A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder.
Inherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff's disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations. (+info)
Separation of molecular species of glucosylceramide by high performance liquid chromatography of their benzoyl derivatives.
The method of separation of glucosylceramide by HPLC was reported. Glucosylceramide was perbenzoylated and separated on a packed muBondapack C18 column, using methanol as eluting solvent. The pattern obtained by HPLC closely resembled that obtained by GLC of the TMS-glucosylceramide, and reflected the molecular species of fatty acid components. This method is reproducible, and sensitive as GLC. This method also can be used for analysis of higher glycolipids. (+info)
Determination of the anomeric configurations of Corbicula ceramide di- and trihexoside by chromium trioxide oxidation.
The anomeric configurations of Corbicula ceramide dihexoside and ceramide trihexoside were determined by chromium trioxide oxidation and the structures of these lipids were shown to be Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide and Man-alpha(1 leads to 4)-Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide. These results are compatible with those obtained by enzymic hydrolysis reported previously. (+info)
MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast.
Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed. (+info)
Regulation of intracellular ceramide content in B16 melanoma cells. Biological implications of ceramide glycosylation.
We previously reported that ceramide released from glycosphingolipids (GSLs) by endoglycoceramidase was directly metabolized to GSLs, and thus the content of GSLs was constantly maintained in B16 melanoma cells (Ito, M., and Komori, H. (1996) J. Biol. Chem. 271, 12655-12660). In this study, the metabolism of ceramide released from sphingomyelin (SM) by bacterial sphingomyelinase (SMase) was examined using B16 cells and their GSL-deficient mutant counterpart GM95 cells. Treatment of B16 melanoma cells with bacterial SMase effectively hydrolyzed SM on the plasma membrane. Under these conditions, NeuAcalpha2,3Galbeta1, 4Glcbeta1,1ceramide was significantly increased. Interestingly, UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1) activity and GSL synthesis, but not SM synthesis or sphingosine generation, were found to be up-regulated by SMase treatment. The up-regulation of GSL synthesis seemed to occur at both the transcriptional and post-translational steps of GlcT-1 synthesis. Accumulation of ceramide by bacterial SMase was much higher in GM95 cells than in the parental cells. When the enzyme was removed from the culture medium, the intracellular ceramide level in B16 cells, but not that in the mutant cells, normalized. No rapid restoration of SM in either of the cell lines was observed after removal of the enzyme. SMase treatment strongly inhibited DNA synthesis in GM95 cells but not that in B16 cells. In the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, SMase treatment markedly increased the ceramide content and thus inhibited DNA synthesis in B16 cells. Our study provides the first evidence that GlcT-1 functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess. (+info)
Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis.
We report that apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells associates with accumulation of monohexosylsphingolipids produced not only by short-chain ceramide glycosylation but also through glycosylation of a ceramide pool endogenously produced. By high-performance thin layer chromatography on borate silica gel plates, newly formed monohexosylsphingolipids were identified as glucosylceramides (GluCer); however, accumulation of lactosylceramide or higher-order glycosphingolipids was not observed. GluCer accumulation was fully suppressed by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; moreover, while this inhibitor had no effect on cell viability when administered alone, it markedly potentiated the apoptotic effect of C6-Cer. These results provide evidence that activation of GluCer synthesis is an important mechanism through which CHP-100 cells attempt to escape ceramide-induced apoptosis. (+info)
Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells.
To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally alpha2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix. (+info)
Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells.
The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells. (+info)