Ceramide-1-Phosphate, in Contrast to Ceramide, Is Not Segregated into Lateral Lipid Domains in Phosphatidylcholine Bilayers

Sphingolipids are key lipid regulators of cell viability: ceramide is one of the key molecules in inducing programmed cell death (apoptosis), whereas other sphingolipids, such as ceramide 1-phosphate, are mitogenic. The thermotropic and structural behavior of binary systems of N-hexadecanoyl-D-erythro-ceramide (C16-ceramide) or N-hexadecanoyl-D-erythro-ceramide-1-phosphate (C16-ceramide-1-phosphate; C16-C1P) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with DSC and deuterium nuclear magnetic resonance (2H-NMR). Partial-phase diagrams (up to a mole fraction of sphingolipids X = 0.40) for both mixtures were constructed based on DSC and 2H-NMR observations. For C16-ceramide-containing bilayers DSC heating scans showed already at Xcer = 0.025 a complex structure of the main-phase transition peak suggestive of lateral-phase separation. The transition width increased significantly upon increasing Xcer, and the upper-phase boundary temperature of the mixture shifted to ∼65°C at Xcer = 0.40. The temperature range over which 2H-NMR spectra of C16-ceramide/DPPC-d62 mixtures displayed coexistence of gel and liquid crystalline domains increased from ∼10° for Xcer = 0.1 to ∼21° for Xcer = 0.4. For C16-C1P/DPPC mixtures, DSC and 2H-NMR observations indicated that two-phase coexistence was limited to significantly narrower temperature ranges for corresponding C1P concentrations. To complement these findings, C16-ceramide/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and C16-C1P/POPC mixtures were also studied by 2H-NMR and fluorescence techniques. These observations indicate that DPPC and POPC bilayers are significantly less perturbed by C16-C1P than by C16-ceramide and that C16-C1P is miscible within DPPC bilayers at least up to XC1P = 0.30.