Differences in the domain forming properties of N-palmitoylated neutral glycosphingolipids in bilayer membranes

We have compared the domain forming properties of three neutral acyl chain defined glycosphingolipids differing in their head group structures. The aim of the study was to explore if glycosphingolipids and sterols exist in the same lateral domains in bilayer membranes and how the structure of the head group influences the capacity of the glycosphingolipids to colocalize with cholesterol. The glycosphingolipids used in the study were galactosyl-, glucosyl- and lactosylceramides with a palmitic acid in the N-linked position. Domain formation in mixed bilayer vesicles was examined using fluorescent reporter molecules associating with ordered domains, together with a fluorescence quencher lipid in the disordered membrane phase. Our results show that the glycosphingolipids studied were poor in forming sterol-enriched domains compared to palmitoyl-sphingomyelin as detected by cholestatrienol quenching. However, the tendency to associate with cholesterol was clearly dependent on the carbohydrate structure of the glycosphingolipids, also when two glycosphingolipids with different head groups were mixed in the bilayer. All palmitoylated glycosphingolipids associated with palmitoyl-sphingomyelin/cholesterol domains. Our results show that the head group structures of neutral glycosphingolipids markedly affect their domain forming properties in bilayers both with and without cholesterol. The most striking observation being that large differences in domain forming properties were seen even between glucosylceramide and galactosylceramide, which differ only in the stereochemistry of one hydroxyl group in the carbohydrate head group.     

How the molecular features of glycosphingolipids affect domain formation in fluid membranes

Glycosphingolipids, sphingomyelin and cholesterol are often all found in the detergent resistant fraction of biological membranes and are therefore recognized as raft components, but they do not necessarily co-localize in the same lateral domains. From cell biological studies it is evident that different sphingolipid species can be found in different lateral regions within the same cellular membrane. Biophysical studies have shown that their tendency to co-localize with each other and with other membrane components is largely governed by structural features of all lipids present. Glycosphingolipids form gel-phase like domains in fluid lipid bilayers. Sphingomyelin readily associates with cholesterol, forming liquid-ordered phase domains, but glycosphingolipids do not readily form cholesterol-enriched domains by themselves. However, mixed sphingomyelin- and glycosphingolipid-rich domains appear to incorporate cholesterol. Recent studies indicate that the ceramide backbone structure as well as the number of sugar units and presence of charge in the glycosphingolipid head group will influence the partitioning of these lipids between lateral membrane domains. The properties of the domains will be largely influenced by the presence of glycosphingolipids, which have very high melting temperatures. The lateral partitioning of glycosphingolipid molecular species has only recently been studied more intensively, and a lot remains to be done in this field of research.     

Is a fluid-mosaic model of biological membranes fully relevant? Studies on lipid organization in model and biological membranes

The basic concept of the fluid-mosaic model of Singer and Nicolson, an essential point of which is that the membrane proteins are floating in a sea of excess lipid molecules organized in the lipid bilayer, may be misleading in understanding the movement of membrane components in biological membranes that show distinct domain structure. It seems that the lipid bilayer is an active factor in forming the membrane structure, and the lipid composition is responsible for the presence of domains in the membrane. The main role in the process of domain formation is played by cholesterol and sphingolipids. The results presented here show that in a binary mixture of cholesterol and unsaturated phospholipids, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase. This forms cholesterol-enriched domains or clustered cholesterol domains due to the lateral nonconformability between the rigid planar ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at double bond position. These cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by specific proteins which selectively locate in these domains and stabilize them as a result of protein-protein interaction. Such lipid domains are called "rafts" and have been shown to be responsible both for signal transduction to and from the cell and for protein sorting. We also looked at whether polar carotenoids, compounds showing some similarities to cholesterol and affecting membrane properties in a similar way, would also promote domain formation and locate preferentially in one of the lipid phases. Our preliminary data show that in the presence of cholesterol, lutein (a polar carotenoid) may segregate out from saturated lipid regions (liquid-ordered phase) and accumulate in the regions rich in unsaturated phospholipids forming carotenoid-rich domains there. Conventional and pulse EPR (electron paramagnetic resonance) spin labeling techniques were employed to assess the molecular organization and dynamics of the raft-constituent molecules and of the raft itself in the membrane.     

N-palmitoyl-sulfatide participates in lateral domain formation in complex lipid bilayers

Sulfatides (galactosylceramidesulfates) are negatively charged glycosphingolipids that are important constituents of brain myelin membranes. These membranes are also highly enriched in galactosylceramide and cholesterol. It has been implicated that sulfatides, together with other sphingolipids, take part in lateral domain formation in biological membranes. This study was conducted to characterize the lateral phase behavior of N-palmitoyl-sulfatide in mixed bilayer membranes. Going from simple lipid mixtures with sulfatide as the only sphingolipid in a fluid matrix of POPC, to more complex membranes including other sphingolipids, we have examined 1) ordered domain formation with sulfatide, 2) sterol enrichment in such domains and 3) stabilization of the domains against temperature by the addition of calcium. Using two distinct phase selective fluorescent probes, trans-parinaric acid and cholestatrienol, together with a quencher in the fluid phase, we were able to distinguish between ordered domains in general and ordered domains enriched in sterol. We found that N-palmitoyl-sulfatide formed ordered domains when present as the only sphingolipid in a fluid phospholipid bilayer, but these domains did not contain sterol and their stability was unaffected by calcium. However, at low, physiologically relevant concentrations, sulfatide partitioned favorably into domains enriched in other sphingolipids and cholesterol. These domains were stabilized against temperature in the presence of divalent cations. We conclude that sulfatides are likely to affect the lateral organization of biomembranes.     

N-palmitoyl-sulfatide participates in lateral domain formation in complex lipid bilayers

Sulfatides (galactosylceramidesulfates) are negatively charged glycosphingolipids that are important constituents of brain myelin membranes. These membranes are also highly enriched in galactosylceramide and cholesterol. It has been implicated that sulfatides, together with other sphingolipids, take part in lateral domain formation in biological membranes. This study was conducted to characterize the lateral phase behavior of N-palmitoyl-sulfatide in mixed bilayer membranes. Going from simple lipid mixtures with sulfatide as the only sphingolipid in a fluid matrix of POPC, to more complex membranes including other sphingolipids, we have examined 1) ordered domain formation with sulfatide, 2) sterol enrichment in such domains and 3) stabilization of the domains against temperature by the addition of calcium. Using two distinct phase selective fluorescent probes, trans-parinaric acid and cholestatrienol, together with a quencher in the fluid phase, we were able to distinguish between ordered domains in general and ordered domains enriched in sterol. We found that N-palmitoyl-sulfatide formed ordered domains when present as the only sphingolipid in a fluid phospholipid bilayer, but these domains did not contain sterol and their stability was unaffected by calcium. However, at low, physiologically relevant concentrations, sulfatide partitioned favorably into domains enriched in other sphingolipids and cholesterol. These domains were stabilized against temperature in the presence of divalent cations. We conclude that sulfatides are likely to affect the lateral organization of biomembranes.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Sphingolipids and the formation of sterol-enriched ordered membrane domains

This review is focused on the formation of lateral domains in model bilayer membranes, with an emphasis on sphingolipids and their interaction with cholesterol. Sphingolipids in general show a preference for partitioning into ordered domains. One of the roles of cholesterol is apparently to modulate the fluidity of the sphingolipid domains and also to help segregate the domains for functional purposes. Cholesterol shows a preference for sphingomyelin over phosphatidylcholine with corresponding acyl chains. The interaction of cholesterol with different sphingolipids is largely dependent on the molecular properties of the particular sphingolipid in question. Small head group size clearly has a destabilizing effect on sphingolipid/cholesterol interaction, as exemplified by studies with ceramide and ceramide phosphoethanolamine. Ceramides actually displace sterol from ordered domains formed with saturated phosphatidylcholine or sphingomyelin. The N-linked acyl chain is known to be an important stabilizer of the sphingolipid/cholesterol interaction. However, N-acyl phosphatidylethanolamines failed to interact favorably with cholesterol and to form cholesterol-enriched lateral domains in bilayer membranes. Glycosphingolipids also form ordered domains in membranes but do not show a strong preference for interacting with cholesterol. It is clear from the studies reviewed here that small changes in the structure of sphingolipids alter their partitioning between lateral domains substantially.     

Modulation of phospholipase A2 by electrostatic fields and dipole potential of glycosphingolipids in monolayers

Phospholipase A2 activity against mixed monolayers of dilauroylphosphatidic acid or dilauroylphosphatidylcholine with glycosphingolipids can be reversibly modulated by external constant electrostatic fields. The changes of enzymatic activity are correlated to the depolarization or hyperpolarization of the film caused by specific dipolar properties of glycosphingolipids. Hyperpolarizing fields enhance the enzymatic activity against pure dilauroylphosphatidic acid while depolarizing fields induce a decrease of activity. Compared to the pure substrate, the interface of mixed films containing neutral glycosphingolipids or gangliosides is already partially depolarized and the magnitude of activation induced by an external hyperpolarizing field is decreased; conversely, depolarizing fields cause an increased inhibition of activity. Differing from gangliosides, sulfatides bring about a hyperpolarization of the mixed lipid monolayer and external hyperpolarizing or depolarizing fields cause enhanced activation and reduced inhibition, respectively. The effects of glycosphingolipids depend on their relative proportion in the monolayer. Results were similar with dilauroylphosphosphatidylcholine but the field effects were less than half of those found with dilauroylphosphatidic acid. Our work shows that the activity of phospholipase A2 in addition to responding reversibly to external electrostatic fields, is directly modulated by the polarity and magnitude of the lipid polar head group dipole moments.     

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the ²H NMR spectra of 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.     

A New Aspect in Glycolipid Biology: Glycosphingolipids as Antigens Recognized by T Lymphocytes

T cells may recognize a large variety of ligands with different chemical structures. Recently, glycosphingolipids have also been shown to stimulate human T lymphocytes. Recognition of glycosphingolipids is restricted by the nonpolymorphic CD1 molecules, expressed by professional antigen-presenting cells and by macrophages infiltrating inflammatory sites. CD1 molecules have a structure resembling that of classical MHC class I molecules, with the terminal extracellular domains characterized by two antiparallel a helices placed on two hydrophobic pockets. The glycosphingolipids bound to CD1 insert the lipid tails in the two pockets and position the hydrophilic head on the external part of CD1. The TCR interacts with aminoacids present in the two a helices and with residues provided by the carbohydrate moiety of glycosphingolipids and discriminates their structural variations. T cells recognizing self-glycosphingolipids release proinflammatory cytokines and may have a pathogenetic role in autoimmune diseases such as multiple sclerosis.     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

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.     

Biophysics of sphingolipids II. Glycosphingolipids: An assortment of multiple structural information transducers at the membrane surface

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.     

Biophysics of sphingolipids II. Glycosphingolipids: an assortment of multiple structural information transducers at the membrane surface

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.     

Sphingolipids and the formation of sterol-enriched ordered membrane domains

This review is focused on the formation of lateral domains in model bilayer membranes, with an emphasis on sphingolipids and their interaction with cholesterol. Sphingolipids in general show a preference for partitioning into ordered domains. One of the roles of cholesterol is apparently to modulate the fluidity of the sphingolipid domains and also to help segregate the domains for functional purposes. Cholesterol shows a preference for sphingomyelin over phosphatidylcholine with corresponding acyl chains. The interaction of cholesterol with different sphingolipids is largely dependent on the molecular properties of the particular sphingolipid in question. Small head group size clearly has a destabilizing effect on sphingolipid/cholesterol interaction, as exemplified by studies with ceramide and ceramide phosphoethanolamine. Ceramides actually displace sterol from ordered domains formed with saturated phosphatidylcholine or sphingomyelin. The N-linked acyl chain is known to be an important stabilizer of the sphingolipid/cholesterol interaction. However, N-acyl phosphatidylethanolamines failed to interact favorably with cholesterol and to form cholesterol-enriched lateral domains in bilayer membranes. Glycosphingolipids also form ordered domains in membranes but do not show a strong preference for interacting with cholesterol. It is clear from the studies reviewed here that small changes in the structure of sphingolipids alter their partitioning between lateral domains substantially.     

Glycolipid Sensing and Innate Immunity in Paracoccidioidomycosis

Distinct glycolipid profiles are described in microorganisms, which have been shown to modulate the innate immune system. We tested the hypothesis that glycosphingolipids from Paracoccidioides brasiliensis have immunomodulatory properties on monocytes and dendritic cells of two groups of healthy individuals, one cured of paracoccidioidomycosis in the past (CUR-I) and the other nonexposed to P. brasiliensis (HNE-I). Two classes of glycosphingolipids purified from yeast cells were evaluated: a neutral glycosphingolipid, monohexosylceramide (CMH), and acidic glycosylinositolphosphorylceramides (GIPCs). Both glycosphingolipids affected the functioning of innate immunity cells, interfering with the antigen presenting process: P. brasiliensis yeast cells phagocytosis, IL-10 secretion, and costimulatory molecules and recognition receptors expression by monocytes were altered, while dendritic cell antigen presentation to autologous T cells was markedly down-modulated as shown by reduced T-cell proliferative responses. The mechanisms by which CMH and GIPCs exert their effects differ since the target cells did not always respond similarly to the challenge with the glycosphingolipids. Moreover, CUR-I and HNE-I presented different responses to the glycosphingolipids. Differences not only in the glycosphingolipid structure (such as the polar head group or the ceramide moiety), but also in the innate immunity properties of CUR-I and HNE-I, may underlie these differences and contribute to individual’s susceptibility or resistance to develop paracoccidioidomycosis.     

Protein-induced formation of cholesterol-rich domains

A major protein of neuronal rafts, NAP-22, binds specifically to cholesterol. We demonstrate by circular dichroism that NAP-22 contains a significant amount of beta-structure that is not sensitive to binding of the protein to membranes, suggesting that a major portion of the protein does not insert deeply into the membrane. The free energy of binding of NAP-22 to liposomes of dioleoylphosphatidylcholine with 40% cholesterol is -7.3 +/- 0.5 kcal/mol. NAP-22 mixed with dipalmitoylphosphatidylcholine and 40% cholesterol partitions into the detergent insoluble fraction in the presence of 1% Triton X-100. NAP-22 also causes this insoluble fraction to become enriched in cholesterol relative to phospholipid, again demonstrating the ability of this protein to segregate cholesterol and phospholipids into domains. Differential scanning calorimetry results demonstrate that NAP-22 promotes domain formation in liposomes composed of cholesterol and phosphatidylcholine. This is shown by NAP-22-promoted changes in the shape and enthalpy of the phase transition of phosphatidylcholine as well as by the appearance of cholesterol crystallite transitions in membranes composed of phosphatidylcholine with either saturated or unsaturated acyl chains. In situ atomic force microscopy revealed a marked change in the surface morphology of a supported bilayer of dioleoylphosphatidylcholine with 0.4 mole fraction of cholesterol upon addition of NAP-22. Prior to the addition of the protein, the bilayer appears to be a molecularly smooth structure with uniform thickness. Addition of NAP-22 resulted in the rapid formation of localized raised bilayer domains. Remarkably, there was no gross disruption or erosion of the bilayer but rather simply an apparent rearrangement of the lipid bilayer structure due to the interaction of NAP-22 with the lipid. Our results demonstrate that NAP-22 can induce the formation of cholesterol-rich domains in membranes. This is likely to be relevant in neuronal membrane domains that are rich in NAP-22.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Thermotropic behavior of glycosphingolipids in aqueous dispersions

The thermotropic behavior of 20 chemically related glycosphingolipids (GSLs) of high purity, containing neutral and anionic carbohydrate residues in their oligosaccharide chains, was studied by high-sensitivity differential scanning calorimetry. In general, the polar head group of GSLs appears to be one of the major determinants of their phase behavior. Compared to phospholipids, the presence of the carbohydrate rather than the phosphorylcholine moiety in the polar head group and a sphingosine base in the hydrocarbon portion of GSLs reduces the effect on the transition temperature (Tm) brought about by increasing the number of methylene groups in the amide-linked fatty acyl chains. For simple neutral GSLs, the Tm's were 20-40 degrees C higher than those of phospholipids with comparable hydrocarbon chains. As the oligosaccharide chain of GSLs becomes more complex, the excess heat capacity, Tm, enthalpy (delta Hcal), and entropy of the transition decrease proportionally to the number of carbohydrate residues present in the polar head group. The Tm and delta Hcal for anionic GSLs were 16-25 degrees C and 1-3 kcal mol-1 lower than those of neutral GSLs with comparable oligosaccharide chains. A linear dependence of delta Hcal with Tm was found. However, the slopes of these plots were different for neutral and for anionic GSLs, suggesting different types of intermolecular organizations for the two. The Tm and delta Hcal were linearly dependent on the molecular area of both neutral and anionic GSLs; this indicated that the influence of the complexity of the polar head group in GSLs for establishing the thermodynamic behavior may be mediated by the intermolecular spacings.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

The fusogenic tilted peptide (67-78) of α-synuclein is a cholesterol binding domain

Parkinson's disease-associated α-synuclein is an amyloidogenic protein not only expressed in the cytoplasm of neurons, but also secreted in the extracellular space and internalized into glial cells through a lipid raft-dependent process. We previously showed that α-synuclein interacts with raft glycosphingolipids through a structural motif common to various viral and amyloidogenic proteins. Here we report that α-synuclein also interacts with cholesterol, as assessed by surface pressure measurements of cholesterol-containing monolayers. Using a panel of recombinant fragments and synthetic peptides, we identified two distinct cholesterol-binding domains in α-synuclein. One of these domains, which corresponds to the tilted peptide of α-synuclein (67-78), bound cholesterol with high affinity and was toxic for cultured astrocytes. Molecular modeling suggested that cholesterol binds to this peptide with a tilt angle of 46°. α-synuclein also contains a cholesterol recognition consensus motif, which had a lower affinity for cholesterol and was devoid of toxicity. This motif is encased in the glycosphingolipid-binding domain (34-45) of α-synuclein. In raft-like model membranes containing both cholesterol and glycosphingolipids, the head groups of glycosphingolipids prevented the accessibility of cholesterol to exogenous ligands. Nevertheless, cholesterol appeared to 'signal' its presence by tuning glycosphingolipid conformation, thereby facilitating α-synuclein binding to raft-like membranes. We propose that the association of α-synuclein with lipid rafts involves both the binding of α-synuclein (34-45) to glycosphingolipids, and the interaction of the fusogenic tilted peptide (67-78) with cholesterol. Coincidentally, a similar mechanism is used by viruses (HIV-1, HTLV-I, Ebola) which display a tilted peptide and fuse with host cell membranes through a sphingolipid/cholesterol-dependent process.