Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.     

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.     

Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin. Role of p-glycoprotein in glycolipid synthesis

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E. coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.     

Cloning of Gb3 synthase, the key enzyme in globo-series glycosphingolipid synthesis, predicts a family of alpha 1, 4-glycosyltransferases conserved in plants, insects, and mammals

We have cloned Gb(3) synthase, the key alpha1, 4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb(3) synthase, the alpha1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Galbeta1,4Glcbeta1Cer (LacCer), to produce Gb(3) (Galalpha1,4LacCer) or iGb(3) (Galalpha1, 3LacCer), respectively. GalNAc can be added sequentially to either Gb(3) or iGb(3) yielding globoside and Forssman from Gb(3), and isogloboside and isoForssman from iGb(3). Gb(3) synthase is not homologous to iGb(3) synthase but shows 43% identity to a human alpha1,4GlcNAc transferase that transfers a UDP-sugar in an alpha1, 4-linkage to a beta-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb(3) synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an alpha1,4-glycosyltransferase, possibly Gb(3) synthase, throughout evolution. The isolated Gb(3) synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb(3) synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb(3) glycolipid, also called P(k) antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb(3) and are resistant to verotoxin become susceptible to the toxin following transfection with Gb(3) synthase cDNA.     

Synergistic effect of two cell recognition systems: glycosphingolipid-glycosphingolipid interaction and integrin receptor interaction with pericellular matrix protein.

GM3-expressing cells adhere, spread and migrate on plastic plates coated with Gg3, LacCer and Gb4, but not with other glycosphingolipids (GSLs). Thus, cell adhesion, spreading and migration through GSL-GSL interaction occur in an analogous fashion to the interaction of cells with adhesive matrix proteins [AP, e.g. fibronectin (FN), laminin (LN)] through their integrin receptors. In this study, the adhesion of two GM3-expressing cell lines (B16 melanoma and HEL299 fibroblast) on plastic plates co-coated with GSL plus AP is compared with adhesion on plates coated with GSL (Gg3 or LacCer) alone, or coated with AP alone. Results show that: (i) cell adhesion on GSL-coated plates takes place earlier in the incubation period than that on AP-coated plates; (ii) cell adhesion, as well as spreading, was greatly enhanced (in terms of strength and rapidity) on plates co-coated with GSL plus AP; (iii) repulsion (negative adhesion) of cells was observed on plates co-coated with AP plus N-acetyl-GM3 (NAcGM3) and was presumably based on repulsive NAcGM3-NAcGM3 interaction; (iv) GM3-dependent cell adhesion on GSL-coated plates, as well as synergistic promotion of cell adhesion (based on the GSL-GSL and AP-integrin systems), was suppressed by incubation of cells with anti-GM3 monoclonal antibody DH2 or sialidase. Synergistic adhesion of cells on GSL/AP co-coated plates was less inhibited by incubation with peptide sequences RGDS or YIGSR than was adhesion on plates coated with AP alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia

The content of glycosphingolipids (GSL) was studied in the urinary sediments (24-hr specimens) from seven normal subjects, a patient with Fabry's disease, and five homozygotes with familial hypercholesterolemia (FH). Normal urinary sediments contained very small amounts of GalCer, GlcCer, GaOse(2)Cer, LacCer, GbOse(3)Cer, and GbOse(4)Cer. In Fabry urinary sediment, the levels (nmole glucose/24 hr) of GaOse(2)Cer and of GbOse(3)Cer were 389 and 550, respectively. In urinary sediments from the FH subjects, the mean contents (nmol glucose/mg protein per 24 hr) of GlcCer, GalCer, and LacCer were 2.7, 1.9, and 15.8 times higher, respectively, than in normals. The mean contents ( micro g/mg protein per 24 hr) of total cholesterol and phospholipid in the urinary sediment of FH (1.1 and 224, respectively) and normals (0.8 and 220) were similar. The mean contents of GlcCer, GalCer, and LacCer, expressed in terms of the cholesterol content of urinary sediment (nmol glucose/ micro g cholesterol per 24 hr), were increased 3.4-, 1.6-, and 5.4-fold, respectively, in the FH homozygotes. Of the five FH homozygotes, only one, who had undergone a portacaval shunt and was also receiving lipid-lowering therapy, had a normal value of LacCer. The other four FH homozygotes had levels of LacCer that were 3- to 55-fold higher (nmol glucose/mg protein per 24 hr) and 5.5- to 7.3-fold higher (nmol glucose/ micro g cholesterol per 24 hr) than the mean of the normals. One homozygote underwent plasma exchange therapy that reduced both the baseline urinary (nmol glucose/24 hr) and plasma (nmol/100 ml) LacCer levels from 86 to 7 and from 1491 to 852, respectively. Eleven days after plasma exchange, the urinary LacCer levels approached pre-exchange levels (59 nmol glucose/24 hr). The data indicate that there is an abnormality of GSL metabolism associated with familial hyper-cholesterolemia and that the urinary excretion of GSL can be modified by plasma exchange therapy.-Chatterjee, S., C. S. Sekerke, and P. O. Kwiterovich, Jr. Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia.     

Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.     

A Major Fraction of Glycosphingolipids in Model and Cellular Cholesterol-containing Membranes Is Undetectable by Their Binding Proteins

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based “aglycone” interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb₃), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb_3/cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb₃, HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb₃, more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb₃/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb₃, suggesting GSL-GSL interaction can counter cholesterol masking of Gb₃. The similar separation of Vero cell membrane-derived vesicles into minor “binding,” and major “non-binding” fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of “available” and “cholesterol-masked” plasma membrane Gb₃ pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.     

Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.     

Glycosphingolipid composition of a renal cell line (MDCK) and its ouabain-resistant mutant

Glycosphingolipids were isolated from a canine kidney cell line (MDCK) and its ouabain-resistant mutant (MDCK-OR) by solvent extraction, mild alkaline methanolysis, a DEAE-Sephadex column, and preparative TLC. The glycolipids were characterized by their mobilities on TLC, an analysis of carbohydrates as trimethylsilyl methyl glycosides and acetates of partially methylated alditols, as well as by treatment with specific glycosidases. In the neutral glycolipid fraction of both cell lines, galactosylceramide (GalCer), glucosylceramide (GlcCer), lactosylceramide (LacCer), digalactosylceramide (Ga2Cer), globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), and the Forssman antigen (IV3GalNAc alpha-Gb4Cer) were identified. The contents of Ga2Cer (4.4 nmol/mg protein), Gb3Cer (0.6), Gb4Cer (2.9), and IV3GalNac alpha-Gb4Cer (19.5) in MDCK-OR were 1.4- to 2.1-fold higher than those in MDCK, while the concentrations of GlcCer (5.3) and LacCer (1.4) in MDCK-OR were about half of those in MDCK. Among acidic glycolipids of MDCK-OR, galactosyl sulfatide (GalCer-I3-sulfate) and lactosyl sulfatide (LacCer-II3-sulfate) were increased to 1.9 (2.7-fold) and 0.2 nmol/mg protein (2.0-fold), respectively, as compared to MDCK. However, N-acetylneuraminosyllactosylceramide (GM3), the predominant ganglioside in both cell lines, was decreased to about one third of the level (1.5 nmol/mg protein) in the parent MDCK (4.7 nmol/mg protein). The fatty acid of the glycolipids in both cell lines consisted mainly of saturated acids of 16, 18, 22, and 24 carbons.     

Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus.

Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.     

Glucosylceramide Synthesized In Vitro from Endogenous Ceramide is Uncoupled from Synthesis of Lactosylceramide in Golgi Membranes from Chicken Embryo Neural Retina Cells

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[³H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[³H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [³H]GlcCer from those enriched in [³H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.     

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44⁺/ESA⁺/CD24⁻ markers, Gb3 activates c-Src/β-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/β-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.     

A novel soluble analog of the HIV-1 fusion cofactor,globotriaosylceramide (Gb(3)), eliminates the cholesterol requirement for high affinity gp120/Gb(3) interaction

We have analyzed the interaction of adamantyl Gb(3) (adaGb(3)), a semi-synthetic soluble analog of Gb(3), with HIV-1 surface envelope glycoprotein gp120. In this analog, which was orginally designed to inhibit verotoxin binding to its glycolipid receptor, Gb(3), the fatty acid chain is replaced with a rigid globular hydrocarbon frame (adamantane). Despite its solubility, adaGb(3) forms monolayers at an air-water interface. Compression isotherms of such monolayers demonstrated that the adamantane substitution resulted in a larger minimum molecular area and a more rigid, less compressible film than Gb(3). Insertion of gp120 into adaGb(3) monolayers was exponential whereas the gp120/Gb(3) interaction curve was sigmoidal with a lag phase of 40 min. Adding cholesterol into authentic Gb(3) monolayers abrogated the lag phase and increased the initial rate of interaction with gp120. This effect of cholesterol was not observed with phosphatidylcholine or sphingomyelin. In addition, verotoxin-bound adaGb(3) or Gb(3) plus cholesterol was recovered in fractions of comparable low density after ultracentrifugation through sucrose-density gradients in the presence of Triton X-100. The unique biological and physico-chemical properties of adaGb(3) suggest that this analog may be a potent soluble mimic of Gb(3), providing a novel concept for developing GSL-derived viral fusion inhibitors.     

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.     

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.     

Glycosphingolipid synthesis is essential for MDCK cell differentiation

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.     

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.