Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications

Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and-?4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.     

Association of glycosphingolipids with intermediate filaments of human umbilical vein endothelial cells

Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside GM3 are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and GM3 antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and GM3 with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-gamma decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on GM3 distribution. Less abundant HUVEC GSLs, including Gb3, nLc4, IV2FucnLc4, and IV3NeuAcnLc4, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs.     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

Cloning of a mouse beta 1,3 N-acetylglucosaminyltransferase GlcNAc(beta 1,3)Gal(beta 1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the beta1,3 N-acetylglucosaminyltransferase GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the beta1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(beta1,4)GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminal N-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situ hybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.     

Presence of an unusual GM2 derivative,taurine-conjugated GM2, in Tay-Sachs brain

Tay-Sachs disease (TSD) is a classical glycosphingolipid (GSL) storage disease. Although the genetic and biochemical bases for a massive cerebral accumulation of ganglioside GM2 in TSD have been well established, the mechanism for the neural dysfunction in TSD remains elusive. Upon analysis of GSLs from a variant B TS brain, we have detected a novel GSL that has not been previously revealed. We have isolated this GSL in pure form. Using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this unusual GSL was established to be a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. Using a rabbit anti-tauro-GM2 serum, we also detected the presence of tauro-GM2 in three other small brain samples from one variant B and two variant O TSD patients. On the other hand, tauro-GM2 was not found in three normal human brain samples. The presence of tauro-GM2 in TS brains, but not in normal brains, indicates the possible association of this unusual GM2 derivative with the pathogenesis of TSD. Our findings point to taurine conjugation as a heretofore unelucidated mechanism for TS brain to cope with water-insoluble GM2.     

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.     

Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.     

Novel class of glycosphingolipids involved in male fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.     

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.     

Solubilized glycosyltransferases and biosynthesis in vitro of glycolipids

The assembly of most of the ceramide-linked glycolipids (GSLs) in eukaryotic cells occurs in Golgi bodies. At least 18 different glycolipid:glycosyltransferases (GSL:GLTs) have been characterized, 10 of which have been solubilized. These GLTs can be classified into 2 distinct groups: 1) GLTs dedicated to either Dol-P-P-sugar(s) or ceramide-linked sugar(s); and 2) GLTs with dual loyalties (i.e., they compete with glycolipid- and glycoprotein-bound oligosaccharides). Studies with solubilized and purified GalNAcT-1 and GalNAcT-2 from embryonic chicken brains prove that GalNAcT-1 (UDP-GalNAc:GM3 beta 1-4GalNAcT) is specific for GSL, whereas GalNAcT-2 (UDP-GalNAc:Gb3 beta 1-3GalNAcT) can transfer to an oligosaccharide containing the alpha-linked terminal galactose. Similarly, GalT-3 (UDP-Gal:GM2 beta 1-3GalT) is more specific for ganglio-oligosaccharide and GalT-4 (UDP-Gal:Lc3 beta 1-4GalT) can transfer galactose to N-acetylglucosamine linked to p-nitrophenol, glycolipid or glycoprotein. Both GalT-3 and GalT-4 have been separated and purified from embryonic chicken brains. Studies with solubilized SAT-4 and SAT-3, from bovine spleen and embryonic chicken brains, respectively, suggest the existence of 2 different gene-expressed alpha 2-3SATs. The newly discovered FucT-3 (GDP-Fuc:NeuGc-iLc6-alpha 1-3FucT) from human colon carcinoma (Colo-205) has also been solubilized and separated from other GSL:GLTs. Using a new activity gel-Western blot combined technique, the molecular mass of this FucT-3 was determined to be 105 kDa.     

Dynamic modulation of the glycosphingolipid content in supported lipid bilayers by glycolipid transfer protein

Supported lipid bilayers (SLBs) are popular models of cell membranes. Owing to the importance of glycosphingolipids (GSLs) in modulating structure and function of membranes and membrane proteins, methods to tune the GSL content in SLBs would be desirable. Glycolipid transfer protein (GLTP) can selectively transfer GSLs between membrane compartments. Using the ganglioside GM1 as a model GSL, and two mass-sensitive and label-free characterization techniques—quartz crystal microbalance with dissipation monitoring and ellipsometry—we demonstrate that GLTP is an efficient and robust biochemical tool to dynamically modulate the GSL content of SLBs up to 10 mol % GM1, and to quantitatively control the GSL content in the bulk-facing SLB leaflet. By exploiting what we believe to be a novel tool, we provide evidence that GM1 distributes highly asymmetrically in silica-supported lipid bilayers, with ∼85% of the ganglioside being present in the bulk-facing membrane leaflet. We report also that the pentameric B-subunit of cholera toxin binds with close-to-maximal stoichiometry to GM1 in SLBs over a large range of GM1 concentrations. Furthermore, we quantify the liganding affinity of GLTP for GM1 in an SLB context to be 1.5 μM.     

Expression of glycosphingolipids in lymph nodes of mice lacking TNF receptor 1: biochemical and flow cytometry analysis

The expression of gangliosides and neutral glycosphingolipids (GSLs) in the lymph nodes of mice lacking the gene for the tumour necrosis factor-alpha receptor p55 (TNFR1) has been investigated. GSL expression in the tissues of mice homozygous (TNFR1-/-) or heterozygous (TNFR1+/-) for the gene deletion was analysed by flow cytometry and high-performance thin-layer chromatography (HPTLC) followed by immunostaining with specific antibodies. HPTLC immunostaining revealed that lymph nodes from TNFR1-/- mice had reduced expression of ganglioside GM1b and GalNAc-GM1b, neolacto-series gangliosides, as well as the globo- (Gb3, Gb4 and Gb5) and ganglio-series (Gg3 and Gg4) neutral GSLs. Flow cytometry of freshly isolated lymph node cells showed no significant differences in GSL expression, except for the GalNAc-GM1b ganglioside, which was less abundant on T lymphocytes from TNFR1-/- lymph nodes. In TNFR1-/- mice, GalNAc-GM1b+/CD4+ T cells were twofold less abundant (3.8% vs 7.6% in the control mice), whereas GalNAc-GM1b+/CD8+ T cells were fourfold less abundant (5.0% vs 20.2% in the control mice). This study provides in vivo evidence that TNF signalling via the TNFR1 is important for the activation of GM1b-type ganglioside biosynthetic pathway in CD8 T lymphocytes, suggesting its possible role in the effector T lymphocyte function.     

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.     

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.     

Glycosphingolipid patterns of peripheral blood lymphocytes, monocytes, and granulocytes are cell specific

We analyzed the amounts and types of glycosphingolipids (GSLs) from peripheral blood lymphocytes, monocytes, and granulocytes isolated by counter-current elutriation. The three cell types contained different amounts of neutral and acidic GSLs. The highest amount of neutral GSLs (109 micrograms/10(8) cells) was found in granulocytes, with considerably less found in monocytes (11 micrograms/10(8) cells) and lymphocytes (4 micrograms/10(8) cells). The neutral GSLs were composed of four types of lipids, GL1 through GL4 (mono-, di-, tri-, and tetraosylceramide). The highest percentage of GL1 was detected in lymphocytes and the lowest percentage in granulocytes, with the reverse order observed for GL2. GL3 and GL4, which were minor components of the neutral GSLs, were highly cell specific, with lymphocytes containing GL3 and GL4 of the globo series, granulocytes containing GL3 and GL4 of the lacto or neolacto series, and monocytes containing GL3 and GL4 of both types. The acidic GSL, sialosyl hexaosylceramide (lacto-series), was abundant in granulocytes but not in monocytes or lymphocytes. Another ganglioside, GM3, although present in all three cell types, was most abundant in monocytes and lymphocytes, whereas sialosyl paragloboside was higher in granulocytes than in lymphocytes and monocytes. These results indicate that peripheral blood lymphocytes, monocytes, and granulocytes have distinct "GSL fingerprints."     

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

Secondary accumulation of gangliosides in lysosomal storage disorders

Glycosphingolipids (GSLs) known as gangliosides have been documented to accumulate in a wide range of lysosomal storage disorders, including those with and without primary defects in ganglioside degradation. The same two gangliosides, GM2 and GM3, are often found elevated in diseased neurons whereas in normal mature neurons both are essentially undetectable. Altered expression of these two gangliosides does not appear to result solely from cellularity changes or gliosis since immunocytochemical studies show that both GM2 and GM3 reside in vesicular structures within affected neurons. Elevated expression of one of these gangliosides (GM2) has also been found to closely correlate with the growth of ectopic dendrites on susceptible neurons, a phenomenon that uniquely characterizes many lysosomal diseases. Understanding the precise role of the endosomal-lysosomal system in the overall homeostatic control of GSL expression in neurons can be expected to provide key insight into both the function of gangliosides and the pathogenic mechanisms underlying lysosomal disease.