Factors affecting surface expression of glycolipids: influence of lipid environment and ceramide composition on antibody recognition of cerebroside sulfate in liposomes

The reactivity of the acidic glycolipid cerebroside sulfate (CBS) with antibody was studied as a function of its lipid environment in vesicles and of its ceramide composition. The lipid environment was varied by using phosphatidylcholine of varying chain length with cholesterol in a phosphatidylcholine:cholesterol:cerebroside sulfate molar ratio to glycolipid of 1:0.75:0.1. The ceramide structure of CBS was varied by using synthetic forms containing palmitic acid, lignoceric acid, or the corresponding alpha-hydroxy fatty acids. Reactivity with antibody was determined by measuring complement-mediated lysis of the vesicles containing a spin-label marker, tempocholine chloride. The data were analyzed by a theoretical model which gives relative values for the dissociation constant and concentration of antibodies within the antiserum which are able to bind to the glycolipid. If the phosphatidylcholine chain length was increased, increasing the bilayer thickness, only a small population of high-affinity antibodies were able to bind to cerebroside sulfate, suggesting decreased surface exposure of the glycosyl head group. A larger population of lower affinity antibodies were able to bind to it in a shorter chain length phosphatidylcholine environment. However, if the chain length of the cerebroside sulfate was increased, it could be recognized by more antibodies of lower affinity than the short chain length form, suggesting that an increase in chain length of the glycolipid increased surface exposure. Hydroxylation of the fatty acid inhibited antibody binding; only a smaller population of higher affinity antibodies was able to bind to the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of sphingomyelin bilayer: insight gained through systematic comparison to phosphatidylcholine

Sphingomyelin, one of the main lipid components of biological membranes, is actively involved in various cellular processes such as protein trafficking and signal transduction. In particular, specific lateral domains enriched in sphingomyelin and cholesterol have been proposed to play an important functional role in biomembranes, although their precise characteristics have remained unclear. A thorough understanding of the functional role of membranes requires detailed knowledge of their individual lipid components. Here, we employ molecular dynamics simulations to conduct a systematic comparison of a palmitoylsphingomyelin (PSM, 16:0-SM) bilayer with a membrane that comprises dipalmitoylphosphatidylcholine (DPPC) above the main phase transition temperature. We clarify atomic-scale properties that are specific to sphingomyelin due to its sphingosine moiety, and further discuss their implications for SM-rich membranes. We find that PSM bilayers, and in particular the dynamics of PSM systems, are distinctly different from those of a DPPC bilayer. When compared with DPPC, the strong hydrogen bonding properties characteristic to PSM are observed to lead to considerable structural changes in the polar headgroup and interface regions. The strong ordering of PSM acyl chains and specific ordering effects in the vicinity of a PSM-water interface reflect this issue clearly. The sphingosine moiety and related hydrogen bonding further play a crucial role in the dynamics of PSM bilayers, as most dynamic properties, such as lateral and rotational diffusion, are strongly suppressed. This is most evident in the rotational motion characterized by spin-lattice relaxation times and the decay of hydrogen bond autocorrelation functions that are expected to be important in complexation of SM with other lipids in many-component bilayers. A thorough understanding of SM bilayers would greatly benefit from nuclear magnetic resonance experiments for acyl chain ordering and dynamics, allowing full comparison of these simulations to experiments.     

Imaging of the domain organization in sphingomyelin and phosphatidylcholine monolayers

The lateral organization of biomembranes has gained significant interest when the fluid mosaic model was challenged by the model of "lipid rafts". Several lipid classes like cholesterol and sphingolipids are considered to be essential for their formation. Here we investigate the lateral domain formation in binary mixtures of sphingomyelin and phosphatidylcholine. Both are major lipid components of lipoproteins and mammalian cell membranes at various molar ratios. Surface pressure-area isotherms and surface potential-area isotherms of monolayers composed of these lipids clearly indicated non-ideal mixing. In addition, Brewster angle microscopy provided a well-suited approach to image the formation of lateral domains. These images demonstrated that pure sphingomyelin forms very stable finger-like domains that exhibit a distinct internal organization suggesting an anisotropic orientation of the acyl side chains. Similar behavior was found for mixtures containing more than 60 mol% sphingomyelin. With increasing content of phosphatidylcholine the domain size decreased and the surface pressure, where domain formation occurred, increased. At lower sphingomyelin content (30-60 mol%) rather round-shaped, smaller domains were observed. Thus, the potential of sphingomyelin domains as potentially important building blocks for actual domains that could be building blocks for raft formation is suggested, even without the presence of cholesterol. In addition, these observations may suggest a role for the distinct molar ratio of these key lipids frequently found in physiologically relevant particles such as low and high density lipoproteins or the outer leaflet of the human erythrocyte membrane.     

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers

To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state ³¹P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, T_m, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 °C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 °C were found to be 6.1±0.8 mm ^–1 and 3.7±0.4 mm ^–1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, R_i≤5) mixed micelles are formed over the entire temperature range (20° to 60 °C) for both kinds of lipids.     

A synchrotron X-ray diffraction characterization of the structure of complexes formed between sphingomyelin and cerebroside

Specific lipid-lipid interactions are believed to be responsible for lateral domain formation in the lipid bilayer matrix of cell membranes. The miscibility of glucocerebroside and sphingomyelin extracted from biological tissues has been examined by synchrotron X-ray powder diffraction methods. Fully hydrated binary mixtures of egg-sphingomyelin codispersed with glucosylceramide rich in saturated C22 and C24 N-acyl fatty acids were subjected to heating scans between 20 and 90 °C at 2 °C·min(-1). X-ray scattering intensity profiles were recorded at 1 °C intervals simultaneously in both small-angle and wide-angle scattering regions. A gel phase characterized by a single symmetric peak in the wide-angle scattering region was transformed in all mixtures examined to a fluid phase at about 40 °C, similar to dispersions of pure egg-sphingomyelin. A coexisting lamellar structure was identified at temperatures up to about 75 °C which was characterized by a broad Bragg reflection. The scattering intensity of this structure increased relative to the structure assigned as bilayers of pure sphingomyelin with increasing proportions of glucosylceramide in the mixture. The relationship between the scattering intensities of the two peaks and the relative mass fractions of the two lipids showed that the bilayers assigned to a glucosylceramide-rich structure were composed of sphingomyelin and glucosylceramide in molar ratios of 1 : 1 and 2 : 1, respectively, at temperatures below and above the order-disorder phase transition temperature of the sphingomyelin (40 °C).     

Diversity of the effect of phosphatidylcholine and sphingomyelin on adenylate deaminase from pig brain

Liposomes made of sphingomyelin were found to inhibit both ATP-activated and non-activated AMP deaminase from pig brain, in contrast to liposomes made of egg yolk phosphatidylcholine which exhibited an activating effect on the ATP-activated enzyme, being without effect on AMP deaminase in the absence of ATP. Dioleoylphosphatidylcholine exerted a similar effect as egg yolk phosphatidylcholine but dipalmitoylphosphatidylcholine was without effect.     

Influence of structural modifications on the phase behavior of semi-synthetic cerebroside sulfate

Cerebroside sulfate (galactosylceramide I3-sulfate) containing alpha-hydroxy lignoceric acid (C24:0h-CBS), nervonic acid (C24:1-CBS), or hexacosanoic acid (C26:0-CBS) was prepared by a semi-synthetic procedure and studied by differential scanning calorimetry. The phase behavior of these species in 2 M KCl was compared to that of shorter chain length hydroxy and non-hydroxy fatty acid species reported earlier. All three of the new lipids undergo metastable phase behavior similar but not identical to the other species. In addition, the metastable phase behavior of all of the non-hydroxy fatty acid species was found to be more complex than previously thought, with several phases of high transition temperatures and enthalpies possible. Fatty acid hydroxylation inhibits the transition from the metastable to some of the more stable phases. It also significantly increases the phase transition temperatures of both the metastable and stable phases indicating that it contributes to the hydrogen bonding network formed between the lipid molecules and helps overcome the lateral repulsive effect of the negatively charged sulfate. The C-15 cis double bond significantly lowers the temperature and enthalpy of the phase transition indicating that it increases the lateral separation of the lipid molecules and decreases the intermolecular hydrogen bonding interactions. However, it does not prevent formation of a more stable phase. By comparing the effect of various structural modifications reported here and earlier it could be concluded that fatty acid chain length has little effect on the phase transition temperature and enthalpy. This suggests that the forces between the lipid molecules may be dominated by head group interactions rather than interactions between the lipid chains. However, fatty acid chain length has a significant effect on the tendency of the hydroxy fatty acid species to form the more stable phase. The ease of formation of the stable phase increases with increase in chain length. Thus an increase in chain length helps overcome the kinetic barrier to stable phase formation presented by hydroxylation of the fatty acid.     

Diacetylenic lipid microstructures: structural characterization by X-ray diffraction and comparison with the saturated phosphatidylcholine analogue

Thermotropic and lyotropic mesomorphism in the polymerizable lecithin 1,2-ditricosa-10,12-diynoyl-sn-glycero-3-phosphocholine and its saturated analogue, 1,2-ditricosanoyl-sn-glycero-3-phosphocholine, has been investigated by wide- and low-angle X-ray diffraction of both powder and oriented samples and by differential scanning calorimetry. Previous studies have shown that the hydrated diacetylenic lipid forms novel microstructures (tubules and stacked bilayer sheets) in its low-temperature phase. The diffraction results indicate that at low temperatures fully hydrated tubules and sheets have an identical lamellar repeat size (d001 = 66.4 A) and crystalline-like packing of the acyl chains. Chain packing in the lamellar crystalline phase is hydration independent. A model for the polymerizable lecithin with (1) fully extended all-trans methylene segments, (2) a long-axis tilt of 32 degrees, and (3) minimal chain interdigitation seems most reasonable on energetic grounds, is consistent with the diffraction data (to 3.93-A resolution), and is likely to support facile polymerization. Above the chain "melting" transition the lamellar repeat of the polymerizable lipid increases to 74 A. The conformational similarity between tubules, sheets, and the dry powder is corroborated by calorimetry, which reveals a cooling exotherm at the same temperature where tubules form upon cooling hydrated sheets. The data suggest that although a high degree of conformational order is a pertinent feature of tubules, this character alone is not sufficient to account for tubule formation. The conformation of the corresponding saturated phosphatidylcholine appears to be similar to that of other saturated phosphatidylcholines in the lamellar gel phase. Furthermore, above the main transition temperature, the dry, saturated lipid shows evidence of a P delta phase (112 degrees C), whereas the diacetylenic lipid appears to exhibit a centered rectangular phase, R alpha (55 degrees C).     

Hydrolysis of sphingomyelin and phosphatidylcholine by midgut homogenates of the stable fly

Qualitative and quantitative analyses were made to characterize the enzymatic degradation of sphingomyelin and phosphatidylcholine by midgut homogenates of the adult stable fly, Stomoxys calcitrans (L.). The results indicated that sphingomyelin was hydrolyzed by an enzyme with sphingomyelinase-like properties, and that phosphatidylcholine was hydrolyzed by an enzyme with properties similar to phospholipase C. The optimum pH for the sphingomyelinase was 7.6, and the rate of hydrolysis of sphingomyelin at that pH was linear from 1 to 4 nmol of substrate and 5 to 25 micrograms of enzyme preparation. Dialysis of the homogenates against Tris-HCl and imidazole buffers resulted in a decrease of sphingomyelinase activity by 59% and 98%, respectively, and the original activity was not restored with the addition of Ca++, Mg++, or Mn++.     

Content of phospholipid, cerebroside and cerebroside sulfate in the central nervous system of mice with acute experimental viral demyelination

A study was made of the content of phospholipids, cerebrosides and cerebroside sulfates in the central nervous system of mice with experimental acute viral encephalomyelitis. No considerable changes in phospholipid content were revealed. A significant drop in the content of cerebrosides and cerebroside sulfates was defected in the CNS, being more pronounced in the spinal cord of sick animals. The reduction in the content of glycolipids can be explained by myelin disintegration and by the effect of viruses on the olygodendrocytes in which cerebrosides and cerebroside sulfates are synthesized.     

Importance of phosphatidylcholine on the chloroplast surface

In plant cells, phosphatidylcholine (PC) is a major glycerolipid of most membranes but practically lacking from the plastid internal membranes. In chloroplasts, PC is absent from the thylakoids and the inner envelope membrane. It is however the main component of the outer envelope membrane, where it exclusively distributes in the outer monolayer. This unique distribution is likely related with operational compartmentalization of plant lipid metabolism. In this review, we summarize the different mechanisms involved in homeostasis of PC in plant cells. The specific origin of chloroplast PC is examined and the involvement of the P4-ATPase family of phospholipid flippases (ALA) is considered with a special attention to the recently reported effect of the endoplasmic reticulum-localized ALA10 on modification of chloroplast PC desaturation. The different possible roles of chloroplast PC are then discussed and analyzed in consideration of plant physiology.     

Effect of sphingomyelin versus dipalmitoylphosphatidylcholine on the extent of lipid oxidation

Phospholipids with sites of unsaturation are targets of peroxidation. We investigated the effect of two types of lipids with identical headgroups, sphingomyelins (SMs) and dipalmitoylphosphatidylcholine (DPPC), on the extent of oxidation of stearoyl-arachidonoyl phosphatidylcholine (SAPC) with four double bonds. Peroxidation was induced with tert-butylhydroperoxide and FeCl(2) at 35 degrees C. The decrease of SAPC versus DPPC, or N-palmitoyl SM, or N-stearoyl SM, was monitored by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) at various reaction times. For corresponding molar ratios of DPPC:SAPC and SM:SAPC, SAPC oxidized faster and to a greater extent when DPPC, rather than N-palmitoyl SM or bovine brain SMs, was present. However, at 35 degrees C the hydrophobic tails in SM mixtures were more disordered than in those of DPPC. The slower oxidation of SAPC in SM-rich vesicles may result from the presence of a tight network of H-bonds that bridge neighboring SM molecules and poses a stronger interfacial barrier to the passage of oxidants.     

Investigation of human low-density lipoprotein by (1)H nuclear magnetic resonance spectroscopy: mobility of phosphatidylcholine and sphingomyelin headgroups characterizes the surface layer

The resolution of the trimethyl headgroup resonance of phosphatidylcholine (PC) and sphingomyelin (SM) in the intact human low-density lipoprotein (LDL) (1)H NMR spectrum at 600 MHz enabled the investigation of LDL surface structure and phospholipid-apoB interactions. We have previously shown that a higher proportion of PC headgroups (25-35% of total PC in LDL) compared to SM were tightly bound to apoB and therefore NMR-invisible [Murphy, H. C., et al. (1997) Biochem. Biophys. Res. Commun. 234 (3), 733-737]. In the present study, we have investigated the mobility of phospholipid (PL) headgroups, using (1)H NMR spin-spin (T(2)) relaxation measurements, in LDL isolated from nine volunteers. We show that both PC and SM exist in two additional and distinct environments indicated by the biexponential behavior of the relaxation decays in each case. The data showed that 36% of PC headgroups had a short T(2) component, mean T(2) of 31 ms, and 64% had a longer T(2) component of 54 ms. Approximately 15% of SM headgroups had a short T(2) component (mean T(2) of 27 ms) and 85% had a longer T(2) component of 78 ms. Therefore the majority of SM headgroups (85%) were more mobile than PC (P < 0.001) and since PC headgroups in organic media were more mobile than SM, we conclude that the characteristic high mobility of LDL SM is not an intrinsic property but arises from a high degree of order in molecular packing of the surface PL of human LDL. We suggest that because PC and SM interact differentially with cholesterol and possibly with neighboring phospholipids, this results in the formation of relatively long-lived microdomains of PL in vivo.     

Trifluoperazine stimulates the coordinate degradation of sphingomyelin and phosphatidylcholine in GH3 pituitary cells

Prior studies demonstrated that 1,2-diacylglycerols stimulated degradation of the choline-containing phospholipids, phosphatidylcholine and sphingomyelin, in GH3 pituitary cells by a phospholipase A2 and a sphingomyelinase, respectively (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762). The present studies demonstrate that the phenothiazine trifluoperazine also stimulates degradation of these phospholipids. Trifluoperazine (25 microM) reduced phosphatidylcholine and sphingomyelin levels to 81 and 58% of control, respectively, after 30 min in cells labeled for 48 h with [3H] choline. Choline-containing metabolites were released specifically into the cytosolic fraction. The level of cytosolic phosphocholine, but not choline or CDP-choline, increased to 150% of control. These events were not mediated by inhibition of phosphatidylcholine synthesis. The level of 1,2-diacylglycerols, but not lysophosphatidylcholine or glycerol-3-phosphocholine, also increased. These data are most consistent with phosphatidylcholine degradation via a phospholipase C. Trifluoperazine-stimulated sphingomyelin degradation was accompanied by quantitative generation of ceramides consistent with activation of a sphingomyelinase. In contrast to trifluoperazine, choline-containing metabolites were released into the medium during stimulation by the 1,2-diacylglycerol 1,2-dioctanoyl-glycerol. Although both trifluoperazine and 1,2-dioctanoylglycerol increased ceramide levels, only 1,2-dioctanoylglycerol increased the sphingoid base level from 24 to 43 pmol/10(6) cells. Hence, trifluoperazine appears to deplete an intracellular pool of phosphatidylcholine and sphingomyelin by a different mechanism than 1,2-diacylglycerols. This is the first report of phenothiazine-induced degradation of choline-containing phospholipids.     

Structure of PITPbeta in complex with phosphatidylcholine: comparison of structure and lipid transfer to other PITP isoforms

Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentially binds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids between membranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITPalpha and PITPbeta isoforms (269-270 residues). The crystal structure of rat PITPbeta complexed to dioleoylphosphatidylcholine was determined to 2.18 A resolution with molecular replacement using rat PITPalpha (77% sequence identify) as the phasing model. A structure comparison of the alpha and beta isoforms reveals minimal differences in protein conformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solvent structure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using small unilamellar vesicles with carefully controlled phospholipid composition, indicates that the beta isoforms have minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles contain predominantly PtdCho. When PtdCho and PtdIns are present in equivalent concentrations in donor vesicles, PtdIns transfer occurs at approximately 3-fold the rate of PtdCho. The rat PITPbeta isoform clearly has the most diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutations of two locations within the lipid binding cavity that possess differing biochemical properties were characterized: I84alpha/F83beta and F225alpha/L224beta. The 225/224 locus is more critical in determining substrate specificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specific activity became PITPalpha (F225L) approximately PITPbeta and PITPbeta (L224F) approximately PITPalpha. The 225alpha/224beta locus plays a modest role in the specificity of both isoforms toward CerPCho.     

The role of endogenous phosphatidylcholine and ceramide in the biosynthesis of sphingomyelin in mouse fibroblasts

The intracellular location of sphingomyelin formation via the cholinephosphotransferase reaction from both endogenous an exogenous phosphatidylcholine and ceramide substrates has been studied in the subcellular membrane fractions prepared from mouse fibroblasts. The enzyme was found to be located in both the plasma membrane and the Golgi fractions. Activity in the Golgi fraction was stimulated to a greater extent by the addition of exogenous ceramide than was the activity in the plasma membrane fraction. It is concluded that endogenous phosphatidylcholine is available to the cholinephosphotransferase at saturating concentration and, therefore, is not rate-limiting. In contrast, the very low concentration of endogenous ceramide seems to limit the reaction rate, necessitating supplementation with exogenous material Both endogenous substrates are shown to be utilized in an intramembranous rather than an intermembranous reaction. The capacity of the plasma membrane fraction to synthesize sphingomyelin from endogenous phosphatidylcholine and ceramide was found to be sufficiently high to account for the rate of net synthesis of plasma membrane-bound sphingomyelin observed in the logarithmically multiplying cell culture. In contrast, the Golgi fraction displayed only 26% of the expected capacity, but it was stimulated 6-fold by the addition of exogenous ceramide. These results demonstrate that the total cellular sphingomyelin of the mouse fibroblasts can be provided via the cholinephosphotransferase pathways and that the plasma membrane and the Golgi fraction are most probably the intracellular sites of sphingomyelin biosynthesis.     

The effect of phosphatidylcholine to sphingomyelin mole ratio on the dynamic properties of sheep erythrocyte membrane

Sheep red blood cells are shown to incorporate phosphatidylcholine when incubated in human plasma in the presence of EGTA. This treatment results in up to a 5-fold increase in mol ratio of phosphatidylcholine to sphingomyelin. By replacing EGTA with Ca²⁺ the increase of phosphatidylcholine content is completely inhibited, due to the activation of the membrane bound lecithinase which rapidly degrades the incorporated phosphatidylcholine. Analogous treatments of the isolated erythrocyte membranes resulted in similar phosphatidylcholine incorporation but in the presence of Ca²⁺ a residual phosphatidylcholine uptake was still observed. These results suggest that in the isolated membranes small amounts of phosphatidylcholine can be incorporated into an additional region which is unavailable for the membrane lecithinase. The increase in the phosphatidylcholine to sphingomyelin mol ratio in sheep red blood cells is concomitant with an increase in lipid fluidity, as well as increase in osmotic fragility.