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.     

Alcohols reduce lateral membrane pressures: predictions from molecular theory

We explore the effects of alcohols on fluid lipid bilayers using a molecular theory with a coarse-grained model. We show that the trends predicted from the theory in the changes in area per lipid, alcohol concentration in the bilayer, and area compressibility modulus, as a function of alcohol chain length and of the alcohol concentration in the solvent far from the bilayer, follow those found experimentally. We then use the theory to study the effect of added alcohol on the lateral pressure profile across the membrane, and find that added alcohol reduces the surface tensions at both the headgroup/solvent and headgroup/tailgroup interfaces, as well as the lateral pressures in the headgroup and tailgroup regions. These changes in lateral pressures could affect the conformations of membrane proteins, providing a nonspecific mechanism for the biological effects of alcohols on cells.     

Transbilayer Pores Induced by Thickness Fluctuations

Thermally-induced fluctuations of individual phospholipids in a bilayer lipid membrane (BLM) are converted into collective motions due to the intermolecular interactions. Here, we demonstrate that transbilayer stochastic pores can be generated via collective thermal movements (CTM). Using the elastic theory of continuous media applied to smectic-A liquid crystals, we estimate the pore radius and the energetic requirements for pore appearance. Three types of thermally-induced transbilayer pores could be formed through BLMs: open and stable, open and unstable, and closed. In most of the situations, two open and stable pores with different radii could be generated. Notably, the two pores have the same generation probability. Unstable pores are possible to appear across thin bilayers that contain phospholipids with a large polar headgroup. Closed pores are present throughout the cases that we have inspected. The effects of hydrophobic thickness, polar headgroup size of phospholipids, temperature, surface tension, and elastic compression on the pore formation and pore stability have been examined as well.     

Interaction of myelin basic protein, melittin and bovine serum albumin with gangliosides, sulphatide and neutral glycosphingolipids in mixed monolayers

Some parameters that may regulate the miscibility and stability of mixed lipid-protein monolayers at the air-145 mM NaCl interface were studied employing six glycosphingolipids (acidic or neutral), three different types of proteins (soluble, extrinsic or highly amphipathic) and some phospholipids. The results obtained show that the percentage of the total area occupied by the protein at the interface is an important parameter leading to lateral phase separations; the amount and area contribution of the protein accepted in the film before the components become immiscible increase with the complexity of the polar head group of the glycosphingolipids. The interactions occur with progressive reductions of the intermolecular packing as the polar head group of the glycosphingolipid becomes more complex and this is accompanied by more negative values of the excess free energy of mixing. The lipid component seems to be the major responsible for the reduction in mean molecular area.     

Lateral compressibility of lipid mono- and bilayers. Theory of membrane permeability

The passive sodium permeability of pure lipid vesicles and dispersions has a large peak at the bilayer phase transition temperature. We discuss this anomaly in terms of density fluctuations, which can open up cavities in the headgroup region into which small ions can enter, and which may be large if bilayer conditions at the melting point are similar to those near the critical point which seems to exist in monolayers. We present two arguments, one thermodynamic and one microscopic, which suggest that the permeability is proportional to the lateral compressibility. We then calculate the lateral compressibility for two previously published theoretical models and compare the results with experiment.     

Interactions of pyrethroids with phosphatidylcholine liposomal membranes

Interactions of several pyrethroids with membrane lipids in the form of dipalmitoylphosphatidylcholine (DPPC) liposomes have been studied using fluorescent membrane probes. Fluorescence anisotropy values and lifetimes (determined by phase-shift and demodulation techniques) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, were decreased in gel phase liposomes by pyrethroids at concentrations on the order of 10 microM. The pyrethroids containing a cyano substituent were also observed to cause collisional quenching of diphenylhexatriene fluorescence. Pyrethroids differed in their effectiveness at lowering the phase transition temperature of DPPC, and in their ability to broaden the temperature range of this transition. The fluorescence intensity of DPPC-incorporated chlorophyll a was used to monitor the pretransition of DPPC and the lateral diffusion of a membrane component located in the polar headgroup region. Permethrin did not affect chlorophyll a fluorescence intensity at any temperature. It may be concluded from these results that pyrethroids are preferentially located in the interior hydrophobic regions of the lipid bilayer, and that these compounds can disorder hydrocarbon packing in the bilayer core. However, polar headgroups were not disordered, and diffusion of membrane components in the polar headgroup region was not altered.     

Egghead and brainiac are essential for glycosphingolipid biosynthesis in vivo

The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a beta4-mannosyltransferase, and brainiac encodes a beta3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galbeta1-4Glcbeta1-Cer) using a human beta4-galactosyltransferase (beta4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manbeta1-4Glcbeta1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure.     

Micellar properties of glycosphingolipids in aqueous media

The aggregation properties of neutral glycosphingolipids and gangliosides were investigated by ultracentrifugal sedimentation and gel permeation chromatography. Initially, glycosphingolipids in buffer formed high molecular aggregates. On standing, the glycosphingolipids produced smaller and stable micelles in equilibrium with their monomers at the critical micellar concentration (cmc). The cmc's of monohexaosyl- to tetrahexaosylceramides were on the order of 10(-8) to 10(-7)M. In contrast, values of 10(-8)M for monosialogangliosides and 10(-6)-10(-5)M for di- and trisialogangliosides were found. From estimations of hydrodynamic radii, Svedberg coefficients, and partial specific volumes, relative masses of glycosphingolipid micelles were calculated to be in the range of 300 to 1000 for the neutral glycosphingolipids and 100 to 350 for the gangliosides.     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.     

Hydration of polyethylene glycol-grafted liposomes.

This study aimed to characterize the effect of polyethylene glycol of 2000 molecular weight (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodynamic stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodynamic and hydrational characterization. Using a differential scanning calorimetry technique we showed that each molecule of PEG2000 binds 136 +/- 4 molecules of water. For PEG2000 covalently attached to the lipid molecules organized in micelles, the water binding increases to 210 +/- 6 water molecules. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concentrations over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calculated from ultrasound velocity and density) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concentration up to approximately 7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodynamic stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concentrations, compressibility and partial volume of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five additional water molecules per lipid molecule for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.     

Glycolipids support E-selectin-specific strong cell tethering under flow

This study provides functional evidence that glycosphingolipids constitute ligands for E-selectin but not P-selectin. Chinese hamster ovary (CHO) cells expressing E-selectin (CHO-E) or P-selectin (CHO-P) were perfused over alpha2,3-sialyl Lewis X (alpha2,3-sLe(x)) presented as the hexaosylceramide glycosphingolipid adsorbed in a monolayer containing phosphatidylcholine and cholesterol. CHO-E cells tethered extensively and formed slow, stable rolling interactions with alpha2,3-sLe(x) glycosphingolipid but not with the comparable alpha2,6-sLe(x) glycosphingolipid. Tethering/rolling varied with wall shear stress, selectin density, and ligand density. In contrast, alpha2,3-sLe(x) glycosphingolipid supported only limited, fast CHO-P cell rolling. As calculated from a stochastic model of cell rolling, the step size between successive bond releases from the alpha2,3-sLe(x) glycosphingolipid was smaller for CHO-E than CHO-P cells, whereas the opposite effect was observed for the waiting time between these events. Detachment assays revealed stronger adhesive interactions of CHO-E than CHO-P cells with alpha2,3-sLe(x) glycosphingolipid. These findings indicate that glycosphingolipids expressing an appropriate oligosaccharide mediate cell tethering/rolling via E-selectin but not P-selectin.     

Quantitative analysis of neutral glycosphingolipids from human lymphocyte subpopulations.

The glycosphingolipids of normal human lymphocytes from individual donors were analysed by high-pressure liquid chromatography. In addition, purified T- and B-lymphocytes were examined separately. Lactosylceramide was shown to be the major neutral glycosphingolipid in human lymphocytes, and monohexosylceramide, trihexosylceramide, globoside and paragloboside were all detected in smaller amounts. Analysis of purified B- and T-cell fractions revealed that each of these populations contained a similar qualitative profile for neutral glycosphingolipids, but that quantitatively, B-cells contained several times more of each glycosphingolipid per cell than did T-cells.     

表面活性剂与脂质体相互作用的~(31)P核磁共振研究

(31)~P—NMR实验表明,小的超声囊受表面活性剂Tween80或TritonX—100作用后磷脂头部基团的运动受到限制,可能与磷脂的磷酸根和水化的表面活性剂分子通过氢键的相互作用有关。多层脂质体加Triton X—100后显示各向同性运动,提示表面活性剂可诱发双层结构的改变。     

Drug-membrane interactions studied in phospholipid monolayers adsorbed on nonporous alkylated microspheres

Characterization of interactions with phospholipids is an integral part of the in vitro profiling of drug candidates because of the roles the interactions play in tissue accumulation and passive diffusion. Currently used test systems may inadequately emulate the bilayer core solvation properties (immobilized artificial membranes [IAM]), suffer from potentially slow transport of some chemicals (liposomes in free or immobilized forms), and require a tedious separation (if used for free liposomes). Here the authors introduce a well-defined system overcoming these drawbacks: nonporous octadecylsilica particles coated with a self-assembled phospholipid monolayer. The coating mimics the structure of the headgroup region, as well as the thickness and properties of the hydrocarbon core, more closely than IAM. The monolayer has a similar transition temperature pattern as the corresponding bilayer. The particles can be separated by filtration or a mild centrifugation. The partitioning equilibria of 81 tested chemicals were dissected into the headgroup and core contributions, the latter using the alkane/water partition coefficients. The deconvolution allowed a successful prediction of the bilayer/water partition coefficients with the standard deviation of 0.26 log units. The plate-friendly assay is suitable for high-throughput profiling of drug candidates without sacrificing the quality of analysis or details of the drug-phospholipid interactions.     

Interaction of salt with ether- and ester-linked phospholipid bilayers

A distinguishing feature of Archaeal plasma membranes is that their phospholipids contain ether-links, as opposed to bacterial and eukaryotic plasma membranes where phospholipids primarily contain ester-links. Experiments show that this chemical difference in headgroup-tail linkage does produce distinct differences in model bilayer properties. Here we examine the effects of salt on bilayer structure in the case of an ether-linked lipid bilayer. We use molecular dynamics simulations and compare equilibrium properties of two model lipid bilayers in NaCl salt solution – POPC and its ether-linked analog that we refer to as HOPC. We make the following key observations. The headgroup region of HOPC “adsorbs” fewer ions compared to the headgroup region of POPC. Consistent with this, we note that the Debye screening length in the HOPC system is ∼ 10% shorter than that in the POPC system. Herein, we introduce a protocol to identify the lipid-water interfacial boundary that reproduces the bulk salt distribution consistent with Gouy-Chapman theory. We also note that the HOPC bilayer has excess solvent in the headgroup region when compared to POPC, coinciding with a trough in the electrostatic potential. Waters in this region have longer autocorrelation times and smaller lateral diffusion rates compared to the corresponding region in the POPC bilayer, suggesting that the waters in HOPC are more strongly coordinated to the lipid headgroups. Furthermore, we note that it is this region of tightly coordinated waters in the HOPC system that has a lower density of Na⁺ ions. Based on these observations we conclude that an ether-linked lipid bilayer has a lower binding affinity for Na⁺ compared to an ester-linked lipid bilayer.     

The glycosphingolipid composition of the human hepatoma cell line,Hep-G2

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM3, and lactosyl sulfatide. Four additional minor components (3-fucosyl-lactosamine containing glycolipids, asialo GM2, galactosylgloboside, and ganglioside GM1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.     

Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence.

We have characterized the fluorescence properties of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN) in pure interfaces formed by sphingomyelin and 10 chemically related glycosphingolipids (GSLs).1 The GSLs contain neutral and anionic carbohydrate residues in their oligosaccharide chain. These systems were studied at temperatures below, at, or above the main phase transition temperature of the pure lipid aggregates. The extent of solvent dipolar relaxation around the excited fluorescence probe in the GSLs series increases with the magnitude of the glycosphingolipid polar headgroup below the transition temperature. This conclusion is based on LAURDAN's excitation generalized polarization (GPex) and fluorescence lifetime values found in the different interfaces. A linear dependence between the LAURDAN GPex and the intermolecular spacing among the lipid molecules was found for both neutral and anionic lipids in the GSLs series. This relationship was also followed by phospholipids. We conclude that LAURDAN in these lipid aggregates resides in sites containing different amounts of water. The dimension of these sites increases with the size of the GSLs polar headgroup. The GP function reports on the concentration and dynamics of water molecules in these sites. Upon addition of cholesterol to Gg4Cer, the fluorescence behavior of LAURDAN was similar to that of pure cerebrosides and sphingomyelin vesicles. This observation was attributed to a change in the interfacial hydration as well as changes in the shape and size of the Gg4Cer aggregates in the presence of cholesterol. After the addition of cholesterol to gangliosides, the changes in the LAURDAN's spectral parameters decrease progressively as the polar headgroup of these lipids becomes more complex. This finding suggests that the dehydration effect of cholesterol depends strongly on the curvature radius and the extent of hydration of these lipid aggregates. In the gel phase of phrenosine, GalCer, Gg3Cer, sulfatide, and sphingomyelin, the excitation red band (410 nm) of LAURDAN was reduced with respect to that of LAURDAN in the gel phase of pure phospholipids. This observation indicates a local environment that interacts differently with the ground state of LAURDAN in GSLs when compared with LAURDAN in phospholipids.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15 degrees C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37 degrees C membranes, while 15 degrees C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15 degrees C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15 degrees C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37 degrees C, but only 50% at 15 degrees C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15 degrees C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Glycosphingolipids of normal and leukemic human leukocytes

Summary We have reviewed the studies on neutral glycosphingolipids and gangliosides of normal and leukemia human leukocytes. In this review, we examine (a) the glycosphingolipid composition of various leukocyte populations, (b) the differences in glycosphingolipids found among subsets of these cells, (c) the possible use of these compounds as markers of differentiation, and (d) the changes in glycosphingolipid composition that occur with leukemogenesis.