Ganglioside GM1b Research Articles

Dicalcin suppresses invasion and metastasis of mammalian ovarian cancer cells by regulating the ganglioside-Erk1/2 axis

Metastasis, a multistep process including cancer cell migration and invasion, is the major cause of mortality in patients with cancer. Here, we investigated the effect of dicalcin, a Ca2+-binding protein, on the invasion and metastasis of ovarian cancer (OC) cells. Extracellularly administered dicalcin bound to the membrane of OV2944 cells, mouse OC cells, and suppressed their migration in vitro; however, cell viability or proliferation were unaffected. Repeated intraperitoneal injection of a partial peptide of dicalcin (P6) prolonged the survival, and reduced the number of microcolonies in the livers of cancer-bearing mice. P6 bound to the ganglioside GM1b in a solid-phase assay; treatment with P6 inhibited the constitutive activation of Erk1/2 in OC cells, whereas excess administration of GM1b augmented Erk activity and cancer cell migration in vitro. Thus, dicalcin, a novel suppressor of invasion and metastasis of OC cells, acts via the GM1b-Erk1/2 axis to regulate their migration.

One-pot multienzyme (OPME) chemoenzymatic synthesis of brain ganglioside glycans with human ST3GAL II expressed in E. coli.

A human sialyltransferase ST3GAL II (hST3GAL II) was successfully expressed in Escherichia coli as an active soluble fusion protein with an N-terminal maltose-binding protein (MBP) and a C-terminal hexa-histidine tag. It was used as an efficient catalyst in a one-pot multienzyme (OPME) sialylation system for high-yield production of the glycans of ganglioside GM1b and highly sialylated brain gangliosides GD1a and GT1b. Further sialylation of GM1b and GD1a glycans using a bacterial α2-8-sialyltransferase in another OPME sialylation reaction led to the formation of the glycans of GD1c and brain ganglioside GT1a, respectively. The lower reverse glycosylation activity of the recombinant hST3GAL II compared to its bacterial sialyltransferase counterpart simplifies the handling of enzymatic synthetic reactions and has an advantage for future use in automated chemoenzymatic synthetic processes.

The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Acalpha(2-3)Galbeta(1-3)GalNAcbeta(1-4)Galbeta(1-4)Glc) and its precursor, asialo-GM1 (Galbeta(1-3)GalNAcbeta(1-4)Galbeta(1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the beta-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

An efficient approach for total synthesis of aminopropyl functionalized ganglioside GM1b

A highly efficient protocol for the synthesis of aminopropyl functionalized ganglioside GM1b has been described. The full protected ganglioside GM1b was obtained in 71% yield within 5h. The key feature of the synthetic approach was the use of sialic acid donor that was with a C-5 trichloroacetamide moiety and with a dibenzyl phosphite residue as leaving group at the anomeric carbon. The sialyl donor gave high yields and excellent α-anomeric selectivities with a wide variety of glycosyl acceptors ranging from C-3 or C-6 hydroxyls of galactoside to C-6 hydroxyl of glucosaminoside by using TMSOTf as catalyst in a mixture solution of acetonitrile and methylene chloride.

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-α 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.

Isolated absence of F waves and proximal axonal dysfunction in Guillain-Barré syndrome with antiganglioside antibodies

OBJECTIVESTo investigate the pathophysiology of selective absence of F waves and its relation with antiganglioside antibodies in Guillain-Barré syndrome (GBS). Some patients with GBS show the absence of F waves...

Guillain-Barré syndrome associated with IgG anti-GM1b antibody subsequent to Mycoplasma pneumoniae infection

Sera from patients with Guillain-Barré syndrome (GBS) frequently have antibodies to various gangliosides. We report a girl with GBS after Mycoplasma pneumoniae infection who had serum IgG antibody to GM1b ganglioside as well as the cold agglutinins. The cold agglutinins are polyclonal IgM autoantibodies to `I' antigen on erythrocytes. Ganglioside GM1b contains the terminal moiety shared with sialylated I antigen, a main receptor for M. pneumoniae. In this patient, the anti-GM1b antibody may be elicited in a similar mechanism producing anti-I antibody, and functioned in the development of GBS.

Neuronal expression of a minor monosialosyl ganglioside GM1b in rat brain: immunochemical characterization using a specific monoclonal antibody

Monoclonal antibodies (mAbs) against GM1b ganglioside were raised by immunizing NZB/n mice with the antigen purified from bovine brains, and the details of binding specificity of the mAbs were characterized. Anti-GM1b mAb, termed NA-6, reacted specifically with GM1b (NeuAc) and GM1b(NeuGc). NA-6 antibody did not react with other structurally related gangliosides, indicating that the antibody recognizes NeuAc or NeuGc α2-3Galβ1-4GalNAcβ1-4Gal structure. Using NA-6 antibody, GM1b ganglioside in developing rat brain was investigated by TLC/enzyme-immunostaining and detected first on gestational day 16. The specific content of brain GM1b increased until postnatal day 10, and then gradually decreased in later stage of development. Immunohistochemically GM1b was found in proximal dendrites and cell bodies of neurons in extensive regions of adult rat brain. The immunoreactivity tended to be confined in patch-like clusters on cell membranes, as typically found in the hippocampus and neocortex. The GM1b synthase activity, when assayed in the cerebellar microsome proteins, was significantly reduced in lurcher mutant mouse that is devoid of both cerebellar granule and Purkinje cells. These findings demonstrate that GM1b ganglioside exists in neurons and is actively synthesized during the development in rat brain.

Ganglioside biosynthesis in mouse embryos: sialyltransferase IV and the asialo pathway.

The in vitro activity of sialyltransferase IV (SAT-IV), which catalyzes the transfer of sialic acid to the terminal galactose of different gangliotetraosylceramides (GA1, GM1a and GD1b), was examined in membrane-enriched preparations from mouse embryos at embryonic day 12 (E-12). Gangliosides GD1a and GT1b were the only reaction products using GM1a and GD1b as substrates, respectively. The Km values for GM1a and GD1b were 53 microM and 42 microM, respectively. Competitive inhibition experiments showed that the same enzyme (SAT-IV) catalyzed sialic acid transfer to the terminal galactose residues of both GM1a and GD1b. Two labeled ganglioside products were obtained, however, using GA1 as a substrate. One product was identified as ganglioside GM1b and the enzymatic reaction for its formation was maximal at pH 6.0, similar to that for GD1a and GT1b formation. The second product, synthesized by a different sialyltransferase, was identified as GD1 alpha based on results from TLC immunostaining, neuraminidase digestion, and periodate oxidation-borohydride reduction. The pH dependence curve for GD1 alpha formation had a different shape than that for GM1b formation with a maximum at pH 6.3. GD1 alpha is apparently synthesized from GM1b by an endosialyltransferase that catalyzes the transfer of a second sialic acid to the internal N-acetylgalactosamine of GM1b. The formation of both GM1b and GD1 alpha was linear over protein concentration. The ratio of GD1 alpha/GM1b formation varied from 0.25 to 1.20 depending on the GA1 substrate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Regio- and stereo-selective synthesis of ganglioside GM1b and some positional analogs

Total synthesis of ganglioside GM1b (IV 3NeuAcGgOse 4Cer) and three of its positional analogs are described. Methyl O-(methyl 5-acetamido-4,7,8,9-tetra- O-acetyl-3,5-dideoxy- d- glycero-α- d- galacto-2-nonulopyranosylonate)- (2 → 3)-2,4,6-tri- O-benzoyl-1-thio-β- d-galactopyranoside ( 7) and methyl O-(methyl 5-acetamido-4,7,8,9-tetra- O-acetyl-3,5-dideoxy- d- glycero-α- d- galacto-2-nonulopyranosylonate)-(2→6)- 2,4-di- O-benzoyl-3- O-benzyl-1-thio-β- d-galactopyranoside ( 8) were the key glycosyl donors, prepared according to our reported methods. Coupling of 3,4,6-tri- O-acetyl-2-deoxy-2-phthalimido-α- d-galactopyranosyl bromide and 2-(trimethylsilyl)ethyl O-(2,3,6-tri- O-benzyl-β- d-galactopyranosyl)-(1→4)-2,3,6-tri- O-benzyl-β- d-glucopyranoside gave a trisaccharide, which after removal of O-acetyl and phthaloyl groups was converted separately, by benzylidenation and dibutyltin oxide-mediated selective benzylation, into two glycosyl acceptors. These were suitable respectively for C-3 and C-6 glycosylation reactions, promoted by dimethyl(methylthio)sulfonium triflate (DMTST), with the donors 7 and 8. The four possible coupling reactions gave the corresponding four pentasaccharide derivatives in high yields, and these were transformed into their respective α-trichloroacetimidates. Glycosylation of (2 S,3 R,4 E)-2-azido-3- O-benzoyl-4-octadecene-1,3-diol with the α-trichloroacetimidates gave the corresponding α-glycosides, which on channeling through selective reduction of the azido group, coupling of the thus formed amino group with octadecanoic acid, O-deacetylation, and saponification of the methyl ester group, gave the title compounds.

A facile total synthesis of ganglioside GM1b and its positional analog

A facile total synthesis of ganglioside GM1b and its positional analog

Isolation and characterization of extremely minor gangliosides, GM1b and GD1 alpha, in adult bovine brains as developmentally regulated antigens.

In addition to ganglioside GM1b, an unusual and extremely minor ganglioside, GD1 alpha, was efficiently isolated from bovine brain by combination of Q-Sepharose and Iatrobeads column chromatographies. In the course of purification steps, the presence of the sialidase-labile ganglioside was proved by a highly sensitive TLC/enzyme-immunostaining method. The structure was characterized by gas-liquid chromatography, permethylation study, sialidase degradation, immunostaining with specific antibodies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry. The content of the ganglioside was very small (0.016%) in the total gangliosides. This finding suggests that a synthetic pathway of asialo GM1----GM1b----GD1 alpha may exist in mammalian brains. A monoclonal antibody NA-6 that was obtained by immunizing mice with purified GM1b reacted specifically with GM1b but showed no cross-reactivity with other structurally related gangliosides such as GM1a, GD1a, and so on. Using the method of TLC/immunostaining with NA-6, GM1b was found to be strongly expressed during embryonic days 14-17 in chick brains. Thus, it is assumed that extremely minor gangliosides like GM1b and GD1 alpha found in adult brains are characterized as embryonic molecules.

A stereocontrolled total synthesis of a ganglio-ganglioside GM1b, IV 3NeuAcαGgOse4Cer

A first total synthesis of ganglio ganglioside GM1b. α-D-Neu pAc-(2→3)-β-D-Gal p(1→3)-β-D-Gal pNAc-(1→4)-β-D-Gal p-(1→4)-β-D-Glc p-(1→1)-Cer, was achieved in a stereocontrolled manner.

Glycolipids of mouse erythroleukemia cells (Friend cells) and their alteration during differentiation.

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.

Isolation and characterization of ganglioside GM1b from normal human brain.

A sialidase-susceptible monosialoganglioside was isolated from normal human brain by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The yield of this ganglioside was about 6 mg per whole brain. Its structure was elucidated by sugar analysis, sialidase digestion, permethylation, and proton NMR studies. This ganglioside had carbohydrate, fatty acid, and long-chain base compositions identical to those of brain GM1a. However, the sialosyl residue was found to be linked (alpha 2-3) to the terminal galactosyl residue of the asialo-GM1a backbone. The complete structure of this ganglioside was therefore identified as GM1b or IV3NeuAcGgOse4Cer.

Effects of lectin activation on sialyltransferase activities in human lymphocytes.

The effects of phytohemagglutinin (PHA) stimulation on the activities of sialyltransferase 1 (SAT-1), and sialyltransferase 3 (SAT-3), in human lymphocytes were investigated in vitro. For SAT-1 and SAT-3, respectively, the apparent Km values with variable CMP-NeuAc concentrations were 0.19 and 0.015 mM and with variable LacCer were 0.075 and 0.17 mM. Progressive increases in the activities of SAT-1 and SAT-3 were detected in lymphocytes stimulated with PHA, whereas no increase was observed in control lymphocytes incubated in culture medium alone. These increased activities occurred within 18-36 h of incubation and preceded optimum lymphocyte proliferation. Intact lymphocytes were needed for the lectin-stimulated increase of sialyltransferase activities because neither concanavalin A nor phytohemagglutinin added to the broken cell preparation modulated SAT-1 activity. The glycolipid products formed as a result of these enzymatic reactions in the presence of endogenous and exogenous acceptors were tentatively identified by thin-layer chromatography and autofluorography. The addition of exogenous LacCer to the SAT-1 assay resulted in the radiolabeling of a small amount of ganglioside GM1b (3.4%), but GM3 was the major labeled product (96%). When GgOse4Cer was added to the SAT-3 assay, 32% GM3 and 24.6% GM1b were detected while 44% consisted of glycolipids not labeled in assays performed without exogenous acceptors. Of the radioactivity transferred to endogenous acceptors, 81.3% was in GM3 and 14.6% in GM1b. These results demonstrate that the modulation of sialyltransferase activity occurs earlier than cellular activation.

Ganglioside GM1b as an influenza virus receptor.

Receptor activity toward influenza virus A/Aichi/2/68 (H3N2) of a pair of gangliosides, GM1b (NeuAc alpha 2-3 Ga1 beta 1-3 GalNAc beta 1-4 Gal beta 1-4 GLc beta 1-1'-ceramide) and GM1a (Gal beta 1-3 GalNAc beta 1-4 (NeuAc alpha 2-3) Ga1 beta 1-4 Glc beta 1-1'-ceramide) is described. Receptor activity was monitored by an assay system including incorporation of gangliosides into chicken asialoerythrocytes which had lost the biological response for the virus, and subsequent estimation of recovery of virus-mediated haemagglutination and haemolysis. GM1a and GM1b were incorporated in similar amounts into chicken asialoerythrocytes. It was found that GM1b-erythrocytes revealed distinct recovery of influenza virus A/Aichi-mediated agglutination at 4 degrees C and subsequent haemolysis at 37 degrees C at pH 5.3. However, restoration of biological responsiveness of the erythrocytes to the virus by incorporation of GM1a was very low under the same conditions. The above results indicate that the haemagglutinin of the influenza virus A/Aichi/2/68 (H3N2) recognizes the saliosyl residue linked to nonreducing 'terminal' galactose rather than 'inner' galactose in the ganglio-tetraose core of gangliosides. GM1b could be one of the functional receptors to the influenza virus.