Sphingosine-1-phosphate as an amphipathic metabolite: its properties in aqueous and membrane environments

Abstract

Sphingosine-1-phosphate (S1P) is currently considered to be an important signaling molecule in cell metabolism. We studied a number of relevant biophysical properties of S1P, using mainly Langmuir balance, differential scanning calorimetry, (31)P-NMR, and infrared (IR) spectroscopy. We found that, at variance with other, structurally related sphingolipids that are very hydrophobic, S1P may occur in either an aqueous dispersion or a bilayer environment. S1P behaves in aqueous media as a soluble amphiphile, with a critical micelle concentration of approximately 12 muM. Micelles give rise to larger aggregates (in the micrometer size range) at and above a 1 mM concentration. The aggregates display a thermotropic transition at approximately 60 degrees C, presumably due to the formation of smaller structures at the higher temperatures. S1P can also be studied in mixtures with phospholipids. Studies with dielaidoylphosphatidylethanolamine (DEPE) or deuterated dipalmitoylphosphatidylcholine (DPPC) show that S1P modifies the gel-fluid transition of the glycerophospholipids, shifting it to lower temperatures and decreasing the transition enthalpy. Low (<10 mol %) concentrations of S1P also have a clear effect on the lamellar-to-inverted hexagonal transition of DEPE, i.e., they increase the transition temperature and stabilize the lamellar versus the inverted hexagonal phase. IR spectroscopy of natural S1P mixed with deuterated DPPC allows the independent observation of transitions in each molecule, and demonstrates the existence of molecular interactions between S1P and the phospholipid at the polar headgroup level that lead to increased hydration of the carbonyl group. The combination of calorimetric, IR, and NMR data allowed the construction of a temperature-composition diagram ("partial phase diagram") to facilitate a comparative study of the properties of S1P and other related lipids (ceramide and sphingosine) in membranes. In conclusion, two important differences between S1P and ceramide are that S1P stabilizes the lipid bilayer structure, and physiologically relevant concentrations of S1P can exist dispersed in the cytosol.

Figure 1

(A) CMC of S1P estimated by the surface-pressure method. S1P in PIPES buffer, pH 7.4 (see Materials and Methods), at 21°C. The CMC was measured as the point beyond which further increases in S1P concentration do not lead to increased surface pressures. Average values ± SE of the mean (n = 3). (B) Turbidity (absorbance at 400 nm) of S1P suspensions in PIPES buffer, pH 7.4, at 21°C as a function of S1P concentration. Average values ± SE of the mean (n = 4).

Figure 2

Confocal fluorescence microscopy of a 2.76 mM suspension of S1P. The lipid was doped with 0.2 mol % DiI. Bar = 10 μm.

Figure 3

DSC of S1P in buffer dispersion. Second heating scan. The S1P concentration was 10 mM.

Figure 4

Band positions (maximal frequencies) of the C-H stretching vibrations of S1P as a function of temperature. (A) Symmetric stretching. (B) Asymmetric stretching. (●) Heating run. (○) Cooling run.

Figure 5

Band positions (maximal frequencies) of S1P vibrational bands as a function of temperature. (A) P-O stretching. (B) C-H bending. (●) Heating run. (○) Cooling run.

Figure 6

Static light scattering of a 1 mM S1P dispersion in buffer, as a function of temperature.

Figure 7

Phase transitions of pure DEPE and DEPE:S1P mixtures as detected by DSC. (A) Gel-fluid transitions. (B) Lamellar-hexagonal transitions. Second heating scans. The figures at the right side of each thermogram correspond to the S1P contents, in mole percentages.

Figure 8

Thermodynamic parameters of the phase transitions of pure DEPE and DEPE:S1P mixtures. Data are derived from thermograms as shown in Fig. 6. (A–C) Gel-fluid transitions. (D–F) Lamellar-hexagonal transitions. (A and D) Midpoint transition temperatures. (B and E) Transition enthalpies. (C and F) Transition widths at midheight of the thermograms.

Figure 9

Temperature-composition diagram (“phase diagram”) for the DEPE:S1P system in excess water. Experimental data were derived from DSC data as shown in Fig. 7. L_β, lamellar gel phase. L_α, lamellar fluid, or liquid crystalline phase. H_II, inverted hexagonal phase. Regions of coexisting domains are marked “coexistence”.

Figure 10

³¹P-NMR spectra of DEPE:S1P mixtures in excess water, as a function of temperature. Temperatures are indicated at the right-hand side for each spectrum.

Figure 11

The gel-fluid transitions of pure d₆₂-DPPC, pure S1P, and d₆₂-DPPC:S1P mixtures as detected by IR spectroscopy. The maximal frequencies (band positions) of the corresponding bands are plotted as a function of temperature (data from the second heating scan). (A) C-D symmetric stretching vibrations of d₆₂-DPPC. (B) CO stretching vibrations of d₆₂-DPPC. (C) C-H symmetric stretching vibrations of S1P. (●) Pure d₆₂-DPPC. (○) d₆₂-DPPC:S1P mixture at a 1:1 mol ratio. (▴) Pure S1P.

Figure 12

C=0 stretching region of the deconvoluted d₆₂ DPPC IR spectrum. Dotted line: Pure d₆₂-DPPC. Continuous line: d₆₂-DPPC:S1P mixture at a 1:1 mol ratio. (A) 25°C. (B) 50°C. Deconvolution parameters: bandwidth = 18, enhancement factor = 2.