Nonhydroxy Fatty Acids Research Articles

Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography.

The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glcbeta1Cer), galactosylceramide (Galbeta1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO(3)-3Galbeta1Cer), lactosylceramide (Galbeta1-4Glcbeta1Cer), gangliotriaosylceramide GgO3 (GalNAcbeta1-4Galbeta1-4Glcbeta1Cer), and gangliotetraosylceramide GgO4 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacbeta1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.

Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid–sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.

Drastically abnormal gluco- and galactosylceramide composition does not affect ganglioside metabolism in the brain of mice deficient in galactosylceramide synthase.

Mice that are genetically deficient in UDP-galactose: ceramide galactosyltransferase are unable to synthesize galactosylceramide. Consequently, sulfatide, which can be synthesized only by sulfation of galactosylceramide, is also totally absent in affected mouse brain. Alpha-hydroxy fatty acid-containing glucosylceramide partially replaces the missing galactosylceramide. A substantial proportion of sphingomyelin, which normally contains only non-hydroxy fatty acids, also contains alpha-hydroxy fatty acids. These findings indicate that alpha-hydroxy fatty acid-containing ceramide normally present only in galactosylceramide and sulfatide is diverted to other compounds because they cannot be synthesized into galactosylceramide due to the lack of the galactosyltransferase. We have examined brain gangliosides in order to determine if alpha-hydroxy fatty acid-containing glucosylceramide present in an abnormally high concentration is also incorporated into gangliosides. The brain ganglioside composition, however, is entirely normal in both the total amount and molecular distribution in these mice. One feasible explanation is that UDP-galactose: glucosylceramide galactosyltransferase does not recognize alpha-hydroxy fatty acid-containing glucosylceramide as acceptor. This analytical finding is consistent with the relative sparing of gray matter in the affected mice and provides an insight into sphingolipid metabolism in the mouse brain.

Glycolipids as potential binding sites for HIV: topology in the sperm plasma membrane in relation to the regulation of membrane fusion

Although human sperm cells can bind human immunodeficiency virus (HIV-1), they lack CD4, galactoceramides (GalCer) and sulfogalactoceramides (SGalCer) as gp120 receptors. However, sperm specific glycolipids (sulfogalactosylalkylacylglycerol (SGalAAG) and galactosylalkylacylglycerol (GalAAG)) are structurally closely related to SGalCer and GalCer as predicted by computer simulated molecular modelling. SGalAAG and GalAAG are exclusively localized in the outer leaflet of the human sperm plasma membrane, and therefore we tested whether they could serve as alternative receptors for the gp120. Purified SGalAAG and GalAAG had similar affinities to recombinant gp120 as the hydroxy fatty acid (HFA) SGalCer and HFA-GalCer respectively. However, nonhydroxy fatty acid forms of (S)GalCer, galactosyldiacylglycerol and the deacylated (sulfo)galactosyllipids did not recognize recombinant gp120. Data obtained by surface pressure experiments revealed that the lipid monolayers that contained HFA-GalCer or GalAAG resulted in a similar significant penetration of recombinant gp120 in the monolayer. The penetration was a factor of two lower in monolayers with HFA-SGalCer or SGalAAG. The binding of recombinant gp120 to human sperm cells colocalized with GalAAG and could be blocked with monoclonal antibodies against galactolipids. The possible relevance of gp120 binding to glycolipids for HIV entry in sperm cells is discussed.

The Fate of Ingested Glyceran Esters of Condensed Castor Oil Fatty Acids [Polyglycerol Polyricinoleate (PGPR)] in the Rat

Samples of the emulsifier polyglycerol polyricinoleate (PGPR) were synthesized using the radiolabelled precursors [1- 14C]glycerol ([ 14C]polyglycerol PGPR), [9,10- 3H] or [12- 3H]ricinoleic acid ([ 3H] PGPR) or [1- 14C]stearic acid ([ 14C]stearyl PGPR). The absorption, tissue distribution, metabolism and excretion of these 14C- or tritium-labelled PGPR samples administered to rats was studied. The effects of intestinal and porcine pancreatic lipases on PGPR preparations were examined. Rats were dosed with [1- 14C]glycerol, [ 14C]polyglycerol and ([ 14C]polyglycerol)PGPR by gavage and their urine, faeces and expired CO 2 monitored for 14C. The results from the [1- 14C]glycerol treated animals showed extensive metabolism of glycerol. For [ 14C]polyglycerols, the lower polyglycerols were preferentially absorbed from the intestine and were excreted unchanged in the urine while the higher polyglycerols were found in the faeces. After 4 days, 93% of the dose of polyglycerols was recovered, of which some 30% was found in the urine and 60% in the faeces. Traces of 14C activity were found in depot fat and liver. The excretory pattern and urinary metabolites from ([ 14C]polyglycerol) PGPR was very similar to that of [ 14C]polyglycerol. Analysis of urinary and faecal 14C material indicated that the PGPR polymer was digested to give free polyglycerol and polyricinoleic acid. PGPR was synthesised incorporating [1- 14C]stearic into polyricinoleic acid which was then esterified with polyglycerol. The resulting [ 14C]PGPR or [1- 14C] stearic acid in a dietary slurry was administered to groups of fed or starved rats by gavage. The results indicated complete digestion of PGPR and absorption of the fatty acids. The 14C-material absorbed was extensively laid down in depot fat and some metabolism to 14CO 2 was demonstrated. The fate of the stearic acid was similar whether dosed alone or incorporated into the PGPR polymer. Samples of PGPR were synthesized containing 3H-labelled ricinoleic acid. The resulting [ 3H]PGPR was intubated into rats as a component of a dietary slurry. The results indicated that the polymer is extensively digested and 90% of the administered tritium is absorbed. The absorbed material was extensively metabolized within 24 hr so that large amounts of tritium were present in the aqueous phase of the tissues examined. After 24 hr, less than 5% of the administered material was present as lipid material, of which a large proportion was as non-hydroxy fatty acids. No traces of polymer material were found in the tissues examined. In vitro digestion of PGPR by porcine pancreatic lipase and rat intestinal fractions was demonstrated. The results indicate very extensive digestion of the PGPR polymer to polyglycerols and fatty acids. The fatty acids are metabolized extensively. The mono-, di- and triglycerols are extensively absorbed from the intestinal tract and rapidly excreted in the urine unchanged but the hexa-, penta- and higher polyglycerols are essentially not absorbed and excreted in the faeces unchanged.

A Novel Carbohydrate-Glycosphingolipid Interaction between a β-(1–3)-Glucan Immunomodulator, PGG-glucan, and Lactosylceramide of Human Leukocytes

The immunomodulator Betafectin(R) PGG-glucan is a homopolymer of glucose derived from yeast cell walls which has been demonstrated to enhance leukocyte anti-infective activity in vitro and in vivo, without the induction of proinflammatory cytokines. We report here the purification of a PGG-glucan-binding element from human leukocytes and its identification as lactosylceramide, a major glycosphingolipid of neutrophils, which includes the CDw17 epitope. The binding of radiolabeled PGG-glucan to purified lactosylceramide was saturable, specific, and time- and temperature-dependent. Lactosylceramides from human leukocytes were fractionated by high performance liquid chromatography in order to analyze the effect of ceramide structure on binding. A variety of fatty acid chain lengths with varying degrees of unsaturation were found to support binding to radiolabeled PGG-glucan. However, DL-lactosylceramides containing dihydrosphingosine did not bind. Radiolabeled PGG-glucan bound several other neutral glycosphingolipids with a terminal galactose, including galactosylceramide, globotriaosylceramide, and gangliotetraosylceramide. The binding of radiolabeled PGG-glucan to lactosylceramide was not inhibited by glycogen, dextran, mannan, pustulan, laminarin, or a low molecular weight beta-(1-3)-glucan, but was inhibited by high molecular weight beta-(1-3)-glucans and by a monoclonal antibody to lactosylceramide. Although this glycosphingolipid has been shown in numerous reports to bind various microorganisms, this represents the first report of lactosylceramide binding to a macromolecular carbohydrate.

The lactosylceramide binding specificity of Helicobacter pylori.

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.

Isolation and characterization of a unique sulfated ganglioside, sulfated GM1a, from rat kidney.

A novel class of sulfoglycosphingolipid, a sulfate analog of ganglioside, was isolated from mammals for the first time. This sulfated ganglioside was purified from rat kidney by column chromatographies on anion exchangers and silica beads. One-dimensional 1H NMR, compositional and permethylation analyses showed that this glycolipid has a Gg4Cer core with 1 mol each of sulfate ester and N- glycolylneuraminic acid (NeuGc) at C-3 of galactose. The major ceramide consisted of nonhydroxy fatty acids (24:0 and 22:0) and 4-hydroxysphinganine (t18:0), deduced from the compositional analysis and negative liquid secondary ion mass spectrometry (LSIMS). Mild acid hydrolysis and solvolysis produced compounds which correspond to Gg4Cer IV3-sulfate (SM1b) and II3NeuGcalpha-Gg4Cer (GM1a (NeuGc)), respectively. The abundant ions characteristic for sulfated mono- and disaccharides in high-energy collision-induced dissociation (CID) spectra were consistent with the structure at the non-reducing terminus, HSO3 -O- Hex -O- HexNAc- rather than the alternative structure, NeuGc -O- Hex -O- HexNAc-. The two-dimensional 1H NMR further evidenced the presence of a 3 -O- sulfated galactose in the molecule. From these results the complete structure was proposed to be HSO3-3Galbeta-3GalNAcbeta-4(NeuGcalpha-3)Galb eta-4Glcbeta-1Cer (II3NeuGcalpha-Gg4Cer IV3-sulfate).

Acidic glycosphingolipids of cock testis elucidated by mass spectrometry and NMR spectroscopy.

The main acidic glycosphingolipids (GSLs) of cock testis were identified as GalCer I3-sulfate and gangliosides GM4, GM3, GD3 and GT3. They contained N-acetylneuraminic acid as the major sialic acid, and ceramides composed mainly of sphingosine (dl8:1) and C18-24 non-hydroxy fatty acids. Appreciable amounts of hydroxy fatty acids were detected only in the GM4 preparation.

FTIR Spectroscopy Studies of the Conformational Order and Phase Behavior of Ceramides

Ceramides, the major lipid component of the stratum corneum (SC), provide many of its unique physical properties. Surprisingly, only a few biophysical studies of hydrated ceramides have been reported. The current Fourier transform infrared (FTIR) spectroscopy investigation provides the first detailed study of intermolecular and intramolecular chain and headgroup interactions in hydrated non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) ceramides. Information about NFA and HFA ceramide chain subcell structure and conformational order is derived from the temperature dependence of the methylene stretching, scissoring, and rocking mode frequencies. At low temperatures, NFA ceramide is highly ordered and packed in an orthorhombic subcell structure that undergoes a solid−solid phase transition to a conformationally ordered hexagonal phase at 60 °C. A second transition to a conformationally disordered non-bilayer-like phase occurs at 80 °C. The lipid chains of HFA ceramide undergo a single transition from a conformationally ordered, orthorhombic subcell, phase to a conformationally disordered phase at 76 °C. In NFA ceramide the amide I and II modes of the headgroup are each split into two bands, indicating strong intermolecular headgroup coupling between two NFA ceramide headgroups in a factor group perpendicular to the bilayer plane. In contrast, splitting is not observed for either amide mode in HFA ceramide. However, the presence of strong H bonding indicates an interaction between molecules in the bilayer plane. The contrasting behavior of the headgroups in NFA and HFA ceramide suggests that these molecules make distinct contributions to the structural integrity of the stratum corneum. The implications of these findings to the recently proposed domain mosaic model of the stratum corneum lipid barrier are discussed.

Formation of high-axial-ratio-microstructures from natural and synthetic sphingolipids

Amphiphiles that form high-axial-ratio-microstructures (HARMs) are being considered as novel materials for controlled release of drugs and other biologically functional molecules. HARMs consisting of tubules, ribbons, solid rods and helices are formed from sphingolipids by addition of water to a solution of amphiphile in DMF. Single molecular species of galactocerebroside (GalCer) containing long unsaturated fatty acid chains or natural GalCer containing mixed-length, non-hydroxy fatty acids (NFA-GalCer) or α-hydroxy fatty acids (HFA-GalCer) form cylindrical structures. In contrast, single molecular species of GalCer containing long saturated fatty acids form ribbons and helices. GalCer HARMs are typically under 100 nm in diameter and have lengths of several microns. The importance of the amide of GalCer for HARM formation was evaluated using psychosine, which forms solid fibers, whereas sphingosine and an analog of GalCer in which the amide is reduced to a secondary amine form amorphous aggregates. Single molecular species of ceramide containing long unsaturated fatty acid chains form cylindrical structures, whereas those with long saturated fatty acids form ribbons and helices. Short chain saturated ceramide also forms cylindrical structures. GalCer analogs with N-acetyl-glycine in place of the galactose form fibers whereas those with N-acetyl-proline yield amorphous material. The N-acetyl-proline-containing amphiphile can de doped into pure GalCer or NFA-GalCer without perturbing tubule formation.

Neutral glycosphingolipids induce cell-cell aggregation of a variety of hematopoietic cell lines.

Exogenous neutral glycosphingolipids induced homotypic cell-cell aggregation of a variety of hematopoietic cell lines. A mouse cytotoxic T cell line, CTLL-2, was chosen to study the neutral glycosphingolipid-induced aggregation. Among neutral glycosphingolipids tested, galactosylceramide (GalCer) and glucosylceramide (GlcCer) were potent inducers, followed by lactosylceramide (LacCer); globotriaosylceramide (Gb3Cer) and neolactotetraosylceramide (nLc4Cer) were less effective. GalCer that contained a non-hydroxy fatty acid was more efficient than GalCer containing an alpha-hydroxy fatty acid. The minimum concentration of GalCer containing a non-hydroxy fatty acid that induced aggregation was 1 microM, and maximum aggregation occurred at 10-20 microM within 24 h. Cytochalasin B and a mixture of sodium azide and 2-deoxyglucose inhibited the aggregation, whereas cycloheximide, actinomycin D, and colchicine did not. Aggregated and dispersed cells, which were designated as competent cells, re-aggregated in the absence of neutral glycosphingolipids. Anti-GalCer polyclonal antibody inhibited GalCer-induced reaggregation. Furthermore, competent cells bound and aggregated non-competent cells in the absence of neutral glycosphingolipids. Cell-cell aggregatign was similar for CTLL-2 cells and the other hematopoietic cells that were tested. These findings suggest that the neutral glycosphingolipid-induced cell-cell aggregation of CTLL-2 cells was mediated by heterophilic interaction(s) between glycosphingolipids and other cell surface components. These properties are shared by a variety of hematopoietic cell lines.

N-acyl amino acid biosynthesis in marine bacterium, Deleya marina

We reported previously that the marine bacterium, Deleya marina (ATCC 25374), produced N-acyl leucine and isoleucine, in which nonhydroxy fatty acid was linked to α-amino group of amino acid. Further analysis of bacterium lipids revealed the additional production of N-acyl ornithine. The N-acyl ornithine had a 3-hydroxy fatty acid linked by an amide bond to α-amino group of ornithine and a nonhydroxy fatty acid esterified to the hydroxy group of the 3-hydroxy fatty acid. N-acyl ornithine was located in the cell membrane and N-acyl leucine and isoleucine in cytoplasm. N-acyl ornithine is thought to be a functional analogue of phosphatidylethanolamine (PE) because of their similar structure. PE replacement into N-acyl ornithine in the cell membrane under phosphate-limited conditions was observed with other bacteria, so we anticipated the nonbiosynthesis of N-acyl ornithine under phosphate-sufficient conditions. We did not anticipate that N-acyl leucine and isoleucine in cytoplasm, whose structure is dissimilar to that of PE, would be replaced into PE in the cell membrane. Neither N-acyl leucine, N-acyl isoleucine, nor N-acyl ornithine was biosynthesized under phosphate-sufficient condition. Thus, we report here for the first time that N-acyl amino acids in cytoplasm were not biosynthesized under phosphate-sufficient conditions.

Enrichment of lesquerolic and auricolic acids from hydrolyzed Lesquerella fendleri and L. gordonii oil by crystallization

AbstractLesquerolic and auricolic acids were obtained from hydrolyzed lesquerella oil by a low‐temperature crystallization procedure. The lesquerolic and auricolic fatty acid fraction was enriched from 55–59% to 85–99% with high yields (94%). Washing the free fatty acids with pH 6.0 buffer provided reproducible crystallizations of those hydroxy fatty acids. In contrast, when hydrolyzed oil from Lesquerella fendleri was not buffer‐washed, there was, in most cases, no separation of hydroxy fatty acids by crystallization. This crystallization procedure is suitable for a large‐scale separation process of the hydroxy fatty acids from nonhydroxy fatty acids obtained from hydrolyzed lesquerella oil.

Characterization of the interaction of Ca2+ with hydroxy and non-hydroxy fatty acid species of cerebroside sulfate by Fourier transform infrared spectroscopy and molecular modeling.

Ca2+-mediated interactions between the carbohydrate groups of glycolipids, including that of cerebroside sulfate (galactosylceramide I3-sulfate), have recently been implicated as a basis of cell recognition and adhesion. Hydroxylation of the fatty acid of this lipid has an effect on these interactions. Therefore, FT-IR spectroscopy was used to study the interaction of Ca2+ with semisynthetic hydroxy (HFA) and non-hydroxy fatty acid (NFA) species of cerebroside sulfate (CBS). Ca2+ caused partial dehydration of the sulfate group and reduced hydrogen bonding of the sugar hydroxyls of both species. The amide I and II bands of the lipids in the absence of Ca2+ (NH4+ salt forms) suggested that the N-H of the HFA species is involved in a bent intramolecular hydrogen bond, probably with the fatty acid hydroxyl group and the glycosidic oxygen, while that of the NFA species is involved in a linear intermolecular hydrogen bond with the C=O and/or other oxygens. Ca2+ caused a rearrangement of the hydrogen-bonding network in the interfacial region of the HFA species involving the amide group. The results suggested increased hydrogen bonding of the C=O and a shift in hydrogen bonding of the N-H of the Ca2+ salt form of the HFA species from a bent intramolecular hydrogen bond to a linear intermolecular hydrogen bond, probably with the C=O of neighboring molecules, similar to the NFA species. The involvement of the fatty acid alpha-hydroxyl group in the rearranged network was indicated by a reduction in mobility of the alpha-CH group of the HFA species, in contrast to that of the NFA species. Participation of the alpha-OH group in hydrogen-bonding networks in the interfacial region of both the NH4+ and Ca2+ salt forms caused a significant increase in the interchain packing, as evident from correlation field splitting of the HFA-CBS methylene scissoring mode, while this did not occur for the NFA species. The absence of intramolecular hydrogen bonding of the N-H with the glycosidic oxygen for both salt forms of the NFA species and for the Ca2+ salt form of the HFA species may destabilize the "bent shovel", bilayer planar conformation of the sugar and cause it to be in the extended, bilayer perpendicular conformation. Calculations of the three-dimensional interaction energy of Ca2+ with CBS showed strong binding around the sulfate and the surface of galactose facing the bilayer in the bent shovel conformation. Ca2+ binding at this surface would disrupt intra- and intermolecular hydrogen-bonding interactions of the head group, thus accounting for its effect in inducing a transition to the extended conformation.

A novel fucosyltetrahexosylceramide in plerocercoids of the parasite Spirometra erinacei.

This glycosphingolipid was tentatively designated as GalSEGLx, in which the carbohydrate structure is characterized by an additional galactose molecule attached to the reducing-end galactose of SEGLx [Gal beta-4 (Fuc alpha-3) Glc beta-3 Gal beta Cer], which was previously determined by us [Kawakami, Y., Nakamura, K., Kojima, H., Suzuki, M., Inagaki, F., Suzuki, A., Sonoki, S., Uchida, A., Murata, Y. & Tamai, Y. (1993) J. Biochem. 114, 677-683], through a beta 1-6 linkage. The ceramide contained sphinganine and 4D-hydroxysphinganine in an about equimolar ratio, and a non-hydroxy fatty acid with carbon atoms ranging from 16 to 28, 26:0, 28:0 and 28:1 being major components. Based on the finding that a novel carbohydrate structure. Gal beta-4 Glc beta-3 Gal, was commonly found in glycosphingolipids from the parasite, S. erinacei, we here propose the terms, spirometo series, for this core structure series, and spirometosides, for glycosphingolipids having this carbohydrate structure.

Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase.

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.

Effects of Brefeldin A on Galactosphingolipid Synthesis in an Immortalized Schwann Cell Line: Evidence for Different Intracellular Locations of Galactosylceramide Sulfotransferase and Ceramide Galactosyltransferase Activities

Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6-h labeling period, a dose-dependent inhibition of [35S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 microgram/ml BFA. Labeling of newly synthesized galactosphingolipids with [3H]-palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA-GalCer) to 162% of control values, whereas labeling of GalCer containing 2-hydroxy fatty acids (HFA-GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA-GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA-GalCer synthesis. Examination of [3H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2-hydroxylation or the formation of HFA ceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and thermotropic properties of synthetic C16:0 (palmitoyl) ceramide: effect of hydration.

Differential scanning calorimetry (DSC) and X-ray diffraction techniques have been used to investigate the structure and thermotropic properties of synthetic, non-hydroxy fatty acid (16:0) ceramide (NFA(C16)CER) as a function of hydration. Anhydrous NFA(C16)CER shows a single, broad endothermic transition at 95.4 degrees C (delta H = 10.4 kcal/mol). On hydration, a broad exothermic transition appears at approximately 50-70 degrees C while the main endothermic transition decreases to 90.0 degrees C (delta H = 13.8 kcal/mol). The enthalpy of the exothermic transition increases with hydration to a maximum value, delta H = 4.8 kcal/mol. This polymorphic phase behavior depends on the low temperature incubation time and prior cooling rate. X-ray diffraction of fully hydrated NFA(C16)CER at 26 degrees C, shows a well-ordered lamellar phase with a bilayer periodicity d = 46.9 A. At 68 degrees C, above the first exothermic transition, X-ray diffraction shows again a lamellar phase with reduced bilayer periodicity d = 41.8 A and an increased number of both lamellar and wide-angle reflections indicative of enhanced layer and chain packing order, respectively. At 90.0 degrees C, above the main transition, the diffraction pattern shows a broad, intense reflection at 29.9 A and a diffuse reflection at 4.6 A, indicative of a melted chain phase. On cooling, NFA(C16)CER exhibits polymorphic phase behavior involving the conversion of the melted chain phase to a metastable bilayer phase. On heating, this metastable phase undergoes an exothermic transition to a stable bilayer phase; on further heating, NFA(C16)CER converts endothermically to the melted-chain phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical properties of ceramides: effect of fatty acid hydroxylation.

The structural and thermotropic properties of alpha-hydroxy fatty acid (HFA) and non-hydroxy fatty acid (NFA) ceramides (CER) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction techniques. The DSC of anhydrous HFA-CER shows a single, sharp reversible transition at 95.6 degrees C (delta H = 15.3 kcal/mol). At intermediate hydrations HFA-CER exhibited more complex behavior but at maximum hydration only a single reversible transition is observed at 80.0 degrees C (delta H = 8.5 kcal/mol). X-ray diffraction of hydrated (74% water) HFA-CER at 20 degrees C shows a lamellar structure with a bilayer periodicity d = 60.7 Angstrum; a single wide angle reflection at 4.2 Angstrum is characteristic of hexagonal chain packing. Above the main transition temperature at 91 degrees C, a hexagonal (HII) phase is observed. In contrast, DSC of anhydrous NFA-CER demonstrates two thermal transitions at 81.3 degrees C (delta H = 6.8 kcal/mol) and 85.9 degrees C (delta H = 3.5 kcal/mol). With increasing hydration, both transitions shift towards lower temperatures; at maximum hydration, on heating, the endothermic transitions occur at 72.7 degrees C (delta H = 9.8 kcal/mol) and 81.1 degrees C (delta H = 4.0 kcal/mol). On cooling, there is hysteresis of both transitions. X-ray diffraction of NFA-CER (80% water) at 20 degrees C shows a well-ordered lamellar structure with a bilayer periodicity d = 58.6 Angstrum and three wide-angle reflections at 4.6 Angstrum, 4.2 Angstrum, and 3.8 Angstrum. At 77 degrees C (between the two transitions), again a lamellar structure exists with reduced bilayer periodicity d = 53.1 Angstrum and four wide-angle reflections at 4.6 Angstrum, 4.2 Angstrum, and 3.8 Angstrum are observed. Above the second transition, only a single low angle reflection at 30.0 Angstrum is observed; a diffuse reflection at 4.6 Angstrum is indicative of a melted chain phase. Thus, HFA-CER exhibits a simple phase behavior involving the reversible conversion of a gel phase to a hexagonal phase (L beta-->HII). However, NFA-CER shows a more complex polymorphic phase behavior involving two gel phases.