Natural Ceramides and Lysophospholipids Cosegregate in Fluid Phosphatidylcholine Bilayers

Abstract

The mode of interactions between palmitoyl lysophosphatidylcholine (palmitoyl lyso-PC) or other lysophospholipids (lyso-PLs) and palmitoyl ceramide (PCer) or other ceramide analogs in dioleoylphosphatidylcholine (DOPC) bilayers has been examined. PCer is known to segregate laterally into a ceramide-rich phase at concentrations that depend on the nature of the ceramides and the co-phospholipids. In DOPC bilayers, PCer forms a ceramide-rich phase at concentrations above 10 mol%. In the presence of 20 mol% palmitoyl lyso-PC in the DOPC bilayer, the lateral segregation of PCer was markedly facilitated (segregation at lower PCer concentrations). The thermostability of the PCer-rich phase in the presence of palmitoyl lyso-PC was also increased compared to that in the absence of palmitoyl lyso-PC. Other saturated lyso-PLs (e.g., palmitoyl lyso-phosphatidylethanolamine and lyso-sphingomyelin) also facilitated the lateral segregation of PCer in a similar manner as palmitoyl lyso-PC. When examined in the DOPC bilayer, it appeared that the association between palmitoyl lyso-PC and PCer was equimolar in nature. It is proposed that the interaction of PCer with lyso-PLs was driven by the need of ceramide to obtain a large-headgroup co-lipid, and saturated lyso-PLs were preferred co-lipids over DOPC because of the nature of their acyl chain. Structural analogs of PCer (1- or 3-deoxy-PCer) were also associated with palmitoyl lyso-PC, similarly to PCer, suggesting that the ceramide/lyso-PL interaction was not sensitive to structural alterations in the ceramide molecule. Binary complexes containing palmitoyl lyso-PC and ceramide were prepared, and these had a bilayer structure as ascertained by transmission electron microscopy. It is concluded that ceramides and lyso-PLs associated with each other both in binary bilayers and in ternary systems based on the DOPC bilayers. This association may have biological relevance under conditions in which both sphingomyelinases and phospholipase A₂ enzymes are activated, such as during inflammatory processes.

Figure 1

Chemical structures of some of the lyso-PLs and ceramides used in the study. As a lyso-PC, the sn-1-oleoyl analog was also used (data not shown in figure). As ceramides, N-oleoyl and N-nervonyl analogs were also included (data not shown in figure). trans Parinaric acid (tPA) is also shown.

Figure 2

Effect of palmitoyl lyso-PC on PCer segregation in DOPC bilayers and on gel-phase stability. For the indicated compositions, 1 mol% tPA was included as a reporter molecule. (A) shows the average emission lifetime data for DOPC based bilayers, whereas (B) shows tPA steady-state anisotropy as a temperature function for the same compositions. Each value is average ± SD with n = 3. To see this figure in color, go online.

Figure 3

Lateral segregation of PCer in DOPC bilayers containing the indicated lyso-PLs (equimolar to PCer). The segregation of PCer and the subsequent formation of a gel phase were detected because of the increase in tPA average emission lifetime as a function of PCer concentration. Each value is average ± SD of n = 3. All lyso-PLs had an sn-1 palmitoyl chain except oleoyl lyso-PC. To see this figure in color, go online.

Figure 4

Thermostability of the lyso-PL/PCer gel phase in DOPC bilayers. The gel-phase end melting temperature was determined from the anisotropy of the tPA emission. The bilayer compositions were as follows: DOPC/PCer 85:15 by mol; DOPC/lyso-PL/PCer 70:15:15 by mol. The sn-1 acyl chains of the lyso-PLs were 16:0 (PC and PE) or 18:1 (PC) or had the sphingosine long chain base in lyso-SM. Each value is the average ± SD for n = 3.

Figure 5

Segregation of various ceramide species in the absence and presence of equimolar lyso-PC in DOPC bilayers at 23°C. Each value is the average ± SD of n = 3. Closed symbols contain lyso-PC/ceramide and open symbols only ceramide. (A) Circle = 1-deoxy-PCer; triangle = 3-deoxy-PCer. (B) Circle = 24:1-Cer; triangle = OCer. To see this figure in color, go online.

Figure 6

End of the gel-phase melting temperature in PCer/DOPC bilayers containing increasing proportions of palmitoyl lyso-PC. The initial bilayer composition was DOPC/PCer 90:10 by mol (at zero lyso-PC). The mol% PCer was kept at 10, whereas the lyso-PC mol% increased as indicated. Each value is average ± SD of n = 3.

Figure 7

Extruded aggregates prepared from palmitoyl lyso-PC and oleoyl ceramide were visualized using transmission electron microscopy. The sample contained equimolar amounts of lyso-PC and ceramide, and the total lipid concentration was 0.2 mM. Scale bars, 200 nm in (A) and 50 nm for (B). The large unilamellar vesicle sample was diluted 10× for (B).