Ganglioside Molecular Species Research Articles

Isolation and Structure of Biologically Active Gangliosides from the Sea CucumberHolothuria pervicax

Three ganglioside molecular species, HPG-8, HPG-3, and HPG-1, have been obtained from the polar fraction of the chloroform/methanol extract of the sea cucumber Holothuriapervicax. The structures of these gangliosides have been determined on the basis of chemical and spectroscopic evidence. They represent new ganglioside molecular species. HPG-8 is a sulfated monosialo-ganglioside, while HPG-3 and HPG-1 are disialo-gangliosides possessing 24-linked tandem-type disialosyl moieties. These three gangliosides qualitative neuritogenic activity toward the rat pheochromocytoma cell line, PC-12 cells.

Isolation and Structure of Biologically Active Glycosphingolipids from the Sea CucumberCucumaria echinata

Five cerebrosides, CE-1-1, CE-1-2, CE-1-3, CE-3-1, and CE-3-2, and a ganglioside molecular species CG-1 have been obtained from the less polar and polar fractions, respectively, of the chloroform/methanol extract of the sea cucumber Cucumaria echinata. The structures of these glycosphingolipids have been determined on the basis of chemical and spectroscopic evidence. The cerebrosides show lethality toward brine shrimps. On the other hand, the ganglioside CG-1 exhibits neuritogenic activity toward the rat pheochromocytoma cell line, PC-12 cells.

Biologically Active Glycosides from Asteroidea, 36. Re‐examination of the Structure of Acanthaganglioside C, and the Identification of Three Minor Acanthagangliosides F, G and H

AbstractThe structure of acanthaganglioside C (1) has been re‐examined using MQ‐COSY, TOCSY, NOESY, HSQC, HMBC and HSQC‐TOCSY experiments. As a result, it is clarified that N‐acetyl neuraminic acid (NeuAc) is linked to the C‐3 position, and not the C‐4 position, of the β‐galactopyranose. In addition, three minor acanthagangliosides, F (2), G (3) and H (4), have also been isolated from the ganglioside molecular species AG‐2. Their structures have been determined on the basis of chemical and spectroscopic evidence.

Age-related changes of the ganglioside long-chain base composition in rat cerebellum

The ganglioside mixture from the cerebellum of young, 6 month old and two years old rats, was fractionated by reversed phase high performance liquid chromatography, each ganglioside homogeneous in the oligosaccharide chain as well as in the long-chain base being subsequently quantified. Two long-chain bases, LCB, were components of the five major gangliosides GMI, GD1a, GD1b, GT1b and GQ1b, these being the C18 : 1 LCB and C20 : 1 LCB. The content of C20 : 1 ganglioside molecular species was lower than that of the C18 : 1 one. In very young animals, day 8, the C20 : 1 ganglioside species represented about 8% of the total ganglioside content, then they progressively increased and reached, at 2 years, about 42% of the total. C18 : 1 GD1a and C18 : 1 GT1b, were the major species in young animals and reached their highest content at day 29, being 1.45 and 1.28 nmol/mg protein, respectively. The content of these two species decreased in adult and old animals and at two years it was 0.71 and 0.82 nmol/mg protein, respectively.

Structure determination of the major component of the starfish ganglioside molecular species LG-2 by tandem mass spectrometry

The biologically active ganglioside molecular species, LG-2, which has been obtained from the starfish Astropecten latespinosus, was completely methylated followed by separation using reversed phase HPLC to give a methylated major component, LG-2M-5, in the pure state. The ceramide moiety of LG-2M-5 was indicated by the positive ion fast atom bombardment tandem mass spectrometry (FABMS/MS) of LG-2M-5. This fact was verified from the measurements of the positive ion FABMS/MS of the permethylates prepared from four known starfish cerebrosides.

Biologically active glycosides from asteroidea, XXXII. Glycosphingolipids from the starfish Astropecten latespinosus, 2. Structure of two new ganglioside molecular species and biological activity of the ganglioside

AbstractTwo ganglioside molecular species [LG‐1 (1) and LG‐2 (5)] have been obtained from the water‐soluble lipid fraction of the chloroform/methanol extract of the starfish Astropecten latespinosus. On the basis of chemical and spectroscopic findings, the structure of 1 and 5 have been elucidated. They are new ganglioside molecular species. Negative FAB mass spectrometry provided important information both on the structure of the sugar moiety and on the molecular mass of the gangliosides. Partial hydrolysis by hot water has proved useful for structure elucidation of the complex oligosaccharide moieties. In addition, the major ganglioside molecular species, LG‐2 (5), shows antitumor activity against murine lymphoma L1210 cells in vitro.

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.

Biosynthesis of gangliosides containing C18:1 and C20:1 [3‐14C]sphingosine after administrating [1‐14C]palmitic acid and [1‐14C]stearic acid to rat cerebellar granule cells in culture

The biosynthesis of ganglioside molecular species containing sphingosine of different structure was investigated by administrating rat cerebellar granule cells in culture with [1-14C]palmitic and [1-14C]stearic acids which are the precursors for sphingosine biosynthesis. The incorporation of radioactivity into the sphingosine of the ganglioside species containing C20:1 sphingosine after administrating [1-14C]stearic acid was low in comparison with the incorporation of radioactivity into the sphingosine of ganglioside species containing C18:1 sphingosine after administration of [1-14C]palmitic acid, but the ratio between the radioactivity incorporated in the C20:1 and the C18:1 sphingosine of C20 and C18 ganglioside species progressively increased when the cell culture was prolonged. Ceramide-containing radioactive sphingosine was found after palmitic or stearic acid administration. Ceramide-containing C20:1 sphingosine found after adding stearic acid was about 5% of that synthesized starting from palmitic acid and containing C18:1 sphingosine. Free radioactive C18:1 and C20:1 sphingosine were found after adding radioactive palmitic or stearic acid. This is representative of a catabolic process occurring after biosynthesis of the complex sphingolipid starting from the radioactive precursor. In fact it has been proved that only saturated sphingosines are used for the synthesis of complex sphingolipids, the introduction of the double bond at position four of the sphingoid chain occurring at the level of ceramide [Rother, J., van Echten, G., Schwarzmann, G. & Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14-20]. Saturated sphingosines were not present. The lack of free C20:0 sphingosine confirms the hypothesis that the C20:0 sphingosine synthesis and the process (C20:0 sphingosine-->C20:0 ceramide-->C20:1 ceramide) occur in the correct quantity for the synthesis of C20:1 gangliosides. Moreover, we found only traces of free C20:1 sphingosine, at days 8 and 15 of cell culture when the biosynthesis of complex C20:1 gangliosides and the related catabolic processes occur to a higher extent, thus excluding the idea that a large amount of C20:0 sphingosine can be acylated to C20:0 ceramide and dehydrogenated to C20:1 ceramide which, being not used for ganglioside biosynthesis, is immediately catabolised to C20:1 sphingosine.

Biologically Active Glycosides from Asteroidea, XXXI. Glycosphingolipids from the Starfish Asterias amurensis versicolor sladen, 2. Structure and Biological Activity of Ganglioside Molecular Species

AbstractTwo ganglioside molecular species [GAA‐6 (1) and GAA‐7 (4)] have been obtained from the water‐soluble lipid fraction of the chloroform/methanol extract of the whole bodies of the starfish Asterias amurensis versicolor Sladen. The structures of these ganglioside molecular species have been determined on the basis of chemical and spectroscopic findings. GAA‐6 (1) is a starfish ganglioside molecular species resembling a mammalian brain ganglioside, GM3. In contrast, GAA‐7 (4) is assigned the same structure as that of the main ganglioside[3] from Asterias amurensis[4] for the oligosaccharide moiety. Partial hydrolysis of oligosaccharide moieties with hot water or dilute acetic acid has proved useful for the structure elucidation of the ganglioside molecular species. Moreover, the Mayn ganglioside molecular species, GAA‐7 (4), shows neuritogenic and growth‐inhibitory activities towards the mouse neuroblastoma cell line (Neuro 2a). The structure‐activity relationship of starfish gangliosides is also discussed.

Changes in the Ganglioside Long‐Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture

Changes in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising approximately 8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the "b series" (GD1b, GT1b, and GQ1b) than in the "a series" (GM1 and GD1a). During aging in culture, for 8-22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.

Changes of the human liver GM3 ganglioside molecular species during aging

Sialosyl-lactosylceramide, GM3, is the major ganglioside of human liver, where it constitutes more than 90% of the total lipid-bound sialic acid. When analyzed by thin-layer chromatography, human liver GM3 migrates as two main spots. They are representative of ganglioside molecular species which differ in the acyl moiety. The faster running spot is mainly composed of molecular species with non-hydroxylated C22-C24 acyl chains; the other contains mainly molecular species bearing non-hydroxylated C16-C18 and alpha-hydroxylated C16-C24 acyl chains. In this study the content of the two GM3 molecular species groups was investigated in 31 subjects ranging from 19 to 85 years of age. By thin-layer chromatography we observed that the group of molecular species containing non-hydroxylated C22-C24 acyl chains, decreased linearly with subject age, while that of non-hydroxylated C16-C18 acyl chains and hydroxylated C16-C24 acyl chains increased linearly. Fast-atom-bombardment mass spectrometry performed on seven samples from subjects ranging from 21 to 78 years of age demonstrated that the age-dependent increase of the lower spot is caused by an increase in the hydroxylated fatty acid form of GM3, the content of non-hydroxylated C16-C18 fatty acid species remaining constant with age.

Rat cerebellar granule cells in culture associate and metabolize differently exogenous GM1 ganglioside molecular species containing a C 18 or C 20 long chain base

A study has been made of the association properties of the two GM1 ganglioside molecular species GM1-C 18 and GM1-C 20 (containing C 18 and C 20 long chain bases, respectively) to rat cerebellar granule cells in culture. Both gangliosides recognized, to the same extent, and associated with them to give a form of association, the trypsin-labile form. This form was removed by treatment with trypsin enzyme. Both gangliosides associated stably with the cells to become components of the cell membranes. Although similar amounts of the two gangliosides entered the cells, being then metabolized, the time course of the association was different for the two gangliosides: after 15 h of ganglioside-cell incubation the amount of GM1-C 18 inserted into the cell membrane was 2.43 times higher than that of GM1-C 20.

Evidence for Nonrandom Distribution of GD1 a Ganglioside in Rabbit Brain Microsomal Membranes

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (approximately 9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained approximately 68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing approximately 10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.

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.

Biologically active glycosides from asteroidea, XXV. Glycosphingolipids from the starfish Asterina pectinifera, 2. Structure of three ganglioside molecular species and a homogeneous ganglioside, and biological activity of the ganglioside

AbstractThree ganglioside molecular species and a homogeneous ganglioside [GP‐1a (1), GP‐1b (2), GP‐2 (3) and asterinaganglioside A (4)] were obtained from the water‐soluble lipid fraction of the chloroform/methanol extract of the starfish Asterina pectinifera. On the basis of chemical and spectral evidence, the structure of 1–4 were elucidated. GP‐2 (3) was a known ganglioside molecular species, but the other three compounds were new gangliosides. Partial hydrolysis of oligosaccharide moieties with hot water was useful for structure elucidation of the gangliosides. NOE Correlation Spectroscopy (NOESY) was also useful for characterization of the complex sugar moiety. For the isolation of homogeneous ganglioside from the complex molecular species, reversed‐phase HPLC with methanol/ammonia/water as solvent was effective. Moreover, the main ganglioside molecular species, GP‐2 (3), was found to support the survival of cultured cerebral cortex cells, most of which were neuronal cells, of rat fetuses.

Bioactive gangliosides: analysis of functional structures of the tetrasialoganglioside G Q1b which promotes neurite outgrowth

Previous reports from our laboratory showed that tetrasialoganglioside G Q1b, when exogenously added, can promote cell proliferation and neurite outgrowth in two human neuroblastoma cell lines, GOTO and NB-1 (Tsuji, S., Arita, M. and Nagai, Y. (1983) J. Biochem. 94, 303–306). To clarify the activity-associated structure of G Q1b, we analyzed the structure-activity relationships using the G Q1b, molecule, derivatives of it, and related gangliosides. When the G Q1b molecule was divided into two parts, the ceramide and oligosaccharide moieties, no activity was found with the former, while with the latter the activity could be seen, although the level of activity obtained never exceeded half that of G Q1b itself and an optimal concentration of 100–200 ng/ml of 20–40-times that of native G Q1b (5 ng/ml) was required. Furthermore, the activity of G Q1b was completely abolished by neuraminidase treatment, which converted G Q1b to G M1, so we examined other molecular species of gangliosides having a common gangliotetraose backbone but linked differently with two to four sialic acids (i.e., G D1a, G D1b, G T1a G Q1c G Q1b and G Q1c). Among them, only G Q1b was found to be active. The results disclosed the interesting fact that deletion of any sialic acid residue from either of the two disialosyi residues of G Q1b results in a complete loss of activity and that the mere existence of the tetrasialosyl structure does not lead to activity; this indicates the absolute necessity of the G Q1b oligosaccharide structure for the expression of activity. For full expression of the activity, both the ceramide and oligosaccharide moieties were necessary. It was also found that the G Q1b oligosaccharide inhibited the activity of G Q1b at a concentration a few times greater than that of G Q1b, suggesting the involvement of a receptor-like mechanism in the action of G Q1b at the cell membrane.

Ganglioside GM3: an acidic membrane component that increases during macrophage-like cell differentiation can induce monocytic differentiation of human myeloid and monocytoid leukemic cell lines HL-60 and U937.

When human myeloid and monocytoid leukemic cell lines HL-60 and U937, respectively, were treated with an exogenous sialoglycosphingolipid, ganglioside GM3, in serum-free medium, cell growth was markedly inhibited, and their morphological maturation along a monocytic lineage was observed. In addition to a significant increase in phagocytic and nonspecific esterase activities, marked increase of monocyte-specific surface antigens detectable with monoclonal antibodies such as OKM1 and OKM5 was observed in GM3-fed cells. Other sialoglycosphingolipids with the carbohydrate structure belonging to ganglio-series oligosaccharide, ganglioside GM1 and a brain ganglioside mixture, had no effect on the cell differentiation, showing instead stimulatory actions on the growth of these cell lines. We recently demonstrated that the ganglio-series ganglioside GM3 characteristically increased during macrophage-like cell differentiation of these cell lines. The present results indicate that ganglioside molecular species that specifically increase during monocytic cell differentiation of human myeloid and monocytoid leukemic cell lines may play, in turn, an important role in the differentiation-induction of these cell lines along a monocytic cell lineage.

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.

Antibody response of rabbits to nerve ending gangliosides: Analysis of antibody specificity by liposome lysis and lysis inhibition assays

Rabbits were immunized with nerve ending fraction prepared from guinea pig brain. Serum antibodies to total gangliosides were followed by enzyme-linked immunosorbent assay; their titers were highest at 2 to 3 weeks after immunization and some rabbits showed a response to reinjections. Specific reactivities of the antibodies against each molecular species of gangliosides were analyzed by liposome lysis assay and liposome lysis inhibition assay. Antibody responses were detected against GM1, GD1b, GM3 and GM2, but not against GD1a and GT1b. Natural and immune antibodies to the asialo glycolipids and to galactosylceramide were also observed.

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.