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At least 19 recordsLinked to original sources
PubMed2003

Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease.

In humans, two major beta-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits alpha and beta (60% identity), whereas Hex B is a homodimer of beta-subunits. Interest in human beta-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G(M2)-ganglioside (G(M2)). Hex A degrades G(M2) by removing a terminal N-acetyl-D-galactosamine (beta-GalNAc) residue, and this activity requires the G(M2)-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4A) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2A) or NAG-thiazoline (2.5A). From these, and the known X-ray structure of the G(M2)-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how alpha and beta-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease (beta-subunit mutations) and Tay-Sachs disease (alpha-subunit mutations).

Amino Acid Sequence↗
PubMed1996

Development of a human monoclonal antibody to ganglioside G(M2) with potential for cancer treatment.

A human B-lymphoblastoid cell clone, L55-81, that produces human monoclonal antibody (MAb) to ganglioside G(M2) was established from peripheral blood B lymphocytes of a melanoma patient. L55-81 secretes IgMkappa light chain antibody in a serum-free medium. G(M2) specificity of the antibody was tested by immune adherence assay, TLC immunostaining, and ELISA. Anti-G(M2) antibody was shown to have the ability to kill the G(M2)-rich human melanoma cell line M14 in the presence of human or rabbit complement. A purified L55-81 MAb (>99.5% purity in protein concentration) was biotinylated and tested for its reactivity to various histological-type biopsied tumor and normal tissues in an avidin-biotin detection system. L55-81 MAb (20 microg/ml) reacted with several types of tumor tissues such as melanoma (7 of 10), colon carcinoma (4 of 5), ovary carcinoma (4 of 5), breast carcinoma (1 of 5), kidney carcinoma (1 of 5), and prostate carcinoma (1 of 5). None of the normal tissues derived from 24 different organs and adjacent normal tissues surrounding the cancerous tissues were stained. Production of the antibody in a serum-free medium, the cytotoxic potential with human complement, the inability to react to normal tissues, and the ability to target antigen-specific target cells make L55-81 a potential therapeutic agent for the treatment of cancers expressing ganglioside G(M2).

Antibodies, Monoclonal↗
PubMed1972

Generalized accumulation of neutral glycosphingolipids with G M2 ganglioside accumulation in the brain.

Analyses have been made of glycosphingolipids from visceral organs and brain of a patient with an unusual lipid storage disorder diagnosed initially as classical Tay-Sachs disease. Levels of the lipids from fresh-frozen sections of gray and white matter, kidney, spleen, liver, and heart from this patient were compared with those of normal juvenile controls, and the fatty acid composition of accumulated glycosphingolipids was compared with reference compounds. This patient was found to have abnormally high concentrations of a globoside in liver, kidney, and spleen, asialo G(M2) ganglioside in brain and liver, and G(M2) ganglioside in the brain. On the basis of these findings along with the clinical manifestations of Tay-Sachs disease with visceral involvement (hepatosplenomegaly) and demonstration of total deficiency of both A and B components of beta-N-acetylhexosaminidase activity, this glycosphingolipidosis is the same as two previously reported cases of G(M2) gangliosidosis with globoside accumulation and total beta-N-acetylhexosaminidase deficiency.

Brain↗
PubMed1999

G(M2)-ganglioside metabolism in situ in mucolipidosis IV fibroblasts.

Mucolipidosis IV (ML IV) is an inherited lysosomal disorder for which the primary biochemical defect has not been identified. In order to detect any defect in glycosphingolipid metabolism, we have examined the metabolism of sphingosine-labeled (3H)G(M2) in situ in fibroblasts from patients diagnosed with ML IV. Fibroblasts were exposed for 10 days in medium containing (3H)G(M2) (15 uM; Sp. Act. 35000 cpm/nmole), washed, harvested and analyzed for radioactivity in extracted lipids. Control cells metabolized about half of the internalized ganglioside, mostly to ceramide. In ML IV fibroblasts, 70-80% of the cellular radioactivity was present as G(M2) indicating reduced degradation. This is not as severe as in G(M2) gangliosidosis as a small amount of G(M2) was metabolized in ML IV cells to ceramide. Since there is no defect in the lysosomal enzyme profile in these cells, it is possible that an abnormality in the translocation of membrane constituents to the lysosomes may explain the slower ganglioside metabolism.

Cells, Cultured↗
PubMed2000

Vaccination with a bivalent G(M2) and G(D2) ganglioside conjugate vaccine: a trial comparing doses of G(D2)-keyhole limpet hemocyanin.

Immunization with GMK vaccine (G(M2) ganglioside conjugated to keyhole limpet hemocyanin mixed with QS-21 adjuvant) induces anti-G(M2) antibodies in close to 100% of patients. We found previously that anti-G(D2) antibodies could be induced in some patients using G(D2)-keyhole limpet hemocyanin + QS-21 (GDK). In this trial, we wished: (a) to determine whether immunization with both GMK and GDK vaccines could induce antibodies against both G(M2) and G(D2); and (b) to determine the optimal dose of GDK. Thirty-one patients with melanoma or sarcoma who had no evidence of disease after complete surgical resection were immunized with both GMK (30 microg of G(M2)) and GDK on weeks 1, 2, 3, 4, 12, 24, and 36. Patients were assigned to one of five GDK dose levels (3, 10, 30, 70, or 130 microg of G(D2)). Anti-G(M2) IgM or IgG were induced in 97% of patients. The dose of GDK did not affect the anti-G(M2) response, although at the highest GDK dose level, 3 of 7 patients did not make anti-G(M2) IgG. GDK was less immunogenic; overall 45% of patients developed either IgM or IgG against G(D2). At GDK doses of 30 or 70 microg, 8 of 11 patients (73%) made either IgM or IgG anti-G(D2) antibodies. We conclude that both GMK and GDK vaccines can induce antibodies against G(M2) and G(D2) in a majority of patients and are safe. The optimal dose of GDK appears to be either 30 or 70 microg when administered with GMK vaccine.

Cancer Vaccines↗
PubMed2005

Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: implication of gangliosides.

Alterations in proliferation and hypertrophy of renal mesangial cells are typical features of diabetic nephropathy. The HP (hexosamine pathway) has been proposed as a biochemical hypothesis to explain microvascular alterations due to diabetic nephropathy; however, involvement of HP in the regulation of mesangial cell growth or hypertrophy has been poorly studied. Although gangliosides are known to regulate cell proliferation, their potential role in mesangial cell-growth perturbations has hardly been explored. In the present study, we investigated the effects of the HP activation, mimicked by GlcN (glucosamine) treatment, on mesangial cell growth and hypertrophy and the potential implication of gangliosides in these processes. Our results indicate that GlcN induced hypertrophy of mesangial cells, as measured by an increase in the protein/cell ratio, and it caused cell-cycle arrest by an increase in the expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1). Furthermore, GlcN treatment resulted in a massive increase in the levels of gangliosides G(M2) and G(M1). Treatment of cells with exogenous G(M2) and G(M1) reproduced the effects of 0.5 mM GlcN on p21(Waf1/Cip1) expression, cell-cycle arrest and hypertrophy, suggesting that gangliosides G(M2) and G(M1) are probably involved in mediating GlcN effects. These results document a new role of the HP in the regulation of mesangial cell growth and hypertrophy. They also suggest a potential new mechanism of action of the HP through modulation of ganglioside levels.

Animals↗
PubMed1980

Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside.

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of G(m2) ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem.55, 315-324; Hechtman & Leblanc (1977) Biochem. J.167, 693-701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of G(m2) ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-beta-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of G(m2) ganglioside decrease the K(m) for G(m2) ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and G(m2) ganglioside was obtained by comparing gel-filtration profiles of (3)H-labelled G(M2) ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and G(M2) ganglioside is the preferred substrate for enzymic hydrolysis.

Acetylglucosamine↗
PubMed1977

Purification and properties of the hexosaminidase A-activating protein from human liver.

Human liver extracts contain an activating protein which is required for hexosaminidase A-catalysed hydrolysis of the N-acetylgalactosaminyl linkage of G(M2) ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl) galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of G(M2) ganglioside hydrolase activity is used to assay the activating protein. The proceudres of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analysed by polyacrylamide-gel disc electrophoresis, two closely migrating protein bands are seen. When purified activating protein is used to reconstitute the G(M2) ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for G(M2) ganglioside and and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-G(M2) ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyse hydrolysis of G(M2) ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme-activator complex. The dissociation constant of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the molecular weight of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36000 and 39000 respectively.

Adult↗
PubMed2004

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats.

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.

Animals↗
PubMed2005

Mutation of the GM2 activator protein in a feline model of GM2 gangliosidosis.

The G(M2) activator protein is required for successful degradation of G(M2) ganglioside by the A isozyme of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52). Deficiency of the G(M2) activator protein leads to a relentlessly progressive accumulation of G(M2) ganglioside in neuronal lysosomes and subsequent fatal deterioration of central nervous system function. G(M2) activator deficiency has been described in humans, dogs and mice. This manuscript reports the discovery and characterization of a feline model of G(M2) activator deficiency that exhibits many disease traits typical of the disorder in other species. Cats deficient in the G(M2) activator protein develop clinical signs at approximately 14 months of age, including motor incoordination and exaggerated startle response to sharp sounds. Affected cats exhibit central nervous system abnormalities such as swollen neurons, membranous cytoplasmic bodies, increased sialic acid content and elevated levels of G(M2) ganglioside. As is typical of G(M2) activator deficiency, hexosaminidase A activity in tissue homogenates appears normal when assayed with a commonly used synthetic substrate. When the G(M2) activator cDNA was sequenced from normal and affected cats, a deletion of 4 base pairs was identified as the causative mutation, resulting in alteration of 21 amino acids at the C terminus of the G(M2) activator protein.

Aging↗
PubMed1999

Ferret pyramidal cell dendritogenesis: changes in morphology and ganglioside expression during cortical development.

Pyramidal cell ontogenesis and basilar dendritic differentiation were evaluated concomitantly with ganglioside expression and distribution in ferret cerebral cortex. Layer V neurons began basilar dendritogenesis on postnatal day 1 (P1) with a peak in dendritic arborization occurring at P21. Layer II/III neurons, in contrast, were in early stages of basilar dendritic differentiation at P14, resulting in a complex dendritic arbor at P28. High performance thin-layer chromatography showed numerous changes in ganglioside expression during cortical development, including a decline of GM2 in the mature cortex. The temporal expression and cellular distribution of GM2, GD2, GM1, GD3, and GM3 gangliosides in developing cerebral cortex were determined by immunocytochemistry. GM2 immunoreactivity (IR) was most prominent in layer V neurons between P1 and P21 and in layer II/III neurons between P14 and P28 with staining diminishing to near absent levels in the adult. GM2-IR appeared as punctate structures within the somatodendritic domain and by electron microscopy was shown to be membrane-bound vesicles often in close proximity to the plasmalemma. Expression of GM2, but not of other gangliosides studied, followed two well-documented developmental neurogenic gradients: ventrolateral to dorsomedial and radial (inside-first outside-last). Onset of significant GD2 expression in layer II/III and V pyramidal cells was delayed until P14 and persisted in adult neocortex. GD3 was localized most prominently to glial-like cells, whereas GM1 was primarily localized to white matter. The close temporal and spatial concordance of GM2-IR in cortical pyramidal neurons undergoing dendritogenesis is consistent with its proposed role as a modulator of dendritic differentiation.

Aging↗
PubMed1999

Physiological concentrations of gangliosides GM1, GM2 and GM3 differentially modify basic-fibroblast-growth-factor-induced mitogenesis and the associated signalling pathway in endothelial cells.

It has been suggested that gangliosides can influence the growth of cells by modulation of growth-factor-receptor signalling. The activation of endothelial cells (EC) during angiogenesis is crucial for tumour growth and for metastasis, also for numerous other physiological and pathological situations. Pre-treatment of bovine aortic endothelial cells (BAEC) with GM1 or GM2 (5-20 microM) inhibited basic-fibroblast-growth-factor (bFGF)-induced mitogenesis, but GM3 (0.1-20 microM) acted synergistically, increasing proliferation above that of bFGF alone (p < 0.05). The mitogenic effect of all 3 gangliosides was markedly reduced if the cells were washed to remove excess gangliosides from the medium before addition of bFGF. We further show that GM1 and to a lesser extent GM2 modify bFGF binding to its receptor and inhibit the associated mitogenic signal-transduction pathway of protein-tyrosine phosphorylation of 40 to 120 kDa, PLCgamma1, MAP kinase and protein-kinase-C activation. In contrast, GM3 increased tyrosine phosphorylation and MAP kinase activity, as compared with bFGF alone. The observed differential modulation of bFGF-induced mitogenesis by GM1, GM2 and GM3 was at concentrations routinely occurring in the serum of cancer patients. The results suggest that circulating gangliosides may have a role in regulating solid-tumour growth by modulating angiogenesis.

Animals↗
PubMed1991

High-performance capillary electrophoresis of gangliosides.

Gangliosides are sialic acid-containing glycosphingolipids. In aqueous media, these glycolipids have been shown to exist as stable micelles. Ganglioside micelles could be analyzed by high-performance zonal capillary electrophoresis in uncoated fused-silica capillaries within 10 min. The mass sensitivity determined by monitoring the absorption of ultraviolet light at 195 nm was in the order of 10(-11) mol. Increasing the pH of the running buffer from 3.0 to 7.4 or the voltage from 10 to 30 kV increased the relative mobilities of gangliosides. By contrast, increasing the ionic strength of the buffer decreased the migration and broadened the elution peak widths of gangliosides. Ganglioside* micelles including GM1, GD1b, and GT1b were resolved into separate peaks by capillary electrophoresis at physiological pH shortly after mixing. Upon prolonged incubation, the ganglioside peaks merged to form a single species. The fusion process was temperature-dependent. At 50 degrees C, formation of mixed micelles between polysialogangliosides GD1b and GT1b was complete within 30 min. In contrast, no fusion of the ganglioside peaks was observed at 0 degrees C even after 75 h. Formation of mixed micelles between GD1b and other polysialogangliosides including GD1a, GT1b, and GQ1b at 37 degrees C required 1.5, 3.0, and 2.0 h, respectively. Formation of mixed micelles between monosialoganglioside GM1 and polysialogangliosides were 6- to 36-fold slower. No fusion was observed between monosialogangliosides GM1 and GM2 after 2 days of incubation. These findings indicate that polysialogangliosides may have higher propensities than monosialoganglioside to form mixed micelles.

Electrophoresis↗
PubMed1997

Isolation of gangliosides by cloud-point extraction with a nonionic detergent.

A rapid, simple, and efficient chloroform/methanol-free method of isolating gangliosides is described. The nonionic polyoxyethylene detergent, hexaethyleneglycol mono-n-tetradecyl ether (C14EO6), forms clear micellar solutions in water, but two-phase separation can be achieved by centrifugation in the presence of ammonium sulfate at room temperature. A mixture of pure gangliosides, metabolically labeled gangliosides obtained from cultured hippocampal neurons, and gangliosides from rat cerebellar tissue were quantitatively recovered in the detergent-rich upper (coacervate) phase, with a partition coefficient K > 60. Gangliosides were subsequently separated from the detergent using an Iatrobead column prior to analysis by thin-layer chromatography. The procedure described here is as efficient as other methods of ganglioside extraction, such as that using chloroform/methanol/water/pyridine, but is less time-consuming inasmuch as extraction, purification, and TLC analysis can be completed within 1 day.

Animals↗
PubMed1998

Gangliosides enhance apoptosis of thymocytes.

Monosialogangliosides, normal components of cell membranes, regulate cell development and differentiation in several organs. Our previous observation of dramatic premature thymic involution in cats with feline GM1 gangliosidosis, whose thymocytes have abnormally high cell surface gangliosides, suggested that excess GM1 ganglioside (GM1) could modulate thymocyte apoptosis in this disease (Cox et al., "Thymic Alterations in Feline GM1 Gangliosidosis," submitted). In these studies, we added exogenous GM1 to murine primary thymocyte cultures and demonstrated enhanced apoptosis in treated cells by DNA fragmentation, apoptotic body, and electrophoretic analyses. GM1-enhanced apoptosis was blocked by common apoptotic pathway inhibitors including aurintricarboxylic acid (inhibitor of endonuclease activity), actinomycin D (inhibitor of RNA transcription), and cycloheximide (inhibitor of protein synthesis). GM1 treatment primarily affected the immature CD4+ CD8+ subset, as shown by flow cytometric evaluation of fetal thymic organ culture and primary thymocyte cultures. Apoptosis also could be induced by GM2, GM3, and GT1b, whereas asialo-GM1 failed to do so, suggesting that the sialic acid moiety may play an important role in the induction of thymocyte apoptosis.

Animals↗
PubMed1992

Metabolism of GM1 ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses, sialidoses, and sphingolipid activator protein (SAP, saposin) 1 and prosaposin deficient disorders.

Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with GM1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in GM1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2-4 fold relative to controls. Labelled GM2 ganglioside accumulated in 4 variants of GM2 gangliosidosis, whereas labelled GM3 ganglioside accumulated in sialidosis, galactosialidoses and sphingolipid activator protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C and D) deficient lipidoses. The reduced degradation of GM3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from GM1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.

Child, Preschool↗
PubMed1980

Characterization of two gangliosides of the paragloboside series from chicken skeletal muscle.

Two glucosamine-containing gangliosides, a ceramide pentasaccharide and ceramide heptasaccharide, have been purified from the gangliosides of chicken skeletal muscle. The lipids were extracted with a mixture of tetrahydrofuran and aqueous KCl and purified by anion-exchange and silicic acid column chromatography. Their structures were determined by specific enzymatic hydrolysis and methylation analysis. The proposed structures are sialosyl derivates of: (a) paragloboside, and (b) paragloboside with an additional lactosamine disaccharide: (a) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer, (b) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer. The long base was predominantly sphingosine and the acyl groups were mainly palmitate, stearate and oleate.

Animals↗