Anti-Ganglioside Antibodies Bind with Enhanced Affinity to Gangliosides Containing Very Long Chain Fatty Acids

Gangliosides function in both physiological and pathological molecular recognition. Although much research has focused on the role of ganglioside glycans in recognition, fewer studies have addressed the role of the ceramide moiety. Ceramides of major brain gangliosides are composed predominantly of monounsaturated 18-carbon and 20-carbon long chain bases with a saturated 18-carbon fatty acid amide. In contrast, gangliosides of X-linked adrenoleukodystrophy patients are characterized by abnormal very long chain fatty acids that are proposed to be associated with autoimmune inflammation. In the current study we synthesized and characterized derivatives of the major brain ganglioside GD1a bearing defined very long chain fatty acid amides (C24:0, C24:1, and C26:0). When tested in a solid phase binding assay in the presence of auxiliary membrane lipids, GD1a species with long chain fatty acids were up to 8-fold more potent than normal brain GD1a in binding four different anti-GD1a monoclonal antibodies. These data support the hypothesis that gangliosides bearing very long chain fatty acids are differentially displayed on membranes, which may lead to altered antigenicity.     

Globoside with spin-labelled fatty acid: bilayer lateral distribution and immune recognition

We have critically addressed the question of lateral distribution of glycolipids in bilayer membranes, and the effect of glycolipid fatty acid chain length upon such distribution. For this purpose we synthesised the complex neutral glycosphingolipid, globoside, with spin-labelled fatty acid. Base hydrolysis to remove the natural fatty acid was found to deacetylate the GalNAc residue concomitantly, necessitating application of the synthetic route described for gangliosides by Neuenhofer et al. (Biochemistry 24, 525-532 (1985)). Globosides were produced with 18-carbon and 24-carbon fatty acids bearing a spin label at the C-16 position. Spin-labelled globosides were incorporated at 2 and 10 mol% into rigid, highly cooperative bilayer matrices of 1,2-dipalmitoylglycerophosphocholine (DPPC) and also into semi-fluid, non-cooperative membranes of DPPC/cholesterol. Recorded electron paramagnetic resonance (EPR) spectra were analysed by comparison with a library of standards representing samples of known composition. Spectra were manipulated using a computer program which permitted linear combination of standards to stimulate coexistence of laterally separated domains of different composition. The most important conclusions were as follows: (1) at least 80% of the globoside was definitely not confined to domains highly enriched in glycolipid, although there was evidence of binary-phase separation in the rigid DPPC/globoside matrix; (2) the presence of 33 mol% cholesterol reduced the evidence of globoside phase separation; (3) there was remarkably little difference in results whether the globoside fatty acid chain length was similar to that of the phospholipid host matrix or eight carbons longer. Temperature profiles derived over the phase-transition region of DPPC using spin-labelled globoside or an unattached amphiphilic spin label were consistent with these findings. The same systems lent themselves to consideration of the role of glycolipid fatty acid chan length and cholesterol in determining glycolipid crypticity in membranes: (1) polyclonal anti-globoside IgG bound to globoside in DPPC liposomes without inducing agglutination. (2) The same antibodies did agglutinate DPPC/cholesterol liposomes bearing globoside. (3) The effect of cholesterol probably was upon glycolipid dynamics or attitude in the membrane, rather than upon distribution. (4) These observations were basically unaffected by the choice of 18-carbon vs. 24-carbon glycolipid fatty acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that trans-bilayer interdigitation of glycosphingolipid long chain fatty acids may be a general phenomenon

'Interdigitation' is a term coined to describe the phenomenon whereby pure phosphatidylcholines with intramolecular fatty acid chain length heterogeneity when hydrated to form bilayers may insert the methyl ends of long fatty acids from one side across more than half of the membrane thickness to protrude amongst the acyl chains of the opposite side of the bilayer (Keough, K.M.W. and Davis, P.J. (1979) Biochemistry 18, 1453-1459; Huang, C. and Mason, J.T. (1986) Biochim. Biophys. Acta 864, 423-470). In this article we address the fate of long fatty acid chains of glycosphingolipids present as minor components in membranes of non-interdigitating phosphatidylcholines. In this pursuit, derivatives of galactosyl ceramide, lactosyl ceramide, globoside and GM1 were synthesized having either 18-carbon or 24-carbon fatty acid with a spin label covalently attached at C-16. Labelled glycolipids were incorporated at 1-2 mol% into bilayers of synthetic phosphatidylcholines, their mixtures with cholesterol, or natural egg phosphatidylcholine. In each case the C-16 carbon of the glycolipid long chain fatty acid showed considerably greater 'order' and immobility than did C-16 of the fatty acid which was similar in length to the host matrix phospholipids. We interpret this as strong evidence that the long chain fatty acid interdigitates across the mid point of the bilayer in the systems studied. Clearly this phenomenon did not require that the phospholipid host matrix have mixed chain lengths. Furthermore it was totally independent of glycolipid family: for a given host matrix and (glycolipid) fatty acid chain length the order parameter values found were the same amongst all four glycolipid families tested.     

All fatty acids are not equal: discrimination in plant membrane lipids

Plant membrane lipids are primarily composed of 16-carbon and 18-carbon fatty acids containing up to three double bonds. By contrast, the seed oils of many plant species contain fatty acids with significantly different structures. These unusual fatty acids sometimes accumulate to >90% of the total fatty acid content in the seed triacylglycerols, but are generally excluded from the membrane lipids of the plant, including those of the seed. The reasons for their exclusion and the mechanisms by which this is achieved are not completely understood. Here we discuss recent research that has given new insights into how plants prevent the accumulation of unusual fatty acids in membrane lipids, and how strict this censorship of membrane composition is. We also describe a transgenic experiment that resulted in an excessive buildup of unusual fatty acids in cellular membranes, and clearly illustrated that the control of membrane lipid composition is essential for normal plant growth and development.     

Subject index

Heats of fusion and heat capacities have been measured for saturated, unsaturated and hydroxy fatty acids, differing in degree of unsaturation, geometric isomerism, and position of unsaturated and hydroxy groups. Entropies of fusion are used to draw conclusions concerning molecular structure of fatty acid chains and lateral chain-chain interactions. Position of the functional group on the chain does not seem to significantly affect the entropy values for trans and cis single double bonds and single triple bonds, but differences are noted with hydroxy group position. Whereas single acid triglycerides of saturated acids have entropies which are about three times that of the corresponding acid, cis and trans single acid triglycerides do not show the same relationship with their corresponding acids. Comparing entropies of fusion for certain groups of fatty acids, only differing in carbon number, allows the estimation of chain equivalence with saturated fatty acids. Hence, for example it is shown that a 22 to 23-carbon cis mono-unsaturated fatty acid is equivalent to an 18-carbon saturated fatty acid.     

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected.Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.     

Glycosphingolipid acyl chain orientational order in unsaturated phosphatidylcholine bilayers.

The glycosphingolipid, galactosyl ceramide (GalCer), was studied by 2H nuclear magnetic resonance (NMR) in fluid phospholipid bilayer membranes, with regard to arrangement of its acyl chain. For this purpose, species with perdeuterated 18-carbon fatty acid (18:0[d35]GalCer) or with perdeuterated 24-carbon fatty acid (24:0[d47] GalCer) were dispersed in bilayers of the 18-carbon phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). For 18:0[d35] GalCer, smoothed profiles of the order parameter, SCD, were found to be very similar to one another over the range of glycolipid concentration, 5-40 mol%. In addition, they were very similar to orientational order parameter profiles well known from the literature on phospholipid and glycolipid acyl chains (which deals in general with membranes of homogeneous chain length in the range 14-18 carbons). Corresponding order parameter profiles for the long-chain species, 24:0[d47] GalCer, were also similar to one another for glycolipid concentrations between 5 and 40 mol%. Their shapes, however, were distinctly different from those of the shorter chain analogues. SCD profiles for the two species were quantitatively similar to a membrane depth of C15. SCD values at C16 and C17 were approximately 20 and 30%, respectively, higher for the long-chain glycosphingolipid than for its short-chain analogue in SOPC. Nitroxide spin labels attached rigidly to C16 of the long-chain glycolipid in SOPC gave electron paramagnetic resonance (EPR) order parameters that were twice as high as for a spin label at C16 on the shorter chain glycolipid. Comparison was made between spectra of 24:0[d47] GalCer in SOPC and fully hydrated bilayers of the pure 24:0[d47] GalCer, a system that is considered to be partially interdigitated in fluid and gel phases. The resultant 2H NMR order parameter profiles displayed similar features, indicating that related organizational properties exist in these fluid systems. Effective chain length of 24:0[d47] GalCer within the SOPC membrane was calculated using the method of Schindler and Seelig (1975. Biochemistry, 14:2283-2287). The result suggested that the long-chain fatty acid should protrude roughly one third of the host matrix chain length across the bilayer midplane. However, a treatment of the same order parameters making very few assumptions about chain conformation indicated a high degree of orientational flexibility for the "extra" length of the long chain fatty acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clustering of fatty acids in phospholipid bilayers.

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at pH greater than 11 is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D₂O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D₂O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Direct determination of free fatty acid transport across the adipocyte plasma membrane using quantitative fluorescence microscopy.

Movement of free fatty acids (FFA) across the plasma membrane has been directly measured for the first time, using fluorescent FFA analogs and quantitative fluorescence microscopy. The rate of short chain FFA (less than or equal to 12 carbons) transport from the extracellular medium into intracellular lipid droplets of 3T3F442A adipocytes was more than 40-fold faster than long chain FFA (16 and 18 carbons). The membrane-impermeable amino reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited greater than or equal to 50% of the long chain FFA transport but had no effect on short chain FFA transport. Oleic acid (2 microM) inhibited 90% of the fluorescent oleate transport but had no effect on the 11-carbon analog. These results indicate that a large fraction of long chain FFA uptake is mediated by a plasma membrane protein (s).     

Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Microsomal preparations from plant and yeast acylate free fatty acids without prior activation to acyl-thioesters

Acylation of fatty acids to hydroxy groups in cells generally require activation to a thioester (ACP or CoA) or transacylation from another oxygen ester. We now show that microsomal membranes from Arabidopsis leaves efficiently acylate free fatty acids to long chain alcohols with no activation of the fatty acids to thioesters prior to acylation. Studies of the fatty alcohol and fatty acids specificities of the reaction in membranes from Arabidopsis leaves revealed that long chain (C18-C24) unsaturated fatty alcohols and C18-C22 unsaturated fatty acids were preferred. Microsomal preparations from Arabidopsis roots and leaves and from yeast efficiently synthesized ethyl esters from ethanol and free fatty acids. This reaction also occurred without prior activation of the fatty acid to a thioester. The results presented strongly suggest that wax ester and ethyl ester formation are carried out by separate enzymes. The physiological significance of the reactions in plants is discussed in connection to suberin and cutin synthesis. The results also have implication regarding the interpretation of lipid metabolic experiments done with microsomal fraction.     

Human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Temperature-induced changes in fatty acid composition of myelinated and non-myelinated axon phospholipids

The olfactory (non-myelinated) and trigeminal (myelinated) nerve axons of garfish show changes in phospholipid fatty acid composition when these fish are acclimated to temperatures ranging from 11 to 35 degrees C. Myelinated and non-myelinated nerve axons show similar changes in the percent saturated, percent 16-carbon, percent 18-carbon, and percent 20-carbon-and-greater unsaturated fatty acids. The observed changes in phospholipid fatty acid composition fit a linear regression model suggesting a gradual change in axonal phospholipid fatty acid composition with temperature. The temperature-induced changes in garfish nerve phospholipid fatty acid composition are consistent with the general observation of increased saturated fatty acid residues in plasma membrane phospholipids of organisms acclimated to higher environmental temperatures. The garfish data are similar to data previously obtained for goldfish tissues and Tetrahymena.