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Abstract: Sphingomyelins are important phospholipids in plasma membranes of most cells. Because of their dominantly saturated nature, they affect the lateral structure of membranes, and contribute to the regulation of cholesterol distribution within membranes, and in cells. However, the abundance of molecular species present in cells also implies that sphingomyelins have other, more specific functions. Many of these functions are currently unknown, but are under extensive study. Mostly model membrane studies have shown that sphingomyelins (and other sphingolipids), in contrast to glycerophospholipids, have important hydrogen bonding properties which in several important ways confer specific functional properties to this abundant class of membrane phospholipids. The often very asymmetric nature of sphingomyelins, arising from mismatch in length between the long chain base and N-acyl chains, also impose specific properties (e.g., interdigitation) to sphingomyelins not seen with glycerophospholipids. In this review, the latest sphingomyelin literature will be scrutinized, and an effort will be made to correlate the molecular structure of sphingomyelin with functional properties. In particular, the effects of head group properties, interfacial hydrogen bonding, long chain base hydroxylation, N-acyl chain hydroxylation, and N-acyl chain methyl-branching will be discussed. [Copyright &y& Elsevier]

Abstract: D-ribo-phytosphingosines are biologically significant long-chain bases present in various sphingolipids from yeasts, fungi, plants and mammals. In this study we prepared phytopalmitoylsphingomyelin (phytoPSM) analogs based on the D-ribo-phytosphingosine base. The N-linked acyl chains were either 16:0, 2OH(R)16:0 (natural isomer), or 2OH(S)16:0. The gel-phase of phytoPSM was more stable than that of PSM (Tm 48.6°C and 41.0°C, respectively). The gel-liquid crystalline phase transition enthalpies were 9.1±0.4 and 6.1±0.3kcal/mol for phytoPSM and PSM, respectively. An N-linked 2OH(R)16:0 in phytoPSM destabilized the gel phase relative to phytoPSM (by ~+6°C, based on DPH anisotropy measurements), whereas 2OH(S)16:0 in phytoPSM stabilized it (by ~−6°C). All phytoPSM analogs formed sterol-enriched ordered domains in a fluid ternary bilayer, and those containing phytoPSM or 2OH(S)phytoPSM were more thermostable than the domains containing 2OH(R)phytoPSM or PSM. The affinity of cholestatrienol for POPC bilayers containing 20mol% phytoPSM was higher than for comparable bilayers with an equal amount of PSM. The 2-hydroxylated acyl chains in phytoPSM did not markedly alter sterol affinity. We conclude that phytoPSM is a more ordered sphingolipid than PSM, and is fully capable of interacting with cholesterol. [Copyright &y& Elsevier]

Abstract: Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5°C (ΔH 8kcal/mol), which is about 15°C higher than the phase transition temperature of PSM. The 3O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol. [Copyright &y& Elsevier]

Abstract: Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1Δ4t), and N-linked acyl chains (often 16:0, 18:0 or 24:1Δ15c). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane. [Copyright &y& Elsevier]

Abstract: Sphingolipids have been found to have single methyl branchings both in their long-chain base and in their N-linked acyl chains. In this study we determined how methyl-branching in the N-linked acyl chain of sphingomyelin (SM) affected their membrane properties. SM analogs with a single methyl-branching at carbon 15 (of a 17:0 acyl chain; anteiso) had a lower gel–liquid transition temperature as compared to an iso-branched SM analog. Phytanoyl SM (methyls at carbons 3, 7, 11 and 15) as well as a SM analog with a methyl on carbon 10 in a hexadecanoyl chain failed to show a gel–liquid transition above 10°C. Only the two distally branched SM analogs (iso and anteiso) formed ordered domains with cholesterol in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. However, domains formed by the branched SM analogs appeared to contain less sterol when compared to palmitoyl SM (PSM) as the saturated phospholipid. Sterol-enriched domains formed by the anteiso SM analog were also less stable against temperature than domains formed by PSM. Both the 10-methyl and phytanoyl SM analogs failed to form sterol-enriched domains in the POPC bilayer. Acyl chain branching weakened SM/sterol interactions markedly when compared to PSM, as also evidenced from the decreased affinity of cholestatrienol to bilayers containing branched SM analogs. Our results show that methyl-branching weakened intermolecular interactions in a position-dependent manner. [ABSTRACT FROM AUTHOR]

Abstract: Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane–water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes. [Copyright &y& Elsevier]

Abstract: Sticholysin II (StnII) is a pore-forming toxin from the sea anemone Stichodactyla heliantus which belongs to the large actinoporin family. The toxin binds to sphingomyelin (SM) containing membranes, and shows high binding specificity for this lipid. In this study, we have examined the role of the hydrogen bonding groups of the SM long-chain base (i.e., the 2NH and the 3OH) for StnII recognition. We prepared methylated SM-analogs which had reduced hydrogen bonding capability from 2NH and 3OH. Both surface plasmon resonance experiments, and isothermal titration calorimetry measurements indicated that StnII failed to bind to bilayers containing methylated SM-analogs, whereas clear binding was seen to SM-containing bilayers. StnII also failed to induce calcein release (i.e., pore formation) from vesicles made to contain methylated SM-analogs, but readily induced calcein release from SM-containing vesicles. Molecular modeling of SM docked to the phosphocholine binding site of StnII indicated that the 2NH and 3OH groups were likely to form a hydrogen bond with Tyr135. In addition, it appeared that Tyr111 and Tyr136 could donate hydrogen bonds to phosphate oxygen, thus stabilizing SM binding to the toxin. We conclude that the interfacial hydrogen bonding properties of SM, in addition to the phosphocholine head group, are crucial for high-affinity SM/StnII-interaction. [Copyright &y& Elsevier]

Abstract: In this study we have prepared ceramide phosphoserine (CerPS) and examined its sterol-interacting properties. CerPS is a hydrogen-bonding sphingolipid, but its head group differs from that found in sphingomyelin (SM). Based on diphenylhexatriene steady-state anisotropy measurements, we observed that fully hydrated N-palmitoyl CerPS had a gel-to-liquid crystalline phase transition temperature of about 51°C in 50mM sodium phosphate buffer (pH 7.4). This was close to the Tm measured for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) bilayers (Tm 50.5°C). Based on cholestatrienol (CTL) quenching experiments in liquid disordered ternary bilayers (containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphcholine; POPC), cholesterol/CTL formed sterol-enriched ordered domains with CerPS. These had similar thermostability as the sterol domains formed with N-palmitoyl SM. Cholesterol failed to form sterol-enriched ordered domains with DPPS under comparable conditions. Based on the equilibrium partitioning of CTL, we observed that the affinity of sterol for bilayers containing POPC/CerPS/cholesterol (6:3:1 by mol) was much higher than the affinity measured for control fluid POPC/cholesterol (9:1 by mol) bilayers, but slightly less than seen for comparable PSM-containing bilayers. We conclude that the phosphoserine head group was less efficient than the phosphocholine head group in stabilizing sterol/sphingolipid interaction. However, hydrogen bonding apparently can overcome some of the negative effects of the phosphoserine head group, since CerPS interacted more favorably with cholesterol compared to DPPS. [Copyright &y& Elsevier]

Abstract: The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase–SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH–SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg2+ and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg2+ and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis. [Copyright &y& Elsevier]

Abstract: A sphingomyelin chimera in which the amide-linked acyl chain was replaced with cholesterol carbamate was prepared and its properties examined. The sphingomyelin/cholesterol chimera (N-cholesterol-D-erythro-sphingomyelin) was able to form unilamellar vesicles of defined size when extruded through 200nm pore size membranes. These N-cholesteryl sphingomyelin bilayers were resistant to solubilization by Triton X-100. When N-cholesteryl sphingomyelin was added to N-palmitoyl sphingomyelin (N-palmitoyl- d -erythro-sphingomyelin) bilayers, it increased acyl chain order as determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy. N-cholesteryl sphingomyelin was, however, not as good an inducer of membrane order compared to cholesterol on a molar basis. Differential scanning calorimetry studies further showed that the miscibility of N-cholesteryl sphingomyelin with N-palmitoyl- d -erythro-sphingomyelin bilayers was non-ideal, and the effect of N-cholesteryl sphingomyelin on the N-palmitoyl- d -erythro-sphingomyelin gel–fluid transition enthalpy differed from that seen with cholesterol. Together with N-palmitoyl- d -erythro-sphingomyelin, the N-cholesteryl sphingomyelin chimera was able to form sterol-enriched ordered domains in a fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. N-cholesteryl sphingomyelin in the absence of N-palmitoyl- d -erythro-sphingomyelin was unable to form such sterol-enriched ordered domains in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. However, N-cholesteryl sphingomyelin markedly increased the affinity of cholestatrienol for N-cholesteryl sphingomyelin containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers, suggesting that N-cholesteryl sphingomyelin was able to somehow stabilize sterol interaction in fluid bilayers. Based on our results, we conclude that N-cholesteryl sphingomyelin behaved more like a cholesterol than a sphingolipid in fluid bilayer membranes. Because N-cholesteryl sphingomyelin increased bilayer order, conferred resistance against detergent solubilization, and is not degradable by phospholipases A2, it could constitute a good lipocomplex matrix for drug delivery vehicles. [Copyright &y& Elsevier]

Abstract: Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties. [Copyright &y& Elsevier]

Abstract: We have characterized the molecular properties and membrane behavior of synthetically modified sphingomyelin analogues, modified on the oxygen connecting the phosphocholine group to the ceramide backbone. The oxygen was replaced with an S-atom (S-PSM), an NH-group (NH-PSM) or a CH2-group (CH2-PSM). Diphenylhexatriene and Laurdan anisotropy experiments showed that an S-linkage increased and NH- and CH2-linkages decreased the stability of PSM-analogue bilayer membranes as compared to PSM. When the polarity of the interface was probed using Laurdan, S-PSM appeared to have a lower polarity as compared to PSM whereas NH-PSM and CH2-PSM had higher polarities of their respective interfaces. Fluorescence quenching-studies with cholestatrienol showed that all compounds formed SM/cholesterol-rich domains. The S-PSM/cholesterol and PSM/cholesterol domains displayed a similar thermostability, whereas NH-PSM/cholesterol and CH2-PSM/cholesterol domains were less thermostable. DSC on vesicles containing the PSM-analogues showed a more complex melting behavior as compared to PSM, whereas equimolar mixtures of the PSM-analogues and PSM showed almost ideal mixing with PSM for NH- and S-PSM. Our data show that the properties of the bond linking the phosphocholine head group to the 1-hydroxyl on the ceramide molecule is important for the stability of SM/SM and SM/cholesterol interactions. [Copyright &y& Elsevier]