Influence of Docosahexaenoic Acid and Cholesterol on Lateral Lipid Organization in Phospholipid Mixtures

We investigated lateral lipid organization in membranes with a lipid composition relevant to neural and retinal membranes [phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/phosphatidylserine (PS)/cholesterol, 4/4/1/1, mol/mol/mol/mol]. The mixed-chain phospholipids contained saturated stearic acid (18:0) in the sn-1 position and the monounsaturated oleic acid (18:1) or polyunsaturated docosahexaenoic acid (22:6) in sn-2. Lateral lipid organization was evaluated by 2H NMR order parameter measurements on stearic acid of all individual types of phospholipids in the mixture and, through a novel approach, two-dimensional NOESY 1H NMR spectroscopy with magic angle spinning (MAS). The docosahexaenoic acid chain order was evaluated from 1H NMR chain signal MAS-sideband intensities. Averaged over all lipids, the cholesterol-induced increase in sn-1 chain order is 2-fold larger in monounsaturated than in polyunsaturated lipids, and the order of both saturated and polyunsaturated hydrocarbon chains increases. Addition of cholesterol increases lipid order in the sequence 18:0-18:1 PE > 18:0-18:1 PC > 18:0-18:1 PS for the monounsaturated and 18:0-22:6 PC ≫ 18:0-22:6 PE > 18:0-22:6 PS for polyunsaturated mixtures. The variation of order parameters between lipid species suggests that cholesterol induces the formation of lipid microdomains with a headgroup and chain unsaturation-dependent lipid composition. The preferential interaction between cholesterol and polyunsaturated 18:0-22:6 PC, followed by 18:0-22:6 PE and 18:0-22:6 PS, was confirmed by 1H MAS NOESY cross-relaxation rate differences. Furthermore, cholesterol preferentially associates with saturated chains in mixed-chain lipids reflected by higher saturated chain-to-cholesterol cross-relaxation rates. We propose that cholesterol forms PC-enriched microdomains in the polyunsaturated 18:0-22:6 PC/18:0-22:6 PE/18:0-22:6 PS/cholesterol membranes in which the saturated sn-1 chains are preferentially oriented toward the cholesterol molecules.