Lateral Segregation of Palmitoyl Ceramide-1-Phosphate in Simple and Complex Bilayers.

Ceramide-1-phosphate is a minor sphingolipid with important functions in cell signaling. In this study, we examined the propensity of palmitoyl ceramide-1-phosphate (Cer-1P) to segregate laterally into ordered domains in different bilayer compositions at 23 and 37°C and compared this with segregation of palmitoyl ceramide (PCer) and palmitoyl sphingomyelin (PSM). The ordered-domain formation in the fluid phosphatidylcholine bilayers was determined using the emission lifetime changes of trans-parinaric acid and from differential scanning calorimetry thermograms. The lateral segregation of Cer-1P was examined when hydrated to bilayers in Tris buffer (50 mM Tris, 140 mM NaCl (pH 7.4)). At this pH, Cer-1P was negatively charged. The lateral segregation propensity of Cer-1P in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers was intermediate between PCer and PSM. Based on differential scanning calorimetry analysis, we observed that the gel domains formed by Cer-1P in POPC bilayers (POPC:Cer-1P 70:30 by mol) were less stable (melting interval 16-37°C) than the corresponding POPC and PCer gel domains at equal composition (melting interval 20-55°C). The gel-phase melting enthalpy was also much lower in Cer-1P (1.5 kcal/mol) than in the PCer-containing POPC bilayers (9 kcal/mol). Cer-1P appeared to be at least partially miscible with PCer domains in POPC bilayers. Cer-1P domains were stabilized in the presence of PSM (POPC:PSM 85:15), similarly as seen with PCer-rich domains. In bilayers at 37°C, with an approximate outer-leaflet cell membrane composition (sphingomyelin and cholesterol enriched, aminophospholipid poor), Cer-1P segregation did not lead to the formation of ordered domains, at least when compared with PCer segregation. In bilayers with an approximate inner-leaflet composition (sphingomyelin poor, cholesterol and aminophospholipid enriched), Cer-1P also failed to form ordered domains. PCer segregated into ordered domains only after the PCer/cholesterol ratio exceeded an approximate equimolar ratio.

Natural Ceramides and Lysophospholipids Cosegregate in Fluid Phosphatidylcholine Bilayers.

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.

Sphingomyelin Stereoisomers Reveal That Homophilic Interactions Cause Nanodomain Formation.

Sphingomyelin is an abundant lipid in some cellular membrane domains, such as lipid rafts. Hydrogen bonding and hydrophobic interactions of the lipid with surrounding components such as neighboring sphingomyelin and cholesterol (Cho) are widely considered to stabilize the raft-like liquid-ordered (Lo) domains in membrane bilayers. However, details of their interactions responsible for the formation of Lo domains remain largely unknown. In this study, the enantiomer of stearoyl sphingomyelin (ent-SSM) was prepared, and its physicochemical properties were compared with the natural SSM and the diastereomer of SSM to examine possible stereoselective lipid-lipid interactions. Interestingly, differential scanning calorimetry experiments demonstrated that palmitoyl sphingomyelin, with natural stereochemistry, exhibited higher miscibility with SSM bilayers than with ent-SSM bilayers, indicating that the homophilic sphingomyelin interactions occurred in a stereoselective manner. Solid-state 2H NMR revealed that Cho elicited its ordering effect very similarly on SSM and ent-SSM (and even on the diastereomer of SSM), suggesting that SSM-Cho interactions are not significantly affected by stereospecific hydrogen bonding. SSM and ent-SSM formed gel-like domains with very similar lateral packing in SSM/Cho/palmitoyloleoyl phosphatidylcholine membranes, as shown by fluorescence lifetime experiments. This observation can be explained by a homophilic hydrogen-bond network, which was largely responsible for the formation of gel-like nanodomains of SSMs (or ent-SSM). Our previous study revealed that Cho-poor gel-like domains contributed significantly to the formation of an Lo phase in sphingomyelin/Cho membranes. The results of the study presented here further show that SSM-SSM interactions occur near the headgroup region, whereas hydrophobic SSM-Cho interactions appeared important in the bilayer interior for Lo domain formation. The homophilic interactions of sphingomyelins could be mainly responsible for the formation of the domains of nanometer size, which may correspond to the small sphingomyelin/Cho-based rafts that temporally occur in biological membranes.

Bilayer Interactions among Unsaturated Phospholipids, Sterols, and Ceramide.

Using differential scanning calorimetry and lifetime analysis of trans-parinaric acid fluorescence, we have examined how cholesterol and cholesteryl phosphocholine (CholPC) affect gel-phase properties of palmitoyl ceramide (PCer) in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC) bilayers. By 2H NMR, we also measured fluid-phase interactions among these lipids using deuterated analogs of POPC, PCer, and cholesterol. The PCer-rich gel phase in POPC bilayers (9:1 molar ratio of POPC to PCer) was partially and similarly dissolved (and thermostability decreased) by both cholesterol and CholPC (sterol was present equimolar to PCer, or in fourfold excess). In DOPC bilayers (4:1 DOPC/PCer molar ratio), CholPC was much more efficient in dissolving the PCer-rich gel phase when compared to cholesterol. This can be interpreted as indicating that PCer interaction with POPC was stronger than PCer interaction with DOPC. PCer-CholPC interactions were also more favored in DOPC bilayers compared to POPC bilayers. In the fluid POPC-rich phase, cholesterol increased the order of the acyl chain of d2-PCer much more than did CholPC. In DOPC-rich fluid bilayers, both cholesterol and CholPC increased d2-PCer acyl chain order, and the ordering induced by CholPC was more efficient in DOPC than in POPC bilayers. In fluid POPC bilayers, the ordering of 3-d1-cholesterol by PCer was weak. In summary, we found that in the gel phase, sterol effects on the PCer-rich gel phase were markedly influenced by the acyl chain composition of the fluid PC. The same was true for fluid-phase interactions involving the sterols. Our results further suggest that PCer did not display high affinity toward either of the sterols used. We conclude that the nature of unsaturated phospholipids (POPC versus DOPC) in bilayers has major effects on the properties of ceramide gel phases and on sterol-ceramide-phospholipid interactions in such complex bilayers.

The Long-Chain Sphingoid Base of Ceramides Determines Their Propensity for Lateral Segregation.

We examined how the length of the long-chain base or the N-linked acyl chain of ceramides affected their lateral segregation in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Lateral segregation and ceramide-rich phase formation was ascertained by a lifetime analysis of trans-parinaric acid (tPA) fluorescence. The longer the length of the long-chain base (d16:1, d17:1, d18:1, d19:1, and d20:1 in N-palmitoyl ceramide), the less ceramide was needed for the onset of lateral segregation and ceramide-rich phase formation. A similar but much weaker trend was observed when sphingosine (d18:1)-based ceramide had N-linked acyl chains of increasing length (14:0 and 16:0-20:0 in one-carbon increments). The apparent lateral packing of the ceramide-rich phase, as determined from the longest-lifetime component of tPA fluorescence, also correlated strongly with the long-chain base length, but not as strongly with the N-acyl chain length. Finally, we compared two ceramide analogs with equal carbon numbers (d16:1/17:0 or d20:1/13:0) and observed that the analog with a longer sphingoid base segregated at lower bilayer concentrations to a ceramide-rich phase compared with the shorter sphingoid base analog. The gel phase formed by d20:1/13:0 ceramide also was more thermostable than the gel phase formed by d16:1/17:0 ceramide. 2H NMR data for 10 mol % stearoyl ceramide in POPC also showed that the long-chain base was more ordered than the acyl chain at comparable chain positions and temperatures. We conclude that the long-chain base length of ceramide is more important than the acyl chain length in determining the lateral segregation of the ceramide-rich gel phase and intermolecular interactions therein.

Effect of Phosphatidylcholine Unsaturation on the Lateral Segregation of Palmitoyl Ceramide and Palmitoyl Dihydroceramide in Bilayer Membranes.

To better understand the interactions of saturated ceramides with unsaturated glycerophospholipids in bilayer membranes, we measured how palmitoyl ceramide (PCer) and dihydroceramide (dihydro-PCer, lacking the trans 4 double bond of the sphingoid base of ceramide) can interact with phosphatidylcholines (PCs) with palmitic acid in the sn-1 position and increasingly unsaturated acyl chains in the sn-2 position. The PCs were 16:0/18:1 (POPC), 16:0/18:2 (PLPC), 16:0/20:4 (PAPC), and 16:0(22:6 (PDPC). We also included di-18:1-PC (DOPC) to compare it with POPC. Because the ceramides were expected to segregate laterally to an ordered ceramide-rich phase, we determined the formation of the ordered phase using lifetime analysis of trans-parinaric acid (tPA) fluorescence. The presence of ordered domains, as indicated by tPA lifetime analysis, was verified by an analysis of tPA anisotropy as a function of temperature. The interaction between PCer and POPC was clearly more favored than interactions with DOPC, as seen from a more thermostable gel phase in POPC than in DOPC at equal ceramide content. The concentration needed for PCer gel phase formation was also lower in POPC than in the DOPC bilayers, suggesting that POPC had better miscibility in the ordered phase. The increased unsaturation of the sn-2 acyl chains of the PCs had more clear effects of dihydro-PCer segregation than on PCer segregation, and the dihydro-PCer gel phase became more thermostable as the unsaturation in the PC increased. We conclude that the interactions between ceramides and PCs were complex and affected both by the trans 4 double bond of PCer by the palmitoyl acyl in the sn-1 position and by the overall degree of unsaturation of the sn-2 acyl chain of the PCs.

Regulation of Sticholysin II-Induced Pore Formation by Lipid Bilayer Composition, Phase State, and Interfacial Properties.

Sticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes. After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids, affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation.

The Influence of Hydrogen Bonding on Sphingomyelin/Colipid Interactions in Bilayer Membranes.

The phospholipid acyl chain composition and order, the hydrogen bonding, and properties of the phospholipid headgroup all influence cholesterol/phospholipid interactions in hydrated bilayers. In this study, we examined the influence of hydrogen bonding on sphingomyelin (SM) colipid interactions in fluid uni- and multilamellar vesicles. We have compared the properties of oleoyl or palmitoyl SM with comparable dihydro-SMs, because the hydrogen bonding properties of SM and dihydro-SM differ. The association of cholestatrienol, a fluorescent cholesterol analog, with oleoyl sphingomyelin (OSM) was significantly stronger than its association with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in bilayers with equal acyl chain order. The association of cholestatrienol with dihydro-OSM, which lacks a trans double bond in the sphingoid base, was even stronger than the association with OSM, suggesting an important role for hydrogen bonding in stabilizing sterol/SM interactions. Furthermore, with saturated SM in the presence of 15 mol % cholesterol, cholesterol association with fluid dihydro-palmitoyl SM bilayers was stronger than seen with palmitoyl SM under similar conditions. The different hydrogen bonding properties in OSM and dihydro-OSM bilayers also influenced the segregation of palmitoyl ceramide and dipalmitoylglycerol into an ordered phase. The ordered, palmitoyl ceramide-rich phase started to form above 2 mol % in the dihydro-OSM bilayers but only above 6 mol % in the OSM bilayers. The lateral segregation of dipalmitoylglycerol was also much more pronounced in dihydro-OSM bilayers than in OSM bilayers. The results show that hydrogen bonding is important for sterol/SM and ceramide/SM interactions, as well as for the lateral segregation of a diglyceride. A possible molecular explanation for the different hydrogen bonding in SM and dihydro-SM bilayers is presented and discussed.

Influence of Hydroxylation, Chain Length, and Chain Unsaturation on Bilayer Properties of Ceramides.

Mammalian ceramides constitute a family of at least a few hundred closely related molecules distinguished by small structural differences, giving rise to individual molecular species that are expressed in distinct cellular compartments, or tissue types, in which they are believed to execute distinct functions. We have examined how specific structural details influence the bilayer properties of a selection of biologically relevant ceramides in mixed bilayers together with sphingomyelin, phosphatidylcholine, and cholesterol. The ceramide structure varied with regard to interfacial hydroxylation, the identity of the headgroup, the length of the N-acyl chain, and the position of cis-double bonds in the acyl chains. The interactions of the ceramides with sphingomyelin, their lateral segregation into ceramide-rich domains in phosphatidylcholine bilayers, and the effect of cholesterol on such domains were studied with DSC and various fluorescence-based approaches. The largest differences arose from the presence and relative position of cis-double bonds, causing destabilization of the ceramide's interactions and lateral packing relative to common saturated and hydroxylated species. Less variation was observed as a consequence of interfacial hydroxylation and the N-acyl chain length, although an additional hydroxyl in the sphingoid long-chain base slightly destabilized the ceramide's interactions and packing relative to a nonhydroxyceramide, whereas an additional hydroxyl in the N-acyl chain had the opposite effect. In conclusion, small structural details conferred variance in the bilayer behavior of ceramides, some causing more dramatic changes in the bilayer properties, whereas others imposed only fine adjustments in the interactions of ceramides with other membrane lipids, reflecting possible functional implications in distinct cell or tissue types.