High performance liquid chromatography preparation of the molecular species of GM1 and GD1a gangliosides with homogeneous long chain base composition

A semi-preparative, analytical high performance liquid chromatographic (HPLC) procedure is described for the isolation of molecular species of GM1 and GD1a gangliosides containing a single long chain base, C18 or C20 sphingosine, C18 or C20 sphinganine, each in its natural erythro or unnatural threo form. The threo forms were obtained from 2,3-dichloro-5,6-dicyanobenzoquinone/NaBH4 -treated gangliosides. The ganglioside molecular species separated by HPLC were analyzed for carbohydrate, fatty acid, and long chain base composition. In particular, long chain bases were submitted to gas-liquid chromatographic-mass spectrometric analyses as their trimethylsilyl (TMS) or N-acetyl-TMS derivatives, and chain length, presence or absence of C4-C5 double bond, and C-3 steric configuration were ascertained. The final preparations of individual molecular species of GM1 and GD1a gangliosides were more than 99% homogeneous in their saccharide moiety, contained a single long chain base (homogeneity higher than 99%), and had a fatty acid composition primarily of stearic acid (92 to 97%). All the individual molecular species of GM1 and GD1a gangliosides were also prepared in radioactive form by selective tritiation at C-3 of the long chain base. Their specific radioactivity ranged from 1.3 to 1.45 Ci/mmol. The availability of these molecular species of gangliosides is expected to facilitate studies aimed at ascertaining the role played by the hydrophobic portion in the functional behavior of gangliosides.     

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.     

New chemical trends in ganglioside research

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.     

A photoreactive derivative of radiolabeled GM1 ganglioside: preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with [3H]acetic anhydride of very high specific radioactivity. The fluorenyl group removed by ammonia treatment was substituted by a nitrophenyl azide group. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 and cold natural GM1 (1:10 by mol) for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive: one, of about 30 kDa, interacted with the ganglioside molecules inserted into the outer membrane layer; three, in the region of 46 kDa, interacted with the portion of associated ganglioside able to be released by trypsin treatment. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of the lacto series of gangliosides, especially of disialo-lacto-N-norhexaosyl ceramide isolated from adult bovine nasal cartilage

A disialosylganglioside was isolated from adult bovine nasal cartilage, and its structure was determined by analysis of sugar composition, permethylation analysis, exoglycosidase treatment, and mild acid hydrolysis. The structure of this ganglioside was identified as disialo-lacto-N-norhexaosyl ceramide, NeuNAc(alpha 2-8)NeuNAc-(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(1-4)Glc(1-1)Cer. Furthermore, we also isolated from this cartilage gangliosides whose structures were presumed to be monosialo-lacto-N-norhexaosyl ceramide, and mono- and disialo-lacto-N-neotetraosyl ceramide. The major fatty acids of the four gangliosides isolated were palmitic, stearic, behenic and lignoceric acids. The predominant long chain bases were sphingenine, heptadecasphingenine and hexadecasphingenine.     

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.     

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

Gangliosides of human, bovine, and rabbit plasma

Gangliosides were isolated from human, bovine, and rabbit plasma and were quantified by gas-liquid chromatography. Purification was achieved by sequential use of partitioning in solvents, DEAE-Sephadex chromatography, base treatment, and silicic acid chromatography. Human and bovine plasma yielded slightly more than 1 micro mole of lipid-bound sialic acid/100 ml; for rabbit plasma the value was 0.28 micro mole/100 ml. The total bovine plasma ganglioside fraction contained equal amounts of N-acetylneuraminic and N-glycolylneuraminic acids, rabbit plasma gangliosides had about 1% of the latter, and the human plasma sample contained only the former. Thin-layer chromatography revealed important differences among the plasmas from the three species, but all possessed hematosides and hexosamine-containing gangliosides. The approximate ratios of these two categories, based on sialic acid content, were (hematosides: hexosamine-type): human, 2:1; rabbit, 3:2; and bovine, 2:3. The fatty acid compositions of both categories were characteristic of extraneural gangliosides and included six major acids: palmitic, stearic, behenic, tricosanoic, lignoceric, and nervonic. The major long-chain base in each sample was sphingosine, while only a trace of the C(20) isomer was detected.     

The presence of sialidase-sensitive sialosylgangliotetraosyl ceramide (GM1b) in stimulated murine macrophages. Deficiency of GM1b in Escherichia coli-activated macrophages from the C3H/HeJ mouse

The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated. The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity. N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized. Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties. Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types. Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b. Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved. However, when E. coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain. Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.     

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.     

Analysis of fatty acid composition of anaerobic rumen fungi

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C(18:0)), arachidic (C(20:0)), heneicosanoic (C(21:0)), behenic (C(22:0)), tricosanoic (C(23:0)) and lignoceric (C(24:0)) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C(20:1)), cis-11,14-eicosadienoic (C(20:2)), erucic (C(22:1n9)), cis-13,16-docosadienoic (C(22:2)) and nervonic (C(24:1)) acids in very small amounts but their presence seems to be unique for anaerobic fungi.     

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.     

Influence of glycolipid oligosaccharide and long-chain base composition on the thermotropic properties of dipalmitoylphosphatidylcholine large unilamellar vesicles containing gangliosides

The thermotropic behavior of dipalmitoylphosphatidylcholine large unilamellar vesicles containing gangliosides has been studied by high-sensitivity heating and cooling differential scanning calorimetry. These studies have been directed to identify and evaluate the influence of both the ganglioside lipidic portion and oligosaccharide moiety on the physical properties of phospholipid bilayers containing gangliosides. The influence of the ganglioside lipidic portion has been evaluated by studying the behavior of vesicles containing different GD1a molecular species carrying homogeneous lipid moieties (C20 or C18 sphingosine or sphinganine and stearic acid). The influence of the ganglioside saccharide portion was evaluated by investigating the thermotropic behavior of vesicles containing different gangliosides (GM1, Fuc-GM1, GD1a, GT1b) carrying the same homogeneous long-chain base moiety (C20 sphingosine and stearic acid). These studies, in conjunction with previous studies using homogeneous lipidic portion ganglioside GM1 and phosphatidylcholines of various chain lengths [Masserini, M., & Freire, E. (1986) Biochemistry 25, 1043-1049], indicate that, for a given oligosaccharide composition, gangliosides exhibit lateral phase separation in an extent dependent upon the length and unsaturation difference between the ganglioside long-chain base and phosphatidylcholine acyl chains. For a given ganglioside lipidic composition the extent of phase separation is dependent upon the number of sugar units present in the glycolipid. The addition of Ca2+ induces or enhances phase separation in a manner dependent on the long-chain base and oligosaccharide composition. Cooling differential scanning calorimetry experiments showed that the ganglioside property to form aggregates within the membrane is independent of the initial physical state of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.     

A novel approach for ganglioside structural analysis based on electrospray multiple-stage mass spectrometry.

A powerful method for detailed structural analysis based on electrospray ionization high-capacity ion-trap multiple-stage mass spectrometry (MS) is for the first time introduced in glycolipidomics. The method was optimized for accurate structural elucidation of human brain gangliosides and specifically applied to normal adult human hippocampus-associated structures. The multiple-stage MS experiments reported here allowed for a complete structural characterization of the oligosaccharide moiety of a GM1 ganglioside species. This was achieved by elucidating the sequence and identification of the GM1a structural isomer from the sialic acid attachment site at the neutral oligosaccharide chain. Moreover, the determination of the d18:1/18:0 sphingoid base/fatty acid composition of the ceramide moiety could be confirmed by this method. The novel protocol developed here proves high potential for rapid, reliable, and reproducible investigation of complex lipid-linked carbohydrates such as polysialylated gangliosides or species carrying some other groups that easily cleave off.     

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.     

Lipid composition and fluidity of plasma membranes isolated from corn (Zea mays L.) roots

Although the results of lipid analyses from several plant species have been available for many years a complete characterization of the corn root plasma membrane is still lacking. The present study provides a detailed analysis of individual lipids and a characterization of the membrane fluidity of corn (Zea mays L.) root plasma membranes isolated by phase-partitioning. Phospholipids (43.9 mol%), sterols (40.8 mol%), and sphingolipids in the form of glucocerebroside (6.8 mol%) constitute the major lipid classes. Stigmasterol (19.8 mol%), campesterol (13.0 mol%), phosphatidylcholine 15.8 mol%), and phosphatidylethanolamine (14.2 mol%) represent the most ubiquitous individual lipids. Hydroxy fatty acids make up 80.9 mol% and very long chain fatty acids are almost 78% of fatty acids in glucocerebroside. Hydroxy arachidic acid (20:0 h) and hydroxy lignoceric acid (24:0 h) are most prominent and glucocerebroside from corn root plasma membranes contains virtually no unsaturated fatty acids. Among the phospholipids only phosphatidylserine displayed a high proportion of very long chain fatty acids (e.g., behenic and lignoceric acid). Membrane fluidity was estimated by fluorescence anisotropy. Due to the high sterol content the plasma membrane of corn roots is relatively rigid.     

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.     

The glycosphingolipid receptor for Vibrio trachuri in the red sea bream intestine is a GM4 ganglioside which contains 2-hydroxy fatty acids

Three major glycosphingolipids (tentatively designated IGL-1, 2, and 3) were isolated from the intestine of red sea bream (Pagrus major) and were subjected to a TLC-overlay assay with (35)S-labeled Vibrio trachuri which causes vibriosis of fish. The bacteria adhered to IGL-2, which was determined to be a GM4 ganglioside (NeuAcalpha2-3Galbeta1-ceramide). The fatty acid portion of IGL-2 was composed of 2-hydroxy C22:0, C24:0, and C24:1, in addition to the non-hydroxy C16:0 and C18:0, while the sphingoid base was composed exclusively of sphingenine (d18:1). Among glycosphingolipids tested, V. trachuri adhered to GM4 the most strongly followed by adherence to GM3 and GalCer, but the bacteria did not adhere to GM1a, GM2, LacCer, or GlcCer. V. trachuri was found to aggregate with the erythrocytes coated with GM4, but not with those coated with GM1a or GM2, thus indicating that specific adhesion occurs on intact cells. Interestingly, the dynamics for adhesion of V. trachuri to glycosphingolipids was defined by the structure of not only the sugar moiety but also the ceramide moiety, since the bacteria adhered to GM4 which contained 2-hydroxy fatty acids much more strongly than to that which contained non-hydroxy fatty acids.