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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: 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: 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (OMPC, edelfosine) and 1-hexadecylphosphocholine (HePC, miltefosine) represent two groups of synthetic ether lipid analogues with anti-tumor activity. Because of their hydrophobic nature, they may become incorporated into plasma membranes of cells, and it has been argued that they may act via association with lipid rafts. With the quenching of steady-state fluorescence of probes preferentially partitioning into sterol-rich ordered domains (cholestatrienol and trans-parinaric acid), we showed that OMPC and HePC by themselves did not form sterol-rich domains in fluid model membranes, in contrast to the two chain ether lipid 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine. Nevertheless, all three ether lipids significantly stabilized palmitoyl–sphingomyelin/cholesterol-rich domains against temperature induced melting. In conclusion, this study shows that anti-tumor ether lipids are likely to affect the properties of cholesterol–sphingomyelin domains (i.e., lipid rafts) when incorporated into cell membranes. [Copyright &y& Elsevier]

Sphingomyelin and phosphatidylcholine are important components in the external leaflet of cellular plasma membranes. In this review we compare the structure of these lipid molecules, with emphasis on the differences in hydrogen bonding capacity and membrane properties that arise from the small but significant differences in molecular structure. The membrane properties of sphingomyelins and the implications that these have, or might have, in biological membranes and for raft function are further discussed. [Copyright &y& Elsevier]

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]

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca2+ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca2+ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides. [ABSTRACT FROM AUTHOR]

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy' to combat many various protein-misfolding diseases associated with aging. [ABSTRACT FROM AUTHOR]

Abstract: The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(d)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5–10°C) gel–liquid transition temperatures (T m) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(d)-palmitoyl SM, however, had lower T m (5°C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner. [Copyright &y& Elsevier]

Abstract: Inositol phosphorylceramides (IPCs) are a class of anionic sphingolipids with a single inositol-phosphate head group coupled to ceramide. IPCs and more complex glycosylated IPCs have been identified in fungi, plants and protozoa but not in mammals. IPCs have also been identified in detergent resistant membranes in several organisms. Here we report on the membrane properties of the saturated N-palmitoyl-IPC (P-IPC) in one component bilayers as well as in complex bilayers together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. The membrane properties of P-IPC were shown to be affected by calcium. According to anisotropy changes reported by DPH, the gel-to-liquid transition temperature (T m) of P-IPC was 48 °C. Addition of 5 mM CaCl2 during vesicle preparation markedly increased the T m (65 °C). According to fluorescence quenching experiments in complex lipid mixtures, P-IPC formed sterol containing domains in an otherwise fluid environment. The P-IPC containing domains melted at a lower temperature and appeared to contain less sterol as compared to domains containing N-palmitoyl-sphingomyelin. Calcium further reduced the sterol content of the ordered domains and also increased the thermal stability of the domains. Calcium also induced vesicle aggregation of unilamellar vesicles containing P-IPC, as was observed by 4D confocal microscopy and dynamic light scattering. We believe that IPCs and the calcium induced effects could be important in numerous membrane associated cellular processes such as membrane fusion and in membrane raft linked processes. [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: There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor–ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin–streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules. [Copyright &y& Elsevier]

The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (Tm = 107.4 ± 0.3 °C) than avidin (Tm = 83.5 ± 0.1 °C) or bacterial streptavidin (Tm = 75.5 °C). AVR4/5 also exhibits a high affinity for biotin (Kd ≈ 3.6 × 10-14 M) comparable to that of avidin and streptavidin (Kd ≈ 10-15 M). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4]5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed. [ABSTRACT FROM AUTHOR]

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers. [Display omitted] • FRET discloses lateral interaction of a glycolipid with helical peptide in membrane. • Dimerization of EGF receptor transmembrane peptide is partially disturbed by GM3. • GM3 interacts EGF receptor transmembrane peptide having preferable tilt angle. • Extracellular domain close to the membrane relates to the ganglioside selectivity. [ABSTRACT FROM AUTHOR]