Dramatic alteration of sphingolipid bases of Hansenula ciferrii by exogenous fatty acid

$\text{Hansenula}\text{ciferrii}$, a yeast which normally excretes large amounts of C₁₈-phytosphingosine in the form of its tetra-acetyl derivative, was grown in basal medium containing added pentadecanoic acid (0.1%) and Brij 58 (1%). Analyses of the sphingolipid bases recovered from the culture medium indicated the presence of significant quantities of C₁₇-phytosphingosine (39%) and C₁₉-phytosphingosine (15%) in addition to the normal C₁₈-phytosphingosine (46%). The sphingolipid bases were characterized by gas-liquid chromatography combined gasliquid chromatography-mass spectrometry, and chemical degradation.     

Isolation and characterization of major gangliosides from frog liver

Four major gangliosides isolated from frog liver were characterized by compositional analysis involving GLC and GC-MS, methylation analysis, chromium trioxide oxidation, and enzymatic hydrolysis. The results revealed that the most major ganglioside in the tissue was GM4 containing N-acetylneuraminic acid and the others were GM4 containing N-glycolylneuraminic acid, GD1a, and a fucosyl ganglioside which was tentatively assigned to be alpha-galactosyl alpha-fucosyl GM1. This is the first report describing the presence of GM4 containing N-glycolylneuraminic acid. The fatty acids in both GM4 were mainly alpha-hydroxylated, and those in the fucosyl ganglioside were exclusively nonhydroxy fatty acids. The GD1a contained both nonhydroxy and alpha-hydroxy fatty acids in a ratio of about 3:2. The predominant species were 22:0, 23:0, 24:0, and 24:1 in both species of the fatty acids. The long-chain bases of these four gangliosides consisted of C18-sphingosine and C18-phytosphingosine together with significant amounts of C16 to C19 dihydroxy and trihydroxy bases with iso and anteiso structures.     

Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae

Immune responses induced by glycans upon infection with Schistosoma mansoni may be mediated by either schistosomal glycoproteins or glycosphingolipids. In this study, we have elucidated the structural features of both carbohydrate moieties and respective ceramide units of complex glycosphingolipids from adult S. mansoni. Obtained data revealed a vast structural heterogeneity due to manifold combinations of different oligosaccharides and ceramide entities. Observed carbohydrate moieties included Lewis(X) (Le(X); Gal(β1-4)[Fuc(α1-3)]GlcNAc) as well as, in part, multiply fucosylated LacdiNAc (LDN; GalNAc(β1-4)GlcNAc) carbohydrate epitopes. Corresponding lipid portions comprised predominantly C18-sphingosine as well as C18- and C20-phytosphingosine derivatives. Intriguingly, glycosphingolipids carrying an Le(X) epitope contained predominantly C18-sphingosine, whereas LDN-based species exhibited mostly phytosphingosine derivatives, in addition to C18-sphingosine, indicating that the two classes of glycosphingolipids might be synthesized via different biosynthetic routes. Compared with literature data, adult worm glycosphingolipids with Le(X) epitopes revealed clear structural differences in comparison to corresponding cercarial species which have been shown to exhibit mainly sphinganine bases with 18-21 carbon atoms. Therefore, it may be hypothesized that the divergent structural features of the respective ceramide moieties are responsible for the published observation that only adult worm, but not cercarial glycosphingolipids are able to induce dendritic cell activation skewing the T-cell response toward a Th1 profile.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

Rabbit muscle gangliosides

Four ganglioside fractions were isolated from rabbit muscle: one hematoside and three hexosamine-containing species. They were analyzed for hexoses, hexosamine, sialic acid, fatty acids, and long-chain base content. The molar ratios of sphingosine-hexose-hexosamine-sialic acid were: for hematoside, 1:2:0:1; for the disialogangliosides, 1:3:1:2; and for trisialoganglioside, 1:3:1:3. The carbohydrates were studied by thin-layer and paper chromatography. The hexoses were glucose and galactose; the hexosamine was N-acetylgalactosamine and the sialic acid was N-acetylneuraminic acid. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography. The fatty acid composition was similar in all of the four gangliosides. The most abundant fatty acids were 16:0 and 18:0, but significant amounts of 16:1, 18:1, 20:0, and 22:0 were also found. Hydroxy fatty acids were not detected. In all of the muscle gangliosides the main long-chain bases were C(18)-sphingenine and C(20)-sphingenine. In hematoside there were also measurable amounts of C(18)-sphinganine and C(20)-sphinganine, whereas in the major gangliosides only traces of C(18)-sphinganine were detected.     

Gangliosides from the eggs of the sea urchin, Anthocidaris crassispina

NeuGc alpha 2-6Glc beta 1-1Cer (M5 ganglioside) and HSO3-8NeuGc alpha 2-6Glc beta 1-1Cer (T1 ganglioside) were purified by column chromatographies with DEAE-Sephadex A-25 and silicic acid from the eggs of the sea urchin, Anthocidaris crassispina. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Long-chain base compositions of both gangliosides were almost identical: all the long-chain bases were phytosphingosines, and C18-phytosphingosine accounted for more than 95% of them. Fatty acid compositions were also very similar: the main fatty acids were 22:1, 23:1, 24:1, and their 2-hydroxylated forms, and the 2-hydroxy fatty acids amounted to 65.3 and 74.3% of the fatty acids in M5 and T1 gangliosides, respectively. Proton nuclear magnetic resonance spectroscopic study revealed a downfield-shifted H8 proton signal of NeuGc residue in T1 ganglioside, in agreement with the presence of sulfate ester at the C8 position.     

Developmental changes of monohexosylceramide and free ceramide in the large intestine of the rat

Neutral glycosphingolipids were isolated from the colon of rats between birth and adulthood. The glycolipid concentration was stable during this period. Epithelial cells of the adult colon contained three times more glycolipids than the whole organ. The distribution pattern underwent only minor modifications during development. Free ceramide contributed for 23-27% of the total neutral sphingolipids at all ages. In 6-day-old rats, it was constituted of nonhydroxylated fatty acids linked to C18-sphingenine (57.3% of the bases), C18- and C20-4D-hydroxysphinganine (24.2 and 14.0% of the bases, respectively). This composition was essentially maintained during development. Glucosylceramide was the major glycolipid at all ages (40-50% of the total neutral sphingolipid content). At birth, 40% of its fatty acids were 2-hydroxylated and 93% of the bases were C18-4D-hydroxysphinganine. In adult epithelial cells, 75% of the fatty acids were 2-hydroxylated and C18- and C20-4D-hydroxysphinganine contributed for 66 and 25% of the bases, respectively. A transient increase of the contribution of nonhydroxylated fatty acids and C18-sphingenine was observed during the first week of life. C20-4D-hydroxysphinganine, which was characterized by gas-liquid chromatography of its aldehydes after periodate oxidation and of its N-acetyl O-trimethylsilyl derivatives, appeared after birth and reached 20% of the bases after two weeks. These findings are another example of the specificity of the lipidic part of glucosylceramide during the ontogenic differentiation.     

Free sphingoid bases in normal murine tissues

Free sphingoid bases, which have been considered not to occur naturally, were detected in murine tissues by derivatization with o-phthalaldehyde and the use of high-performance liquid chromatography. The concentrations were 10-30 pmol/mg tissue. The lung contained the largest amounts of sphingoid bases. In the molecular species of sphingoid bases, the most abundant was C18-sphingenine followed by C18-sphinganine, 4-hydroxysphinganine and C20-sphingenine, in that order. The central nervous tissues contained relatively high amounts of C20-sphingenine and there was a high concentration of 4-hydroxysphinganine in the kidney. In addition, galactosylsphingenine was detected simultaneously in the spinal cord and sciatic nerve. Sphingoid bases were purified from normal murine lungs using lipid-extraction, cation-exchange and silicic acid column chromatographies, alkaline saponification and preparative thin-layer chromatography. In the purified sphingoid bases, erythro-C18-sphingenine and erythro-C18-sphinganine were identified using thin-layer chromatography, high-performance liquid chromatography and fast-atom-bombardment mass spectrometry. Free sphingoid bases occurring in normal tissues may be metabolic intermediates required for the synthesis or be products of degradation of the sphingolipids and function to regulate cellular metabolism.     

Developmental changes of the lipidic part of the neutral glycosphingolipids of the rat stomach

Neutral glycolipids were purified from the glandular part of the stomach of rats of different ages from 20 days of gestation to 60 days after birth. The two major glycolipids were identified as glucosylceramide and isogloboside. Free ceramide was also detected. The concentrations of these sphingolipids remained almost stable with development. Monohexosylceramide contained 55 and 68% of 2-hydroxylated fatty acids at 20 and 22 days of gestation, respectively, and 82% in the adult. Its three major bases, C18-sphingenine, C18- and C20-4D-hydroxysphinganine were characterized by gas-liquid chromatography and mass spectrometry of their N-acetyl-O-trimethylsilyl derivatives. The occurrence of the bases changed with development. C18-sphingenine contributed for 26% of the bases at birth and 65% in the adult. Conversely, C18-4D-hydroxysphinganine contributed for 35% of the bases at birth and 9% in the adult. The ceramide part of isogloboside consisted of nonhydroxylated fatty acids and mainly C18-sphingenine throughout development. The percentage of long-chain fatty acids was higher in older animals. These results stressed the specificity of the lipidic part of the rat gastric glycolipids and their specific evolution during the development.     

Renal sulfatides: sphingoid base-dependent localization and region-specific compensation of CerS2-dysfunction

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer­amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound’s function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS² (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS² analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.     

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract.

Sphingomyelins were isolated from mucosal layers of bovine rennet stomach, duodenum, jejunoileum, and colon ascendens. The ceramides obtained after phospholipase degradation were characterized by thin-layer chromatography, mass spectrometry, and gas-liquid chromatography. The main ceramide group from all regions consisted of dihydroxy long-chain bases and normal fatty acids. Sphingosine was the predominant base in all these fractions, and only in rennet stomach were smaller amounts of the C17 and C20 homologs present. Normal saturated C16, C18, C22, and C24 fatty acids were most abundant. In rennet stomach there was in addition a ceramide group having dihydroxy long-chain bases in combination with hydroxy fatty acids. Sphingosine was the predominant long-chain base and the fatty acids were 2-hydroxy C16, C22, C23, and C24. From jejunoileum three minor ceramide fractions were isolated; these consisted of phytosphingosine and normal fatty acids C22-C24), sphingosine and 2-hydroxy fatty acids (C16-C24), and phytosphingosine and 2-hydroxy fatty acids (C22-C24), respectively. No branched paraffin chains were found in significant amounts. Sphingomyelins with trihydroxy long-chain bases and 2-hydroxy fatty acids found in jejunoileum were also detected in bovine kidney and have not been demonstrated before. These sphingomyelins from both kidney and jejunoileum showed a preferential combination of trihydroxy bases and fatty acids with very long chains (C22-C24).     

A facile synthesis of D-ribo-C(20)-phytosphingosine and its C2 epimer from D-ribose

A facile synthetic route to d-ribo-C(20)-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules.     

Glycolipids of the fish testis.

Glycolipids were purified from the total lipid extract of the testis or milt of a kind of puffer (Fugu rubripes rubripes) by adsorption column chromatography using silicic acid and magnesium silicate and by preparative silica gel TLC. The glycolipids were identified as glucosylceramide (116 mug/g wet tissue) and galactosylceramide 26.7 mug/g). Seminolipid, a sulfagalactolipid specific to mammalian testis was not detected, but the presence of a small amount of sulfatide (15.2 mug/g) was demonstrated. The long-chain bases of both cerebrosides were mainly C18-sphingenine, but in sulfatide, C20-sphingenine was more abundant than C18-sphingenine. In both cerebrosides and sulfatide, the fatty acid compositions were similar, with nervonic acid as the predominant component. Two species of gangliosides were also obtained and were identified as N-acetylgalactosaminyl(1 leads to 4)[N-acetylneuraminyl(2 leads to 3)]galactosyl(1leads to 4)glucosylceramide (59.8 mug/g) and N-acetylneuraminyl(2 leads to 3)galactosyl(1 leads to 4)N-acetylglucosaminyl(1 leads to 3)galactosyl(1 leads to 4)glucosylceramide (45.0 mug/g). The long-chain bases of the two gangliosides consisted of C18-spingenine and C20-sphingenine, and the major fatty acids were palmitic and stearic acids.     

Glycosylinositolphosphoceramides in Aspergillus fumigatus

Fungal glycosylinositolphosphoceramides (GIPCs) are involved in cell growth and fungal-host interactions. In this study, six GIPCs from the mycelium of the human pathogen Aspergillus fumigatus were purified and characterized using Q-TOF mass spectrometry and 1H, 13C, and 31P NMR. All structures have the same inositolphosphoceramide moiety with the presence of a C(18:0)-phytosphingosine conjugated to a 2-hydroxylated saturated fatty acid (2-hydroxy-lignoceric acid). The carbohydrate moiety defines two types of GIPC. The first, a mannosylated zwitterionic glycosphingolipid contains a glucosamine residue linked in alpha1-2 to an inositol ring that has been described in only two other fungal pathogens. The second type of GIPC presents an alpha-Manp-(1-->3)-alpha-Manp-(1-->2)-IPC common core. A galactofuranose residue is found in four GIPC structures, mainly at the terminal position via a beta1-2 linkage. Interestingly, this galactofuranose residue could be substituted by a choline-phosphate group, as observed only in the GIPC of Acremonium sp., a plant pathogen.     

Melibiosylceramide as the sole ceramide dihexoside from the eggs of the sea urchin, Anthocidaris crassispina

Melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) was found as the sole ceramide dihexoside from the eggs of the sea urchin, Anthocidaris crassispina. Ceramide monohexoside of the eggs consisted only of glucosylceramide (Glc beta 1-1Cer). These lipids were purified by successive column chromatographies on DEAE-Sephadex A-25, silicic acid and Florisil, and identified by gas-liquid chromatography, negative-ion fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectroscopy as well as methylation analysis. Long-chain base compositions of both lipids were almost identical and comprised n-C18-phytosphingosine and small amounts of its homologs (C17-C19). Fatty acid compositions were qualitatively very similar, but the glucosylceramide contained more 2-hydroxy fatty acid than the melibiosylceramide. Although the chain length of fatty acids was distributed over a wide range, six major fatty acids, namely 22:1, 23:1, 24:1, 22h:1, 23h:1 and 24h:1, constituted more than 92% of the fatty acid content in these lipids.     

Exposure to galactose oxidase of GM1 ganglioside molecular species embedded into phospholipid vesicles

The exposure of GM1 molecular species present in the native ganglioside, carrying C18:1 or C20:1 long-chain bases (LCB), to Dactylium dendroides galactose oxidase was studied. When native GM1 (49.3% C18:1 and 50.7% C20:1 LCB, respectively), was inserted in dipalmitoylphosphatidylcholine vesicles and partially oxidized (10%), the proportion of C18:1 and C20:1 species in the oxidized GM1 was 59.6% and 40.4%, respectively, suggesting a preferential action of the enzyme on the shorter species. The V_max of the enzyme was higher on C18:1 GM1 than on C20:1 GM1. The molecular species were affected without any preference after partial (10%) oxidation of GM1 incorporated in egg phosphatidylcholine vesicles or in micellar form. These data indicate that the exposure of the terminal galactose moiety of GM1 ganglioside to galactose oxidase is affected by the ganglioside ceramide composition as well as the phospholipid environment, that presumably determine the distribution (molecular dispersion, segregation) of the ganglioside within the membrane.     

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Lipid components of sialosylgalactosylceramide of human brain

A ganglioside, previously designated HG-B in our laboratory, was isolated from mixed human brain ganglioside preparations and shown to contain equimolar quantities of sialic acid, galactose, and sphingosine. Treatment of this material with neuraminidase yielded a galactosylceramide. The ganglioside, now referred to as sialosylgalactosylceramide, thus appears to be identical with G(gal) reported by Kuhn and Wiegandt. The fatty acids and long-chain bases of this material were analyzed by gas-liquid chromatography. Approximately equal amounts of normal and hydroxy acids were found. Oleic, palmitic, and stearic acids were the only normal fatty acids present. In the hydroxy series, the C(24) and C(23) saturated acids were the major components. The ratio of C(20) to C(18) long-chain base was approximately 5:3. These data suggest that sialosylgalactosylceramide has no direct metabolic relationship with either the major brain gangliosides or adult brain cerebroside.     

The specific glycosphingolipid composition of human ureteral epithelial cells

Total non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thin-layer chromatograms. The major epithelial cell glycolipids were Glc beta 1-1ceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.