Aberrant fatty acyl alpha-hydroxylation in human neuroblastoma tumor gangliosides

Abnormalities of ganglioside structure characterize the neoplastic state, and aberrant glycosylation has been implicated as underlying many new tumor ganglioside structures. However, variations in ceramide structure can also result in novel tumor gangliosides. To address systematically this aspect of ganglioside metabolism, we have initiated a study of the structures of the ceramide species of an oligosaccharide-homogeneous human tumor-derived ganglioside, GM2. The ganglioside was isolated from neuroblastoma tissue and purified by normal-phase high pressure liquid chromatography. Marked ceramide heterogeneity was observed; 18 individual ceramide species of neuroblastoma GM2 were separated by reversed-phase high pressure liquid chromatography and collected. Their structures were determined by a combination of negative- and positive-ion fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry of the underivatized gangliosides. The striking finding was the detection of alpha-hydroxylation of a significant fraction of each of the major fatty acid species (16:0, 18:0, 20:0, 22:0, and 24:1); alpha-hydroxylated species quantitatively represented almost one-fifth of the total tumor GM2 species. Fatty acyl hydroxylation was also detected in the ceramide of several other human tumor gangliosides. In contrast, as previously known, fatty acyl hydroxylation was not detected in the normal human brain gangliosides GM3, GM2, and GM1. We propose that aberrant fatty acid alpha-hydroxylation is a novel and sometimes quantitatively significant characteristic of human tumor ganglioside metabolism.     

Preparation of GM1 ganglioside molecular species having homogeneous fatty acid and long chain base moieties

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography. The GM1 ganglioside molecular species were analyzed for saccharide, fatty acid, and long chain base composition by chemical and spectrometric analyses. Using a combination of the two procedures, 32 different molecular species of GM1 ganglioside, over 99% homogeneous, have been prepared.     

Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b

Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.     

Characterization of neutral sphingolipids from chicken erythrocytes

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.     

Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry.

Electrospray ionization (ESI) coupled with tandem mass spectrometry has been used in conjunction with microwave-mediated saponification, periodate oxidation, and clostridial sialidase hydrolysis to enable detailed structural characterization of gangliosides and their derivatives present in mullet milt. The gangliosides extracted from mullet milt were determined to be GM3, GM3 lactone, GM3 methyl ester, and 9-O-acetyl GM3. For the major ganglioside GM3 and all GM3 derivatives, the ceramide composition was revealed to be C18:1/C16:0. GM3 with a C18:0/C16:0 ceramide was also found as a minor ganglioside. Both the ganglioside intramolecular ester and the ganglioside methyl ester (lacking carboxylic acid groups) showed dominant chloride attachment peaks (M + Cl)- in negative ion ESI-MS in addition to low intensity peaks corresponding to (M-H)-. GM3 and O-acetyl GM3 bearing carboxylic acid functions showed only (M-H)-. In positive ion ESI, GM3 and O-acetyl GM3 revealed (M + 2Na-H)+ peaks in addition to (M + Na)+, indicating free exchange of the carboxylic acid proton with a sodium cation, while the ganglioside intramolecular ester and ganglioside methyl ester with no acidic protons yielded only (M + Na)+. The strategy of employing ESI-MS to detect products of established wet chemical reactions represents a general approach for elucidation of ganglioside structural details.     

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.     

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.     

Glycosphingolipid composition of human semen

Glycosphingolipids were extracted from human semen and purified. Based on the fluorometric assay of sphingosine, in spermatozoa a content of 4.4 +/- 0.9 nmol/10(8) cells of gangliosides and 22.1 +/- 1.7 nmol/10(8) cells of neutral glycosphingolipids was determined. Seminal plasma contained 4.1 +/- 0.6 nmol gangliosides and 29.3 +/- 1.5 nmol neutral glycosphingolipids per milliliter. The glycosphingolipid component patterns of human spermatozoa and seminal plasma were determined by thin-layer chromatography. Four neutral glycolipids were isolated and their carbohydrate moieties were characterized. All of these glycolipid components belonged to the globo-series. Gas chromatography, combined gas chromatography/mass fragmentography, and exoglycosidase treatments revealed the following structures for the glycosphingolipids of human semen: Glc1-Cer, Gal beta 1-4Glc1-Cer, Gal alpha 1-4Gal beta 1-4Glc1-Cer, and Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc1-Cer. In addition, the occurrence of trace amounts of lactoneotetraosyl- and lactoneohexaosylceramide was detected by immunostaining after thin-layer chromatographic separation. Human spermatozoa, as well as seminal plasma, contained the gangliosides Glac1,Glac2, a sialolactoneotetraosylceramide, and a sialolactoneohexaosylceramide. The gangliosides were identified on the basis of their running characteristics by high-performance thin-layer chromatography, exoglycosidase treatment, and immunostaining after thin-layer chromatography. The ceramide composition of the glycolipids in human spermatozoa, as well as in seminal plasma, was dominated by C22:0-behenic acid and the saturated sphingoid d18:0, sphinganine.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

A NeuGc-containing trisialoganglioside of bovine brain.

A N-glycolyneuraminic acid containing trisialoganglioside was isolated from bovine brains ganglioside mixture using Q-Sepharose. Its chemical structure was characterized as IV3NeuAc, II3NeuAc-NeuGc, Gg4Cer by gas-liquid chromatography, a permethylation study, sialidase degradation, TLC/enzyme-immunostaining, fast atom bombardment-mass spectrometry, fluorometric HPLC and proton nuclear magnetic resonance spectroscopy. This was unique in the mixed sialic acid constituents. (formula; see text) This accounted for 0.78% of the gangliosides. The ceramide structure was almost identical with those of major bovine brain ganglioside, as mainly composed of 18:0 fatty acid (90.9%) and d20:0 sphingosine base.     

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.     

Oxidation of glycolipids in liposomes by galactose oxidase

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.     

Human medulloblastoma gangliosides

To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/10⁸ cells). The major species have been structurallyconfirmed to be G_M2 (65.9%), G_M3 (13.0%), and G_Dla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of G_M2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry     

Different Ceramide Compositions of Gangliosides Between Human Motor and Sensory Nerves

Ganglioside analysis of human motor and sensory nerves revealed that ceramide compositions of sensory nerve GD1a, GD1b, and GM1 differed apparently from those in the motor nerve. These gangliosides from sensory nerve contained a large amount of long-chain fatty acids and d18:1 as a major long chain base. On the contrary, the motor nerve gangliosides contained C16-18 fatty acids and a large amount of d20:1 besides d18:1. Furthermore, these gangliosides were enriched more in the axon fraction than in the myelin fraction. LM1, which was a major ganglioside in myelin from human peripheral nerve, was composed of similar ceramide compositions in the two nerves. The present findings suggest that the characteristic ceramide species of nerve gangliosides may reflect in part properties of their own neurons.     

Cyclic AMP accumulation in HeLa cells induced by cholera toxin. Involvement of the ceramide moiety of GM1 ganglioside.

The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1. The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate. After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored. Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species. Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1. Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.     

Isolation and characterization of a ganglioside containing fucose from boar testis.

A ganglioside containing fucose (fucoganglioside) was obtained from boar testis and purified by silicic acid and DEAE-cellulose column chromatographies and preparative thin layer chromatography. The structure of this ganglioside, determined by chemical and enzymatic methods was: (see article). Its fatty acids were mainly long chain saturated ones (20 : 0, 22 : 0, 24 : 0). Its long chain bases consisted of 27% C(16:1) sphingosine and 68% C(18:1) sphingosine.     

A novel heptasialosyl c-series ganglioside in embryonic chicken brain: its structure and stage-specific expression

A ganglioside of unknown structure (ganglioside X) was purified from chicken brain at embryonic day 12 (E12) and characterized for its structure. Ganglioside X was reactive with a monoclonal antibody A2B5 and migrated below GH1c on thin-layer chromatography (TLC). Extensive treatment of ganglioside X with Clostridium perfringens sialidase produced a single ganglioside product. This ganglioside was identified as GM1 based upon its chromatographic mobility and reactivity to cholera toxin B subunit and anti-GM1 antibody. Partial hydrolysis of ganglioside X by sialidase generated several degradation products including GH1c, GP1c, and GQ1c. Electrospray ionization (ESI)-mass spectrometry (MS) of the permethylated derivative of ganglioside X produced a triple-charged parent ion peak at m/z 1355, which corresponded with the gangliotetraose oligosaccharide structure having seven sialic acids and ceramide with the molecular mass of 566 (as non-methylated form). Collision-induced dissociation (CID)-MS(2) showed fragment ions including those at m/z 1066 and 1931; these two ions matched the structures of (NeuAc)(3)-Gal-Glc-Cer and (NeuAc)(4)-Gal-GalNAc, respectively. These structures were confirmed by CID-MS(3) of the corresponding peaks. Based upon these findings, the structure of ganglioside X was identified as NeuAc-NeuAc-NeuAc-NeuAc-Galbeta1-3GalNAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. This ganglioside was designated as GS1c. A developmental study demonstrated that GS1c was expressed in chicken brain during a period from E6 to E13 and thereafter decreased rapidly in its concentration. The present study suggests that GS1c may play a specific role in early development of chicken brain.     

Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors

Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood.     

Lack of the Ganglioside Molecular Species Containing the C20-Long-Chain Bases in Human, Rat, Mouse, Rabbit, Cat, Dog, and Chicken Brains During Prenatal Life

The long-chain base composition of the total gangliosides from fetal brain of human, rat, mouse, rabbit, cat, dog, and chicken has been investigated during prenatal life. All the analyzed ganglioside mixtures contained the C18:1- and C18:0-long-chain bases, the latter covering 2-5% of the total long-chain base content. The C20-long-chain bases, which are components of the total ganglioside mixture from adult animals, were absent.     

The liver flukes Fasciola gigantica and Fasciola hepatica express the leucocyte cluster of differentiation marker CD77 (globotriaosylceramide) in their tegument

Glycosphingolipids from the parasitic liver flukes Fasciola gigantica and Fasciola hepatica were isolated and their carbohydrate moieties were structurally analysed by methylation analysis, exoglycosidase treatment, on-target exoglycosidase cleavage and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively. From F. gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal beta4-Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal alpha4Gal beta4-Glc1-ceramide, which is designated as either globotriaosylceramide, Pk-blood group antigen or CD77 leucocyte cluster of differentiation antigen. To our knowledge, this is the first report on the expression of globo-series glycosphingolipids in non-mammalian species. Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2-hydroxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines. By the use of an anti-CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F. hepatica cryosections. The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.