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PubMed2004

Ganglioside glycosyltransferases and newly synthesized gangliosides are excluded from detergent-insoluble complexes of Golgi membranes.

GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.

Animals↗
PubMed1992

Cell surface accessibility of individual gangliosides in malignant melanoma cells to antibodies is influenced by the total ganglioside composition of the cells.

The reactivity of a panel of antiganglioside monoclonal antibodies with a number of melanoma cell lines having different ganglioside composition profiles was studied. One cell line synthesized only GM3, one produced both GM3 and GD2, 2 had GM3 and GD3 as their major gangliosides, and 2 others synthesized approximately equal amounts of GM3, GM2, GD3, and GD2 gangliosides. Antibody reactivity with viable cells was analyzed by: (a) flow cytometry on suspension cells; and (b) mixed hemagglutination assays or immune adherence assays on monolayer cells in culture. GM3 was efficiently detected only in the cell line having GM3 as its sole ganglioside. In the other cell lines, GM3 was difficult to detect even in cells in which it made up a high proportion (up to 50%) of the total ganglioside content. GM2 was easily detectable only in JB-RH melanoma cells (which contain only GM3 and GM2). GD3 was the most reactive ganglioside in 2 cell lines and GD2 in 2 other lines. In general, the most complex ganglioside present in a cell was the one most accessible to antibody. The differential exposure at the cell surface of specific gangliosides may have implications for antibody-directed tumor detection and therapy and for cell-protein or cell-cell interactions that involve glycolipids.

Antigens, Surface↗
PubMed1992

Calmodulin, a ganglioside-binding protein. Binding of gangliosides to calmodulin in the presence of calcium.

Ca(2+)-dependent ganglioside-binding protein was isolated from a soluble cytosol fraction of mouse brains using a ganglioside affinity column prepared with a mixture of bovine brain gangliosides. It was identified as calmodulin based on the following features identical with those of calmodulin: molecular weight, pI, chromatographic profile and amino acid sequences of lysyl-endopeptidase digests, and ability to activate cyclic nucleotide phosphodiesterase. Bovine brain calmodulin derivatized with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl-calmodulin), tetramethylrhodamine isothiocyanate, or biotin was also shown to bind to the ganglioside affinity column Ca2+ dependently and elute with gangliosides GD1a, GD1b, GT1b, GQ1b, GM1, and GM2, melittin, and trifluoperazine but not with GgOse4Cer and oligosaccharides of GM1, GD1a, and GT1b. Modification of the Lys94 residue of calmodulin by biotinylation drastically reduced the capacity for ganglioside binding. Ganglioside GD1b caused a blue shift and increase in intensity of the fluorescence emission spectrum of dansyl-calmodulin in the presence of Ca2+. The increment in fluorescence was proportional to the amount of GD1b added and was maximal at the molar ratio of GD1b to calmodulin, approximately 7.8. Gangliosides are thus shown to specifically bind to calmodulin, and this binding may be a general mechanism for regulating calmodulin-dependent enzymes with consequent cellular response, such as cell differentiation.

Amino Acid Sequence↗
PubMed1999

Effect of a chimeric anti-ganglioside GM2 antibody on ganglioside GM2-expressing human solid tumors in vivo.

Ganglioside GM2 is expressed on the surface of neuroblastoma and glioblastoma cells, and may also be detected on lung cancer cells. We reported previously that anti-ganglioside GM2 antibody exhibited strong in vitro anti-tumor activity against adriamycin-resistant cancer cells, which overexpressed ganglioside GM2. In the present study, we examined the in vivo anti-tumor effect of the chimeric anti-ganglioside GM2 antibody, KM966, against human lung and breast carcinoma cells, SBC-3 and MCF-7, and respective adriamycin-resistant clones, SBC-3/ADM and AdrR MCF-7 in BALB/c nu/nu mice. Ratios of tumor volume (T/C) between KM966-treated group and control group were 0.01 for SBC-3, 0.00 for SBC-3/ADM, 0.85 for MCF-7 and 0.34 for AdrR MCF-7 cells, respectively. Nude mice, which were pretreated with anti-asialo GM1 antibody to remove natural killer cells, were transplanted with 4 x 10(7) of SBC-3 and SBC-3/ADM subcutaneously. Seven days later, when tumors had grown to a diameter of over 8 mm, mice began to receive intravenous treatment of 120 microgram/mouse KM966 daily. Fourteen daily treatments induced regression to less than 4-mm diameter in 4/5 SBC-3 tumors and 5/5 of SBC-3/ADM tumors. All SBC-3/ADM tumors disappeared completely, suggesting that KM966 exerts a strong in vivo anti-tumor effect on ganglioside GM2-expressing cancer cells. In KM966-treated mice, the surface of the tumor cells stained positive with anti-human IgG. In addition, numerous leukocytes had infiltrated into the tumor mass. Antibody-dependent cell-mediated cytotoxicity (ADCC) of KM966 against tumor cells was examined in vitro by (51)Cr-release assay and revealed that KM966 induces ADCC activity against ganglioside GM2-expressing tumors. Our results suggest that immunotherapy using KM966 may be useful for the treatment of ganglioside GM2-expressing solid tumors.

Animals↗
PubMed1996

Metabolic processing of gangliosides by normal and Salla human fibroblasts in culture. A study performed by administering radioactive GM3 ganglioside.

Cultured fibroblasts from normal subjects and from subjects affected by Salla disease, characterized by the lack or misfunction of the membrane carrier responsible for the egress of sialic acid from lysosomes, were fed with ganglioside GM3 labeled at the sialic acid acetyl group, [Neu5Ac-3H]GM3, or at C-3 of sphingosine (Sph), [Sph-3H]GM3, or at C-1 of stearoyl chain, [stearoyl-14C]GM3. After a 15-h pulse the total amount of cell-bound GM3 corresponded to about 2% of the endogenous ganglioside content. Cells were then subjected to a 72-h chase, and the radioactive products from both ganglioside catabolism and salvage processes of catabolic fragments were measured. These data indicated that about 50% of the cell-bound ganglioside underwent metabolic processing, suggesting a ganglioside half-life of 2-3 days. [Neu5Ac-3H] formed from [Neu5Ac-3H]GM3 degradation was mostly re-cycled for the biosynthesis of gangliosides and sialoglycoproteins, only a minor part being degraded to [3H]water, which constituted only 1.6% of total metabolite linked radioactivity. [Sph-3H] from the [Sph-3H]GM3 degradation was partly re-cycled for the biosynthesis of gangliosides, neutral glycosphingolipids and sphingomyelin, and partly (about 20% of the total metabolite linked radioactivity) degraded to [3H]water. In Salla fibroblasts metabolic processing of [Neu5Ac-3H]GM3 produced large amounts of free [3H]Neu5Ac, and a reduced incorporation of radioactivity into glycoconjugates (as compared to normal cells). However, the accumulation of free Neu5Ac was not accompanied by an increase of tritiated water. LacCer and Cer from [stearoyl-14C]GM3 catabolism were found to accumulate in Salla fibroblasts, an indication that the enzymes of glycosphingolipid metabolism were affected by the impairment of Neu5Ac egress from lysosomes. Particularly relevant was the accumulation of ceramide which was hardly detectable in control cells.

Adolescent↗
PubMed1984

Cytosolic gangliosides of rat brain: their fractionation into protein-bound complexes of different ganglioside compositions.

The sialic acid moiety of rat brain cytosolic gangliosides was radiolabeled by intracranial injection of N-(3H)acetylmannosamine. Upon ammonium sulphate fractionation, Sepharose 6B gel filtration, and hydroxylapatite-cellulose chromatography, ganglioside-bound radioactivity of brain cytosolic extract followed the behavior of protein and not that of purified gangliosides. This indicates that cytosolic gangliosides occur as ganglioside-protein complexes. By application of hydroxylapatite-cellulose column chromatography, fractions were obtained having different ganglioside composition. In particular, one fraction contained GM1, one GD1a, and one GT1b with a ganglioside homogeneity better than 95% in each fraction. This indicates the occurrence in brain cytosol of a GM1-protein complex, a GD1a-protein complex, and a GT1b-protein complex.

Animals↗
PubMed1994

Facile methods for isolation and determination of gangliosides in a small scale: age-related changes of gangliosides in mouse brain synaptic plasma membranes.

A quantitative isolation method for gangliosides from small sizes of samples has been developed. Total lipids were separated into gangliosides and other lipids using Phenyl Sepharose column chromatography. Gangliosides were recovered in a yield of 99%. Determination of gangliosides was performed by gas chromatography--mass spectrometry using a selected ion-monitoring technique. These combined methods can provide quantitative isolation and determination of gangliosides from as little as 10 mg fresh brain tissues or 0.5 mg protein of membrane fractions. The present methods were successfully applied to the analysis of gangliosides from mouse brain synaptic plasma membranes to reveal that the ganglioside contents and composition remain constant from adult to senescence.

Aging↗
PubMed1995

Ganglioside modulation of the PDGF receptor. A model for ganglioside functions.

Gangliosides are a family of glycolipids that are present at the cell surface of all mammalian cells. Patterns of gangliosides are different in gliomas than normal brain, and exogenously added gangliosides affect the growth of cultured glioma cells. Gangliosides inhibit the activities of several kinases, including protein kinase C (PKC) and cAMP-kinase. U-1242 MG cells (derived from a human malignant glioma) have receptors for platelet-derived growth factor (PDGF) that become phosphorylated on tyrosine when exposed to PDGF. Exposure of these cells to PDGF also causes an increase in intracellular calcium concentration ([Ca2+]i) and induces a translocation of PKC to the membrane. Preincubation of U-1242 MG cells with several species of gangliosides inhibits the increase in ([Ca2+]i) and PKC translocation in response to PDGF, but GM3 is much less effective than other species tested. This is due to a lack of activation of the receptor tyrosine kinase as monitored by phosphorylation of the receptor on tyrosine residues, but is not due to an inhibition of binding of PDGF to its receptors. The lack of activation of the PDGF receptor tyrosine kinase is due to an inhibition of dimerization of the receptor monomers by gangliosides GM1, GM2, GD1a, GT1b, but not GM3. Therefore, gangliosides may be involved in coordinating the activities of multiple trophic factors simultaneously acting on a cell by regulating the dimerization of their respective receptor monomers.

Brain Neoplasms↗
PubMed1988

Ganglioside content of astroglia and neurons isolated from maturing rat brain: consideration of the source of astroglial gangliosides.

Previous biochemical and histochemical studies have failed to clarify the nature or quantity of gangliosides in CNS astrocytes. Using improved methodologies for bulk isolation of both neurons and astrocytes as well as for ganglioside purification, we find significantly higher ganglioside concentration in astrocytes and very similar thin-layer chromatography (TLC) patterns for the two cell types. However, in vivo labeling of glycoconjugates via intracerebral injection of [3H]glucosamine prior to cell isolation revealed a different picture: whereas glycoproteins were well-labeled in both cell types after labeling periods of 1-2 h, gangliosides were appreciably labeled only in neurons. With longer time periods (8-48 h) between injection and sacrifice, there was convergence of specific radioactivity of gangliosides from the two isolated cell preparations. These changes are compared to those observed in synaptosomes and microsomes that were isolated simultaneously. The results suggest limited ganglioside synthetic ability in astrocytes as compared to neurons, a conclusion supported by assay of UDP-galNAc:GM3 N-acetylgalactosaminyltransferase in the isolated cells. Nevertheless, the presence of ganglioside GM1 in a substantial portion of bulk-isolated astrocytes was demonstrated by indirect immunofluorescent detection of cholera toxin binding. Ideas on the reconciliation of these apparently contradictory phenomena, including the possibility of intercellular transfer and/or phagocytosis are discussed.

Animals↗
PubMed1987

Binding of laminin to brain gangliosides and inhibition of laminin-neuron interaction by the gangliosides.

Binding of laminin to glycolipids of neuronal membranes was studied with a thin-layer chromatography overlay assay. The major brain ganglioside GD1A was the main binding component, when chromatograms containing the same molar amount of the different brain gangliosides and the brain sulfatide were incubated with laminin at physiological ionic strength. The possible role of laminin binding to brain gangliosides in laminin-neuron interactions was studied with adhesion assays. It was found that binding of rat brain neurons to laminin is blocked by 10-40 microM brain gangliosides but not by sulfatide. The inhibition by the gangliosides is suggested to be due to competition with the cell surface interaction sites of laminin and not to binding of the gangliosides to the cells. Our findings support the idea that the adhesive and neurite-promoting effect of laminin is dependent on its interaction with gangliosides at the neuronal cell surfaces.

Animals↗
PubMed1992

Immunorecognition of ganglioside epitopes: correlation between affinity and cytotoxicity of ganglioside antibodies.

Cell-surface gangliosides have immunomodulatory effects that are presumed to play a role in tumour growth, progression, metastasis and therapy. To study the epitopes of gangliosides on human malignant melanomas and to search for monoclonal antibodies (Mabs) with superior immunological effector functions, 19 ganglioside antibodies were established. Specificity and affinity of nine antibodies of IgG3 isotype were evaluated by enzyme linked immunosorbent assay and thin layer chromatography with a panel of purified gangliosides. All antibodies recognised the ganglioside GD3, but their epitope specificity divided them into five groups. Their affinity constants for ganglioside GD3 ranged from 4.7 x 10(6) to 2.3 x 10(8), with 2 x 10(7) for Mab R-24. Two antibodies possessed a higher affinity for GD2 than for GD3. The functional properties of the antibodies were investigated in vitro. Differences in the degree of tumour lysis by complement fixation correlated with the affinity constants. Every ganglioside antibody differed in epitope recognition, affinity and cytotoxicity. Therefore some of these antibodies might even be more useful in the immunotherapy of malignant melanoma than Mab R-24.

Antibodies, Monoclonal↗
PubMed2004

Synthesis of radioactive and photoactivable ganglioside derivatives for the study of ganglioside-protein interactions.

The procedures for the preparation of radioactive and photoactivable ganglioside derivatives have been continuously developed from 1989, when for the first time the synthesis of photoactivable tritium labeled GM1 ganglioside was presented. We described previously the synthesis of photoactivable derivatives of GM3 and GM1 gangliosides, tritium-labeled at acetyl group of sugar units, and of photoactivable GM1 and GD1b gangliosides, tritium-labeled at position 6 of the external galactose. These procedures are reviewed in detail in the present paper. The use of these ganglioside derivatives to study the ganglioside-protein interactions and to identify proteins that specifically interact with gangliosides (including GPI-anchored proteins of the outer membrane leaflet, proteins anchored to the cytoplasmic side of the plasma membrane through a fatty acyl chain, transmembrane proteins, and soluble cytoplasmic proteins) is discussed.

Animals↗
PubMed1997

Identification of a ganglioside recognition domain of tetanus toxin using a novel ganglioside photoaffinity ligand.

Tetanus toxin entry into vertebrate motorneurons may involve binding of neuronal surface gangliosides containing the "1b" substructure (a NeuAcalpha2,8NeuAc group on an internal galactose residue). The domains of tetanus toxin involved in ganglioside binding are known to reside within the carboxyl-terminal half of the toxin's heavy chain ("C fragment"). We developed a novel photoaffinity reagent based upon the structure of the 1b ganglioside GD1b (125I-azido-GD1b) to define the ganglioside-binding domains of tetanus toxin. Using this ligand, we observed radiolabeling of tetanus toxin C fragment which could be specifically inhibited by a ganglioside of the 1b series (GT1b), but not by a non-1b series ganglioside (GM3). When tetanus toxin C fragment was proteolyzed with clostripain, whether before or after reaction with 125I-azido-GD1b, a radiolabeled band was observed by SDS-polyacrylamide gel electrophoresis autoradiography, which was selectively inhibited by GT1b. Protein sequencing of proteolyzed tetanus toxin C fragment co-migrating with that band revealed the carboxyl-terminal 34 amino acid residues of tetanus toxin. Matrix-assisted laser desorption/ionization mass spectrometry of a photoaffinity labeled synthetic polypeptide representing the 34-amino acid domain revealed modification at a single residue (His1293). We propose that this domain of tetanus toxin is sufficient for ganglioside binding.

Affinity Labels↗
PubMed1991

Gangliosides inhibit glucosylceramide synthase: a possible role in ganglioside therapy.

Gangliosides stimulate the hydrolysis of glucosylceramide (GlcCer), their precursor, and therefore may lower the level of cellular GlcCer and exert a feedback control effect to slow the formation of gangliosides. Tests were made to see if a similar effect on GlcCer levels can be exerted by the action of gangliosides on GlcCer synthesis. Using a new assay procedure, we showed that gangliosides do inhibit the synthase in brain membranes quite effectively, the most active being those lipids with more sugar and sialic acid moieties. Mice injected with a mixture of brain gangliosides for 5 days were found to have a lower level of ceramide:UDP-Glc glucosyltransferase activity in brain, liver, and kidney. The inhibition seems to be exerted by competition for the active site and binding to effector site(s) on the enzyme. It is possible that the reported therapeutic actions of gangliosides on the nervous system are, in part, the result of lowered levels of GlcCer. Malignant tumors shed gangliosides into the extracellular fluid, which are believed to block the generation of antibodies by the host's immunodefense system; this effect also may be due, in part, to reduction in the GlcCer level of immunogenic cells. A new finding is that a ceramide containing phytosphingosine is a markedly better substrate for GlcCer synthase than one containing the more common base.

Animals↗
PubMed1991

Isolation and structural elucidation of a novel type of ganglioside, deaminated neuraminic acid (KDN)-containing glycosphingolipid, from rainbow trout sperm. The first example of the natural occurrence of KDN-ganglioside, (KDN)GM3.

Rainbow trout sperm contained almost exclusively monoanionic ganglioside fraction as a major acidic glycosphingolipid. Two monoacidic gangliosides were isolated and purified in this study and designated as sperm ganglioside 1 and 2 (sg-1 and sg-2). The two gangliosides, sg-1 and sg-2, contained the same neutral sugars, galactose and glucose in molar ratio of 1:1 and no GalNAc except for the presence of N-acetyl-neuraminic acid (NeuAc) in sg-1 and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in sg-2. The complete structures of these gangliosides were determined by a combination of methylation analysis, fast atom bombardment mass spectrometry, 400-MHz one- and two-dimensional 1H nuclear magnetic resonance spectroscopy, fatty acid analysis, and endoglycoceramidase digestion NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-1 [(NeuAc)GM3] KDN alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-2 [(KDN)GM3] where, for both sg-1 and sg-2, the ceramide moieties (Cer) were found to be made up of 4-sphingenine and mainly C16:0 fatty acid (palmitate; 95%) with a minor amount of C24:1 fatty acyl chain (nervonate, 5%). The structure of sg-2 is novel and represents the first example of a new class of gangliosides, i.e. KDN-gangliosides.

Animals↗
PubMed1987

Developmental changes of gangliosides of the rat stomach. Appearance of a blood group B-active ganglioside.

Rat stomach gangliosides were purified and their distribution in the different tissue compartments was established. Three major monosialogangliosides were found: GM3, GM1, and a ganglioheptaosylceramide carrying a blood group B determinant. This latter structure was characterized by exoglycosidase degradation, immunostaining with a monoclonal anti-blood group B antibody on thin layer chromatogram, permethylation analysis, electron-impact mass spectrometry of the permethylated-reduced and trimethylsilylated molecule, and 1H NMR spectroscopy of the native ganglioside. It was found to be (Formula: see text) i.e. a GM1 structure substituted with the blood group B determinant and was called B-GM1. A similar structure has been previously identified in precancerous rat liver and chemically induced rat hepatoma (Holmes, E. H., and Hakomori, S. (1982) J. Biol. Chem. 257, 7698-7703). Fucosyl-GM1 was also detected as a minor ganglioside in rat gastric mucosa. The ganglioside profile was modified during the postnatal development. The contribution of GM3 and GD3, which accounted for 95% of the ganglioside sialic acid at birth, decreased during the first 3 weeks of life. GM1, fucosyl-GM1, and B-GM1 were not detected at birth. The concentration of the fucogangliosides increased during the 2nd and 3rd weeks after birth, was stable during the 4th week and then decreased, whereas that of GM1 increased steadily between 6 days and 2 months of age. B-GM1, which has been defined as a tumor-associated ganglioside in the rat liver, was found to be a developmentally regulated antigen of the normal rat stomach.

ABO Blood-Group System↗
PubMed1990

Gangliosides noncovalently bound to DEAE-Sephadex: application to purification of anti-ganglioside antibodies.

A simple, rapid, effective, and inexpensive method for the purification of ligands having high affinity for gangliosides has been developed. DEAE-Sephadex has a high capacity for binding gangliosides (approx 1/1.6, w/w). The gangliosides, bound to the support by electrostatic and hydrophobic interactions, showed a high resistance, in an aqueous environment, to being detached by eluants commonly employed to desorb ligands (i.e., low or high pH or chaotropic agent solutions) or by nonionic detergent solutions as well as by organic solvents. The DEAE-Sephadex-ganglioside complex was assayed as an immunoadsorbent for purifying anti-GM1 ganglioside antibodies from serum of an immunized rabbit. The specific activity of the purified antibodies was 200- to 400-fold higher, and the recovery of the anti-ganglioside activity was above 50%, with respect to the untreated antiserum. The preparation of the complex and the purification of the antibodies can be done in less than 5 h. The glycolipids from the complex can be recovered by elution with organic solvents containing salt or volatile base solutions, and reused. In principle, this method can be adapted for other anionic amphipathic receptor molecules to purify ligands which bind to them.

Adsorption↗
PubMed1989

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression.

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphocytes. Saturation binding by monosialylated [3H]-GM1 to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (KD) of 2.2 +/- 1.4 microM and a binding capacity near 2 fmoles/cell. Competitive inhibition of [3H]-GM1 binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM1-congeneric gangliosides. A comparison between the results of these binding studies and ganglioside-induced decrease of CD4 expression demonstrated that every aspect of [3H]-GM1 binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of ganglioside binding to specific sites on CD4+ T-helper lymphocytes.

Animals↗