Regulation of Transmembrane Signaling by Ganglioside GM1 :

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.     

Ganglioside GM1 beta-galactosidase: studies in human liver and brain

A microcolumn assay for ganglioside GM₁ β-galactosidase (EC 3.2.1.23) has been developed using GM₁ tritiated exclusively in the terminal galactose residue. The reaction is stimulated up to 100-fold by anionic and cationic detergents; this stimulation is inhibited by neutral detergents. 4-Methylumbelliferyl β-d-galactopyranoside is hydrolyzed about seven times more rapidly than GM₁ in human brain (gray matter) and liver. Agarose gel filtration separated two forms of GM₁ β-galactosidase in both brain and liver. The major form (ganglioside GM₁ β-galactosidase A) had a molecular weight of 60–70 × 10³ and the minor form (ganglioside GM₁ β-galactosidase B) 600–800 × 10³. The liver and brain GM₁ β-galactosidases and 4-methylumbelliferyl β-galactosidase A cochromatographed on fractionation. The two forms of the enzyme in liver isolated by gel filtration corresponded to the two major forms found on starch gel electrophoresis and were converted to electrophoretically slower-moving forms after treatment with neuraminidase (EC 3.2.1.8, Cl. perfringens) suggesting that both are sialylated glycoproteins. The activity of GM₁ β-galactosidase in the brain and liver tissue of patients with GM₁ gangliosidosis Types I and II was less than 2% of control values. The mutation in each GM₁ gangliosidosis appears to result in a severe reduction of activity of two ganglioside GM₁ β-galactosidases.     

Ganglioside GM1 mimics: lipophilic substituents improve affinity for cholera toxin

Ganglioside GM1 mimics including (R)-2-hydroxy-3-cyclohexylpropionic acid or (R)-2-hydroxy-3-phenylpropionic acid as replacements for NeuAc are stronger cholera toxin binders than the parent ligand 2, which includes (R)-2-hydroxy-propionic acid.     

Clinical Studies with Dexfenfluramine: From Past to Future

d Fenfluramine (dF) (15 mg twice daily) has been studied in controlled trials in human obesity. It has been shown to increase adherence to weight lowering programs, to double the number of patients losing 10 kg or more when compared with a fairly efficient placebo plus dietary counselling, and to prevent weight regain when continued over a 1 year period. Weight loss after 1 month and 4 months is likely to predict subsequent outcome. Also, significant improvement in metabolic risk factors and blood pressure were clearly demonstrated, even more markedly in some obesity-associated diseases, when body weight is maintained at a lower level. Even moderate but sustained weight loss of some 10 % of starting weight or less has been confirmed to be of medical value. Tolerance and safety of dF can be considered acceptable, even if longer term follow-up is clearly needed. These studies support the concept that long-term pharmacotherapy with this serotoninergic drug might help achieve better outcome in the management of many obese patients, particularly in preventing relapse. The long-term managerial strategies to be developed for each patient might thus include dF together with dietary advice, behavioral modification and physical exercise, either simultaneously or sequentially.     

Ganglioside GM3 and its biological functions

Metabolism, topology, and possible mechanisms for regulation of the ganglioside GM3 content in the cell are reviewed. Under consideration are biological functions of GM3, such as involvement in cell differentiation, proliferation, oncogenesis, and apoptosis.     

Ganglioside GM1 protection from apoptosis of rat heart fibroblasts.

Ceramide is involved as a mediator of apoptosis induced by a variety of signaling molecules or stressful events. Ceramide-derived sphingosine 1-phosphate behaves as an antiapoptotic agent. The ganglioside GM1 is known to protect neuronal cell lines from apoptosis induced by serum/growth factor withdrawal and its effect is mediated in part by the direct activation of the trkA NGF receptor [G. Ferrari et al. (1995) J. Biol. Chem. 270, 3074-3080]. We show that GM1, similarly to sphingosine 1-phosphate, protects rat heart fibroblasts from apoptosis induced by the protein kinase C inhibitor staurosporine and by C2-ceramide. Furthermore, we show that GM1 induces the synthesis of sphingosine 1-phosphate and that this effect is partially prevented by the sphingosine kinase inhibitor N,N-dimethylsphingosine. We conclude that the antiapoptotic action of GM1 is largely to be ascribed to an increased sphingosine kinase activity.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

GM1 Ganglioside Activates the High-Affinity Nerve Growth Factor Receptor trkA

Abstract: The monosialoganglioside GM1 has been shown to possess neurotrophic activity in vitro and in vivo and is now used as an experimental treatment for a variety of neurological disorders and trauma. Little is known about the mechanism of action used by GM1. Because GM1 appears to enhance nerve growth factor (NGF) activity, we have used C6trk⁺ cells, a derivative of C6-2B glioma cells that express the high-affinity receptor for NGF trkA , to determine whether the neurotrophic effects of GM1 occurs through induction of trkA activity. Exposure of C6trk⁺ cells to NGF (10–50 ng/ml) resulted in a five- to 10-fold increase in trkA tyrosine phosphorylation within 5 min. Incubation of cells with GM1 resulted in a threefold increase in trkA phosphorylation beginning within 1 h and peaking between 3 and 6 h. Optimal responses to GM1 were obtained using 80–100 µ M concentrations. Moreover, tyrosine phosphorylation of known trkA target proteins, such as extracellular signal-regulated kinases, and suc -associated neurotrophic factor-induced tyrosine-phosphorylated target, were activated upon stimulation of C6trk⁺ cells with GM1. In addition, GM1 potentiated the NGF-mediated activation of tyrosine phosphorylation of trkA . GM1 failed to induce phosphorylation of trkA and target proteins in mock transfected cells. Thus, our data demonstrate that GM1 mimics some of the effects of NGF and suggest that the neurotrophic properties of GM1 may be attributed to its activation of trkA signal transduction.     

GM1 Ganglioside: In Vivo and In Vitro Trophic Actions on Central Neurotransmitter Systems

Abstract: There is now substantial evidence that GM1 ganglioside is effective in partially correcting the consequences of neuroinjury in a number of in vivo and in vitro model systems. Although the molecular mechanism(s) and the substrates for the neurotrophic activity of the gangliosides are not fully understood, the published experimental work suggests that GM1 has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. This review focuses attention on studies reporting that GM1 restores neuronal integrity and function in the brain of lesioned young as well as aged animals. Critical analysis of these studies can provide guidance for future ganglioside research and may point to novel approaches for treating neuroinjury and a variety of degenerative conditions, including aging.     

Different Metabolic Effects of Ganglioside GM1 in Brain Synaptosomes and Phagocytic Cells

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe²⁺-H₂O₂ system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of ⁴⁵Ca²⁺ influx and oxidative inactivation of Na⁺,K⁺-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10^–11–10^–8 M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10^–11–10^–10 M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.     

Ganglioside GM3 derivatives and their immunomodulating effect

The derivatives of ganglioside GM3-NeuLacCer. NeuLacSph and NeuAcLacSphAc-were obtained and their immunomodulating properties studied. These substances are shown to inhibit lymphocyte blast-transformation independently of their ceramide structure. On the contrary, the stimulation by the above GM3-derivatives of Con A-induced T-suppressor activity depends significantly on the structure of their ceramide moiety.     

GM1 Ganglioside is Involved in Epigenetic Activation Loci of Neuronal Cells

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and are mainly regulated through stage-specific expression of glycosyltransferase (ganglioside synthase) genes. We previously demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase) gene promoter resulted in recruitment of trans-activation factors. In addition, we reported that epigenetic activation of the GalNAcT gene was also detected as accompanied by an apparent induction of neuronal differentiation in neural stem cells responding to an exogenous supplement of ganglioside GM1. Here, we present evidence supporting the concept that nuclear GM1 is associated with gene regulation in neuronal cells. We found that nuclear GM1 binds acetylated histones on the promoters of the GalNAcT and NeuroD1 genes in differentiated neurons. Our study demonstrates for the first time that GM1 interacts with chromatin via acetylated histones at the nuclear periphery of neuronal cells.     

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.     

FXR1真核表达载体的构建与表达及其对神经节苷脂浓度的影响

目的:构建脆性X相关基因1(FXR1)的真核表达载体并检测其对神经节苷脂(GM1)浓度的影响。方法:以pYESTrp3-FXR1为模板,利用PCR扩增FXR1基因,PCR产物经EcoR I和Xho I双酶切后插入真核表达载体pcDNA3.1(-)中,获得的阳性克隆进一步酶切及测序鉴定;将构建成功的pcDNA3.1(-)-FXR1转染SH-SY5Y细胞后,采用Western blot检测FXR1的表达情况,同时采用ELISA试剂盒检测细胞内GM1的浓度。结果:PCR扩增产物为1.9 Kb的片段,与FXR1基因大小相符,阳性克隆经双酶切后获得两条分别为5.4 Kb和1.9 Kb的片段,测序结果与GeneBank中序列相同。构建成功的重组质粒pcDNA3.1(-)-FXR1转染SH-SY5Y细胞后,细胞中FXR1的表达增加,同时有效提高了细胞内GM1的浓度(P0.05)。结论:成功构建了真核表达载体pcDNA3.1(-)-FXR1,FXR1的表达增加可以提高SH-SY5Y细胞中的GM1浓度,这些为后续深入研究FXR1基因在神经组织发育中的调控功能及其在脆性X综合征(FXS)中的作用机制奠定了基础。     

Ganglioside GM1 mediates decapacitation effects of SVS2 on murine spermatozoa

Prior to fertilization, mammalian spermatozoa need to acquire fertilizing ability (capacitation) in the female reproductive tract. On the other hand, capacitated spermatozoa reversibly lose their capacitated state when treated with seminal plasma (decapacitation). Previously, we demonstrated that a mouse seminal plasma protein, SVS2, is a decapacitation factor and regulates sperm fertilizing ability in vivo. Here, we examined the mechanisms of regulation of fertilizing ability by SVS2. Capacitation appears to be mediated by dynamic changes in lipid rafts since release of the cholesterol components of lipid rafts in the sperm plasma membrane is indispensable for capacitation. When the ejaculated spermatozoa were stained with a cholera toxin subunit B (CTB) that preferably interacts with ganglioside GM1, another member of the lipid rafts, the staining pattern of the sperm was the same as the binding pattern of SVS2. Interestingly, SVS2 and CTB competitively bound to the sperm surface with each other, suggesting that the binding targets of both molecules are the same, that is, GM1. Molecular interaction studies by the overlay assay and the quartz crystal microbalance analysis revealed that SVS2 selectively interacts with GM1 rather than with other gangliosides. Furthermore, external addition of GM1 nullified SVS2-induced sperm decapacitation. Thus, ganglioside GM1 is a receptor of SVS2 and plays a crucial role in capacitation in vivo.     

Ganglioside GM1 increases line tension at raft boundary in model membranes

Gangliosides are significant participants in suppression of immune system during tumor processes. It was shown that they can induce apoptosis of T-lymphocytes in a raft-dependent manner. Fluorescence confocal microscopy was used to study distribution and influence of ganglioside GM1 on raft properties in giant unilamellar vesicles. Both raft and non-raft phase markers were utilized. No visible phase separation was observed without GM1 unless lateral tension was applied to the membrane. At 2 mol % of GM1 large domains appeared indicating macroscopic phase separation. Increase of GM1 content to 5 mol % resulted in shape transformation of the domains consistent with growth of line tension at the domain boundary. At 10 mol % of GM1 almost all domains were pinched out from vesicles, forming their own homogeneous liposomes. Estimations showed that the change of the GM1 content from 2 to 5–10 mol % resulted in a several-fold increase of line tension. This finding provides a possible mechanism of apoptosis induction by GM1. Incorporation of GM1 into a membrane leads to an increase of the line tension. This results in a growth of the average size of rafts due to coalescence or merger of small domains. Thus, necessary proteins can find themselves in one common raft and start the corresponding cascade of reactions. The article is published in the original.     

Synthesis of New Photoaffine Probes on the Basis of Ganglioside GM1

New photoaffine probes, photoreactive derivatives of ganglioside GM1 bearing a carbene-generating diazocyclopentadien-2-ylcarbonyl group at various distances from the carbohydrate moiety in their molecules were synthesized.     

Liposome Fluidity Alters Interactions Between the Ganglioside GM1 and Cholera Toxin B Subunit

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.