X-RAY-DIFFRACTION ANALYSIS OF ISOLATED SKIN LIPIDS - RECONSTITUTION OF INTERCELLULAR LIPID DOMAINS

Low- and wide-angle X-ray diffraction were used to determine the struc tural organization of lipids isolated from the stratum corneum extrace llular matrix that forms the major water permeability barrier in mamma lian epidermis. Hydrated pig skin ceramides gave a single low-angle re flection of about 62 Angstrom and a wide-angle reflection at 4.15 Angs trom. The addition of either cholesterol or fatty acid, the other majo r lipid components of the skin stratum corneum extracellular matrix, m odified this diffraction pattern, depending on the lipid mole ratios. In the absence of water, lipid mixtures exhibited lipid phase separati on, as shown by low- and wide-angle reflections typical of a separate cholesterol phase. However, a hydrated 2:1:1 mole ratio of ceramide:ch olesterol:palmitic acid (similar to that found in stratum corneum) pro duced a diffraction pattern with a single sharp wide-angle reflection at 4.10 Angstrom and low-angle reflections which indexed as the first eight orders of a single repeat period of 130 Angstrom. The repeat per iod and intensity distribution of the low-angle data were similar to t hose found in intact stratum corneum [White et al. (1988) Biochemistry 27, 3725-3732; Bouwstra et al. (1994) Biochim. Biophys. Acta 1212, 18 3-192]. Higher concentrations of cholesterol or palmitic acid resulted in lipid phase separations. The 130 Angstrom repeat period decreased only about 3 Angstrom as water was removed by incubation in low-relati ve humidity atmospheres. The 130 Angstrom repeat period depended on th e presence of a particular ceramide, N-(omega-acyloxy)-acylsphingosine , which is found only in the epidermis, In contrast, 2:1:1 mixtures of brain ceramide: cholesterol:palmitic acid gave reflections of 56 and 34 Angstrom. These results indicate that a structure with dimensions s imilar to those of the lamellar repeating unit found in skin stratum c orneum does not depend on the presence of protein but does depend on t he presence of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid.