-α-phosphatidylcholine; ESM, egg yolk sphingomyelin; L_β, gel phase; L_d, liquid-disordered phase; L_o, liquid-ordered phase; S_zz, order parameter; T(C), temperature °C.