Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GD1a and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo-GM2 and asialo-GM1, respectively, by thin-layer chromatography.     

Metabolism of exogenous gangliosides GM1 and chemically modified GM1 in mice

Ganglioside GM1(NeuAc), labeled at the C-3 position of sphingosine with tritium, was injected into C3H/He, C57BL/10, B10.AQR mice intraperitoneally. The incorporation and the distribution of the radioactivity in various organs were examined. The injected [3H]GM1(NeuAc) was mainly incorporated in the liver and hydrolyzed sequentially. Sialic acid of ganglioside GM1(NeuAc) and metabolites was converted to N-glycolyl type from N-acetyl type. An appreciable amount of the sphingosine moiety in the administered GM1(NeuAc), moreover, was reutilized, being converted to sphingomyelin, and incorporated into alkyl chain of the ether lipid in phosphatidylethanolamine. The distributions of radioactivity in the metabolites of GM1(NeuAc) administered to the three strains of mice were different from each other. In other organs, GM1(NeuAc) was incorporated and metabolized only slightly. The N-methylamide, at the carboxyl group of the sialic acid, of the labeled ganglioside GM1(GM1(NeuAc)-NMe) was injected into C3H/He mice. Most of the administered [3H]GM1(NeuAc)-NMe was incorporated in the liver, and was metabolized to GM3(NeuAc)-NMe, via GM2(NeuAc)-NMe, within 24 h. GM3(NeuAc)-NMe was the only radioactive compound in the subsequent 10 weeks, but disappeared from the liver gradually. N-Methylamide-modified gangliosides were resistant to hydrolysis by mouse hepatic sialidase, to elongation by glycosyltransferase and to N-glycolylation at N-acetylneuraminic acid by monooxygenase.     

GM1b and GM1b-GalNAc: major gangliosides of murine-derived macrophage-like WEHI-3 cells.

WEHI-3 cells, derived from a BALB/c mouse, are a myelomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas-liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%. The two major components were identified as (NeuAc)GM1b and (NeuAc)GM1b-GalNAc and the minor component as (NeuAc)GM1b-GalNAc-Gal. The presence of GM1b in this myelomonocytic cell line is consistent with its presence in other murine immune cells and tissues. GM1b-GalNAc and GM1b-GalNAc-Gal have been reported in T-lineage cells but not in resident or stimulated murine macrophages. Each of these monosialogangliosides belongs to the asialoGM1 synthetic pathway. Preliminary results indicate a disialo member of this pathway, GDlc, may also be present as a minor component. This ganglioside pathway, containing species which are not sialylated on the internal galactose, appears to be dominant in and may be characteristic of murine immune cells.     

GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells

The apical domain of epithelial cells is composed of distinct subdomains such as microvilli, primary cilia and a non-protruding region. Using the cholesterol-binding protein prominin-1 as a specific marker of plasma membrane protrusions we have previously proposed the co-existence of different cholesterol-based lipid microdomains (lipid rafts) within the apical domain [R?per, K., Corbeil, D. and Huttner, W.B. (2000), Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582-592]. To substantiate the hypothesis that the microvillar plasma membrane subdomains contain a distinct set of lipids compared to the planar portion we have investigated the distribution of prominin-1 and two raft-associated gangliosides GM(1) and GM(3) by fluorescence microscopy. GM(1) was found to co-localize with prominin-1 on microvilli whereas GM(3) was segregated from there suggesting its localization in the planar region. Regarding the primary cilium, overlapping fluorescent signals of GM(1) or GM(3) and prominin-1 were observed. Thus, our data demonstrate that specific ganglioside-enriched rafts are found in different apical subdomains and reveal that two plasma membrane protrusions with different structural bases (actin for the microvillus and tubulin for the cilium) are composed of distinct types of lipid.     

Differential expression of biofunctional GM1 and GM3 gangliosides within the plastic-adherent multipotent mesenchymal stromal cell population

Background aimsIt is unclear whether the plastic-adherent multipotent mesenchymal stromal cells (MSC) isolated from human bone marrow (BM) represent a uniform cell population or are heterogeneous in terms of cell-surface constituents and hence functionality.MethodsWe investigated the expression profile of certain biofunctional lipids by plastic-adherent MSC, focusing particularly on two membrane microdomain (lipid raft)-associated monosialogangliosides, GM1 and GM3, using indirect confocal laser scanning fluorescence microscopy and flow cytometry.ResultsPhenotypically, we observed a differential expression where certain MSC subsets exhibited GM1, GM3 or both at the plasma membrane. Furthermore, disialoganglioside GD2 detection increased the complexity of the expression patterns, giving rise to seven identifiable cell phenotypes. Variation of standard culture conditions, such as the number of cell passage and period in culture, as well as donors, did not influence the heterologous ganglioside expression profile. In contrast, the binding of various lectins appeared homogeneous throughout the MSC population, indicating that the general glycosylation pattern remained common. Morphologically, the expression of a given ganglioside-based phenotype was not related to a cell with particular size or shape. Interestingly, a segregation of GM1 and GM3 clusters was observed, GM3 being mostly excluded from the highly curved plasma membrane protrusions.ConclusionsThese data highlight the phenotypic heterogeneity of plastic-adherent MSC in terms of certain lipid constituents of the plasma membrane, and the presence and/or absence of distinct ganglioside-based membrane microdomains suggest their potential functional diversity.     

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.     

Modified GM3 gangliosides produced by metabolic oligosaccharide engineering

Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.     

Differential uPAR recruitment in caveolar‐lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.     

Distribution of ganglioside GM3 in the rat ovary after gonadotropin stimulation.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Ovaries have been shown to contain GM3 as a major ganglioside. To study GM3 distribution during gonadotropin stimulation in the hypophysectomized rat ovary, ovarian sections and cultured granulosa cells were stained with specific monoclonal antibody against GM3. Interstitial cells of follicles of immature hypophysectomized rat ovary expressed ganglioside GM3. Theca cells of early antral follicles but not primary follicles expressed GM3. No granulosa cells of these follicles expressed GM3. When a surge dose of FSH/LH was injected, Graafian follicles were formed and GM3 expression was detected in granulosa cells of these follicles. After ovulation, cumulus cells kept expressing GM3 in the ampulla region of ovulated oviduct. The follicles did not show GM3 expression in their granulosa cells after an ovulatory dose of FSH/LH. At 48 h after in vitro culture with FSH/LH of granulosa cells from preantral follicles, GM3 was expressed to a detectable extent on the outer part of the granulosa layer. Finally, at 72 h after culture, all granulosa cells became positive to anti-GM3 antibody. These data suggest that the expression of ganglioside GM3 in the hypophysectomized rat ovary is spatiotemporally regulated by FSH/LH during follicular development and ovulation.     

Presence of activator proteins for the enzymic hydrolysis of GM1 and GM2 gangliosides in normal human urine

Normal human urine has been found to contain activator proteins that stimulate the enzymic hydrolysis of GM1 and GM2 gangliosides. These two activators were partially purified by Sephadex G-200 filtration and DEAE-Sephadex A-50 chromatography. The presence of these two activators was assayed by demonstrating the stimulation of the in vitro hydrolysis of GM1 and GM2 gangliosides. As little as 50 ml of urine is sufficient to detect the presence of these two activators. The crude activator preparation from normal urine was also found to stimulate the hydrolysis of galactosylceramide sulfate by arylsulfatase A.     

Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers

Lateral assemblies of sphingolipids, glycosphingolipids and cholesterol, termed rafts, are postulated to be present in biological membranes and to function in important cellular phenomena. We probed whether rafts are heterogeneous by determining the relative distribution of two gangliosides, GM1 and GD3, in artificial supported monolayers, in intact rat primary cerebellar granule neurones, and in membrane rafts isolated from rat cerebellum. Fluorescence resonance energy transfer (FRET) using fluorophore-labelled cholera toxin B subunit (which binds GM1) and mAb R24 (which binds GD3) revealed that GM1 spontaneously self-associates but does not co-cluster with GD3 in supported monolayers and on intact neurones. Cholera toxin and immunocytochemical labelling of isolated membrane rafts from rat cerebellum further demonstrated that GM1 does not co-localise with GD3. Furthermore, whereas the membrane raft resident proteins Lyn and caveolin both co-localise with GD3 in isolated membrane rafts, GM1 appears in separate and distinct aggregates. These data support prior reports that membrane rafts are heterogeneous, although the mechanisms for establishing and maintaining such heterogeneity remain to be determined.     

Formation of tritium-labeled polysialylated gangliosides in the cytosol of rat cerebellar granule cells in culture following administration of [3H]GM1 ganglioside

GM1 ganglioside tritium-labeled at C-3 of sphingosine has been administered to rat cerebellar granule cells. Tritiated polysialylated gangliosides were observed in the cytosol of the cells, where they resulted in a higher amount after a short period of chase. This, together with the data showing an increase of the tritiated polysialylated gangliosides in the total particulate fraction in parallel to the prolonging of the chase period, suggests that cytosolic gangliosides could be a way of transporting neosynthesized gangliosides from the Golgi apparatus to the plasma membranes.     

Age-related changes in GM1, GD1a, GT1b components of gangliosides in Wistar albino rats

In this study, age-related changes of GM1, GD1a, GT1b fractions of gangliosides were investigated in whole brain of male Wistar albino rats. Insignificant increases were detected in GM1 values from the third to the 24th month, whereas GD1a and GT1b concentrations of ganglioside in 24-month-old rats decreased significantly as compared to 6-month-old rats. Although there were no significant differences in the GD1a/GT1b ratio of any groups, GM1/GD1a and GM1/GT1b ratios were significantly increased as compared to 6-month-old rats. The increase in the ratios of gangliosides are not due to an increase of GM1 fractions; they result from a decrease of GD1a and GT1b fractions of gangliosides. In conclusion, the concentration of ganglioside decreased with ageing.     

Mechanistic study of modulation of SR Ca2+-ATPase activity by gangliosides GM1 and GM3 through some biophysical measurements

On the basis of confirming the antagonistic effects of GM1 and GM3 on the activity of Ca²⁺-ATPase, we further demonstrated that some of the components of these two gangliosides, including sialic acid (NeuNAc), asialo-GM1, asialo-GM3 and ceramide, failed to show any effects on the activity of Ca²⁺-ATPase. Thus it is apparent that the intact molecules of these two gangliosides with their specific conformations were needed to perform their effects on Ca²⁺-ATPase. From the fluorescence resonance energy transfer measurements, the energy transfer between Cys 670/674 and Lys 515 was decreased by GM1 and increased by GM3, indicating GM1 induced the conformation of the hydrophilic region of Ca²⁺-ATPase to be less compact, while GM3 induced it to be more compact. From the CD spectra measurements, GM1 and GM3 both reduced the content of -helical structures of Ca²⁺-ATPase, but GM1 caused a stronger decrease than that of GM3. Using DPH as the probe, we found that the membrane lipid fluidity of the proteoliposomes containing Ca²⁺-ATPase was decreased by GM1 and tend to increase by GM3.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.     

Comparison of the 13C-n.m.r. spectra of gangliosides GM1 with those of GM1-oligosaccharide and asialo-GM1

The ¹³C-n.m.r. spectra of asialo-G_M1 and G_M1-oligosaccharide are completely assigned and compared to those previously found for intact G_M1 and for the series G_M4, G_M3, G_M2, G_M1, G_D1a, G_D1b, and G_T1b. Removal of the ceramide residue from G_M1 liberated a free, reducing aldehyde group, which was reflected in a doubling of the ¹³C-n.m.r. signals assignable to the d-glucose residue because of α,β equilibrium. The spectrum of asialo-G_M1 lacks the resonances from the sialic acid residue, as expected; in addition, several resonances from the neutral gangliotetraglycosyl residue shifted to different field positions after removal of sialic acid from G_M1. These resonances include that of C-4 of the inner β-d-galactosyl residue, and C-1 of the 2-acetamido-2-deoxy-d-galactosyl residue that is near the site of attachment of the sialosyl residue. The differences between the chemical shifts of the carbon resonances of oligomeric and monomeric saccharides, termed linkage shifts, provide a quantitative assignment aid. They are ～ $\text{1}\text{3}$ of those for residues linked to sialic acid than those for residues linked to the neutral hexose chain. Correlations among linkage shifts for pairs of glycosidically-linked carbon atoms for asialo-G_M1 and G_M1-oligosaccharide were compared with those for the series of gangliosides G_M4 to G_T1b, and differences are noted for resonances for carbon atoms near the sialic acid residue. The spectrum of ganglioside G_M1b, a positional isomer of G_M1 whose ¹³C-n.m.r. spectrum has not yet been observed, is predicted.     

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

GM2/GD2 and GM3 gangliosides have no effect on cellular cholesterol pools or turnover in normal or NPC1 mice

These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.