Three-dimensional structure of the oligosaccharide chain of GM1 ganglioside revealed by a distance-mapping procedure: a rotating and laboratory frame nuclear overhauser enhancement investigation of native glycolipid in dimethyl sulfoxide and in water-dodecylphosphocholine solutions

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In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose-and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells.

Section: Methodsmentioning

confidence: 99%

Altered Sphingolipid Metabolism inN-(4-Hydroxyphenyl)- retinamide-resistant A2780 Human Ovarian Carcinoma Cells

Prinetti

Basso

Appierto

et al. 2003

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“…Proton inter-and intra-residual contacts were evaluated by NOE experiments in the rotating frame ROESY (17) with a spin lock pulse strength of 2.6 kH applied at one end of the spectrum to avoid scalar transfer (18,19). Temperature was varied in the range of 305-323 K and mixing time between 100 -200 ms.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Resistance of Tay-Sachs Ganglioside GM2 to Enzymatic Degradation

Hasegawa

et al. 1999

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To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAc␤134(NeuAc␣233)Gal␤134Glc␤1-1Cer) are refractory to ␤-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6GM2 (GalNAc␤136(NeuAc␣233)Gal␤134Glc␤1-1Cer). While GM2 has GalNAc␤134Gal linkage, 6-GM2 has GalNAc␤136Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAc␤134Gal␤13 4Glc␤1-1Cer) and 6GM2. In addition, the conformational properties of both GM2 and 6GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6GM2 were readily hydrolyzed by ␤-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6GM2, only the oligosaccharide from GM2 was resistant to ␤-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.Tay-Sachs disease is caused by the impaired catabolism of ganglioside GM2 1 (GalNAc␤134(Neu5Ac␣233)Gal␤134Glc-Cer). It has been shown that the terminal GalNAc in GM2 is resistant to ␤-hexosaminidase A (Hex A), and that a specific protein cofactor, GM2 activator, is required to assist the hydrolysis (1-4). The disease, therefore, can be caused by the deficiency of either Hex A (5-7) or GM2 activator protein (8 -10). The reason for the resistance of GM2 to Hex A is still an enigma.We have shown that, in addition to the GalNAc, the enzymatic hydrolysis of the Neu5Ac in GM2 also requires GM2 activator protein (11). In GM2, both the GalNAc and the Neu5Ac are linked to the Gal to form GalNAc␤134(Neu5Ac␣233)-Gal␤-, a branched trisaccharide (GM2-epitope). We have rationalized that the resistance of the GalNAc and the Neu5Ac in GM2 to enzymatic hydrolysis may be because of the specific rigid structural conformation of the GM2-epitope and that GM2 activator may interact with the GM2-epitope to make the GalNAc and the Neu5Ac accessible to Hex A and sialidase, respectively (11). To show that this is the case, we have recently synthesized a GM2 analog, 6ЈGM2 (GalNAc␤136(Neu5Ac␣233)-Gal␤134GlcCer), in which the GalNAc is linked ␤136 to the Gal (12). Here, we report that, in sharp contrast to GM2, the GalNAc and the Neu5Ac in 6ЈGM2 were readily hydrolyzed by Hex A and clostridial sialidase, respectively, independent of GM2 activator protein. NMR analysis and molecular mechanics computation revealed that the trisaccharide head group of 6ЈGM2 is much more flexib...

“…Their structural and homogeneity characterization was performed as described elsewhere (27,28). [ 3 H]GM1 and [ 3 H]GM2 containing erythro-C18-sphingosine isotopically tritium-labeled at position 3 were prepared from GM1 and GM2 by the dichloro-dicyano-benzoquinone/ sodium boro-[ 3 H]hydride method followed by reversed-phase high pressure liquid chromatography purification (26,29) (homogeneity over 99%, specific radioactivity of 1.2 and 1.3 Ci/mmol, respectively).…”

Section: Methodsmentioning

confidence: 99%

“…After a chase of 15 h, cells were washed twice with phosphate-buffered saline, scraped off with a rubber policeman, and centrifuged at 1000 ϫ g for 10 min. The pelleted cells were subjected to lipid extraction (27) resulting in a delipidized pellet and a total lipid mixture. The radioactivity imaging of thin layer chromatography plates of lipid extracts was acquired with a ␤-imager 2000 instrument (Biospace, Paris, France).…”

Section: Methodsmentioning

confidence: 99%

Absence of Metabolic Cross-correction in Tay-Sachs Cells

Martino¹,

Emiliani²,

Tancini³

et al. 2002

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We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme ␤-hexosaminidase A (HexA) (␤-N-acetylhexosaminidase, EC 3.2.1.52). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the L␣HexTN retroviral vector carrying the cDNA encoding for the human Hex ␣-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells.