g(m1)-ganglioside and safingol

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, a common contaminant of corn worldwide. FB1 disrupts sphingolipid biosynthesis by inhibiting the enzyme ceramide synthase, resulting in an elevation of free sphingoid bases and depletion of downstream glycosphingolipids. A relationship between maternal ingestion of FB1-contaminated corn during early pregnancy and increased risk for neural tube defects (NTDs) has recently been proposed in human populations around the world where corn is a dietary staple. The current studies provide an in vivo mouse model of FB1 teratogenicity.. Pregnant LM/Bc mice were injected with increasing doses of FB1 on GD 7.5 and 8.5, and exposed fetuses were examined for malformations. Sphingolipid profiles and (3)H-folate concentrations were measured in maternal and fetal tissues. Immunohistochemical expression of the GPI-anchored folate receptor (Folbp1) and its association with the lipid raft component, ganglioside GM1, were characterized. Rescue experiments were performed with maternal folate supplementation or administration of gangliosides.. Maternal FB1 administration (20 mg/kg of body weight) during early gestation resulted in 79% NTDs in exposed fetuses. Sphingolipid profiles were significantly altered in maternal and embryonic tissues following exposure, and (3)H-folate levels and immunohistochemical expression of Folbp1 were reduced. Maternal folate supplementation partially rescued the NTD phenotype, whereas GM1 significantly restored folate concentrations and afforded almost complete protection against FB1-induced NTDs.. Maternal FB1 exposure altered sphingolipid metabolism and folate concentrations in LM/Bc mice, resulting in a dose-dependent increase in NTDs that could be prevented when adequate folate levels were maintained.

Topics: Animals; Carrier Proteins; Dose-Response Relationship, Drug; Female; Folate Receptors, GPI-Anchored; Folic Acid; Fumonisins; G(M1) Ganglioside; Immunohistochemistry; Male; Maternal-Fetal Exchange; Mice; Mycotoxins; Neural Tube Defects; Placenta; Pregnancy; Pregnancy, Animal; Receptors, Cell Surface; Sphingolipids; Sphingosine; Teratogens

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. Recently, sphingolipids metabolites, ceramide, sphingosine and sphingosine-1-phosphate, have been implicated as second messengers in cell growth regulation and differentiation. In this paper, we examined the possibility that interaction of the B subunit with membrane GM1 leads to alterations in metabolism of glycosphingolipids and that increased levels of sphingolipids metabolites may mediate the biological effects of the B subunit. While the B subunit did not induce a change in the level of ceramide or sphingosine, the level of sphingosine-1-phosphate was rapidly and transiently increased. The B subunit also transiently activated cytosolic sphingosine kinase activity, which catalyzes the phosphorylation of the primary hydroxyl group of sphingosine to produce sphingosine-1-phosphate. To determine whether the increase in sphingosine-1-phosphate level plays a role in B subunit-induced mitogenicity, we used a competitive inhibitor of sphingosine kinase, D,L-threo-dihydrosphingosine. D,L-thereo-Dihydrosphingosine not only inhibited B subunit-induced DNA synthesis by 26%, it also reduced its ability to stimulate DNA-binding activity of the transcription factor AP-1. This sphingosine kinase inhibitor also inhibited B subunit-induced increases in the activity of cell cycle-regulated, cyclin-dependent serine/threonine kinases, cdk2 and p34cdc2. These findings suggest that sphingosine-1-phosphate may play a role in the signal transduction pathways activated by binding of the B subunit to endogenous ganglioside GM1.

Topics: 3T3 Cells; Animals; Antibodies; Binding, Competitive; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Division; Ceramides; Cholera Toxin; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; G(M1) Ganglioside; Lysophospholipids; Mice; Mitogens; Peptide Fragments; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Sphingolipids; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

We reported previously that the incorporation of sugars into glycosphingolipids (GSL) is diminished in SW13 cells that lack a vimentin intermediate filament (IF) network (vim-) compared to vim+ cells. To further analyze the nature of this abnormality, we double-labeled cells with 3H-serine and 14C-sugars. There was no difference between vim+ and vim- cells in the incorporation of serine into GSL, although the usual difference in sugar incorporation was observed. This indicated that the defect in vim- cells was not in the incorporation of sugars into ceramide synthesized de novo by acylation of sphinganine (pathway 1). Sugars can also be incorporated into ceramide synthesized from sphingosine that is derived from catabolism of sphingolipids (pathway 2), and into GSL that recycle through the Golgi apparatus from endosomes (pathway 3). The amount of galactose and glucosamine incorporated into GSL in these three pathways was analyzed by the use of two inhibitors of sphingolipid biosynthesis. beta-Chloroalanine inhibits the de novo synthesis of sphinganine (pathway 1), and fumonisin B1 inhibits the acylation of sphinganine and sphingosine (pathways 1 and 2). We were surprised to observe that in both vim+ and vim- cells only 20-40% of sugar incorporation into GSL took place in pathway 1, and 60-80% of sugar incorporation took place in the recycling pathways. Moreover, in contrast to larger GSL, GlcCer was not synthesized in pathway 3. Our observations indicate that vimentin IF facilitate the recycling of GSL and sphingosine, and that the differences between vim+ and vim- cells are predominantly in pathways 2 and 3. Furthermore, although it is generally believed that virtually all GSL are synthesized in the de novo pathway, these data indicate that the recycling pathways predominate in the incorporation of sugars into GSL in SW13 cells.

Topics: Acylation; Adrenal Cortex Neoplasms; Animals; Carcinoma, Small Cell; Ceramides; Endosomes; G(M1) Ganglioside; Galactose; Glucosamine; Glycosphingolipids; Golgi Apparatus; Humans; Mice; Serine; Sphingosine; Tumor Cells, Cultured; Vimentin

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.

Topics: Chromatography, High Pressure Liquid; Fatty Acids; G(M1) Ganglioside; Gangliosides; Mass Spectrometry; Sphingosine