GM1 promotes TrkA-mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA.

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA-GM1 oligosaccharide complex at the cell surface. To study the TrkA-GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross-linked TrkA-GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA-GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent-resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by 'flopping' down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1.

Ceramides as possible nutraceutical compounds: characterization of the ceramides of the Moro blood orange ( Citrus sinensis ).

Ceramides are presented as nutraceutical compounds for protection of colon carcinoma and as important cosmetic preparation components, increasing absorption through the skin. Therefore, the ceramide (Cer) content of Moro blood oranges was determined by mass spectrometry. A total of 114 Cer species were identified: ∼160 mg in the peels and ∼140 mg in the pulp per kilogram of oranges, expressed as "milligram equivalents of d18:1,17:0 Cer". The predominant ceramides contained 4-hydroxy-8-sphingenine (t18:1(Δ8)) and 4-hydroxysphinganine (t18:0) as long-chain bases (LCBs) and fatty acids (FAs) with different structures. In the pulp, t18:1(Δ8)- and t18:0-containing Cer species comprised 50.5 and 33.5% of the total, respectively, 11.5 and 3.5% non-hydroxylated FAs, respectively, 32.0 and 21.0% α-hydroxylated FAs, respectively, and 7.0 and 9.0% α,ß-hydroxylated FAs, respectively. In the peels, t18:1(Δ8)- and t18:0-containing species comprised 49.5 and 34.5% of the total, respectively, 16.0 and 1.5% non-hydroxylated FAs, respectively, 31.5 and 29.0% α-hydroxylated FAs, respectively, and 2.0 and 4.0% α,ß-hydroxylated FAs, respectively.

Anti-GM1/GD1a complex antibodies in GBS sera specifically recognize the hybrid dimer GM1-GD1a.

It is now emerging the new concept that the antibodies from some patients with Guillain-Barré syndrome (GBS) recognize an antigenic epitope formed by two different gangliosides, a ganglioside complex (GSC). We prepared the dimeric GM1-GD1a hybrid ganglioside derivative that contains two structurally different oligosaccharide chains to mimic the GSC. We use this compound to analyze sera from GBS patients by high-performance thin-layer chromatography immunostaining and enzyme-linked immunosorbent assay. We also synthesized the dimeric GM1-GM1 and GD1a-GD1a compounds that were used in control experiments together with natural gangliosides. The hybrid dimeric GM1-GD1a was specifically recognized by human sera from GBS patients that developed anti-oligosaccharide antibodies specific for grouped complex oligosaccharides, confirming the information that GBS patients developed antibodies against a GSC. High-resolution (1)H-(13)C heteronuclear single-quantum coherence-nuclear overhauser effect spectroscopy nuclear magnetic resonance experiments showed an interaction between the IV Gal-H1 of GM1 and the IV Gal-H2 of GD1a suggesting that the two oligosaccharide chains of the dimeric ganglioside form a single epitope recognized by a single-antibody domain. The availability of a method capable to prepare several hybrid gangliosides, and the availability of simple analytical approaches, opens new perspectives for the understanding and the therapy of several neuropathies.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells.

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Ceramide and sphingomyelin species of fibroblasts and neurons in culture.

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

Interactions between gangliosides and proteins in the exoplasmic leaflet of neuronal plasma membranes: a study performed with a tritium-labeled GM1 derivative containing a photoactivable group linked to the oligosaccharide chain.

Interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains can be studied by ganglioside photolabeling combined with cell surface biotin labeling. In the present paper, we report on the results obtained using a novel radioactive photoactivable derivative of GM1 ganglioside, carrying the photoactivable nitrophenylazide group at the external galactose. After cell photolabeling with the radioactive photoactivable derivative of GM1 and cell surface biotin labeling, sphingolipid-enriched domains were prepared from rat cerebellar neurons differentiated in culture and further purified by immunoprecipitation with streptavidin-coupled beads. Among proteins belonging to the sphingolipid-enriched domains that were biotin labeled, thus bearing an exoplasmic domain, a few were also cross-linked by the radioactive photoactivable ganglioside. In particular, two protein bands showing apparent molecular mass of 135 and 35 kDa were intensely photolabeled. The 135 kDa protein was immunologically identified as the GPI-anchored neural cell adhesion molecule TAG-1. These data suggest that hydrophilic interaction between the exoplasmic domains of the protein and the ganglioside sialooligosaccharide chain could exist.

Procedure for separation of GM2 ganglioside species with different ceramide structures by a flash reversed-phase silica gel liquid chromatography.

GM2 ganglioside, beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)-]beta-Gal-(1-4)-beta-Glc-(1-1)-Cer, is the main ganglioside in the brain of Tay-Sachs patients. In this work, GM2 ganglioside was extracted from a Variant B Tay-Sachs human brain, purified to homogeneity of the oligosaccharide moiety by silica gel chromatography. It was further fractionated for the first time into the molecular species differing in the ceramide structures by reverse-phase flash chromatography. The GM2 ganglioside species were characterized by gas-chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. The major GM2 species contained the ceramides with d18:1-18:0 (40.5% of the total GM2 species), d20:1-18:0 (31%) and d18:1-20:0 (12%). We also found minor GM2 species with the ceramides with d18:1-24:1 (4%), d18:1-22:0 (2%) and d18:2-24:1 (1%), which have not been reported previously.