Cell growth arrest by sialic acid clusters in ganglioside GM3 mimetic polymers

Ganglioside GM3, one of the sialic acid containing glycosphingolipids, is known to form clusters in lipid microdomains, which serve as platforms for effective signal transduction. In an attempt to clarify the GM3 cluster effect, we enzymatically synthesized GM3 mimetic polymer (GM3-p), with an acrylamide backbone from LacCer mimetic polymer (LacCer-p). Interestingly, GM3-p, but not LacCer-p, reversibly inhibited proliferation of NIH3T3 cells, which are normally resistant to exogenously added GM3. Moreover, we found that the introduction of carbonic acid into the acrylamide chain aided well-oriented cluster formation and enhanced the inhibitory effect of GM3-p. Since sialyllactosyl polymer and GM4 mimetic polymer, but not GM2 mimetic polymer, also inhibited cell proliferation, sialic acid-galactose units must be essential for the biological activity of GM3-p. These results suggest that the formation of sialic acid-galactose clusters is necessary for the suppressive effect of GM3-p. GM3-p treatment did not affect the serum-dependent activation of ERK1/2 or c-fos expression, but caused a reduction in the gene and/or protein expression of cyclin D1, cyclin E, cyclin-dependent kinase (cdk)4, and cdk2, which are involved in the cell cycle. Therefore, GM3-p inhibits cell proliferation by reducing cyclin D1-cdk4 and cyclin E-cdk2 complexes without affecting growth factor signaling from serum to c-fos.     

Synthesis and enzymatic susceptibility of a series of novel GM2 analogs

A series of GM2 analogs in which GM2 epitope was coupled to a variety of glycosyl lipids were designed and synthesized to investigate the mechanism of enzymatic hydrolysis of GM2 ganglioside. The coupling of N-Troc-protected sialic acid and p-methoxyphenyl galactoside acceptor gave the crystalline disaccharide, which was further coupled with galactosamine donor to give the desired GM2 epitope trisaccharide. After conversion into the corresponding glycosyl donor, the trisaccharide was coupled with galactose, glucose and artificial ceramide (B30) to give the final compounds. The result on hydrolysis of GM2 analogs indicates that GM2 activator protein requires one spacer sugar between GM2 epitope and the lipid moiety to assist the hydrolysis of the terminal GalNAc residue. Synthetic studies on sialoglycoconjugates, Part 140. For part 139, see Ref [1].     

Synthesis of a GM3 ganglioside analogue carrying a phytoceramide moiety by intramolecular glycosylation as a key step

A novel analogue of ganglioside GM3, in which sphingosine was replaced with a phytosphingosine moiety, was synthesized by intramolecular glycosylation as a key step. Glucose, a reducing terminal of the saccharide, and phytoceramide were first tethered by succinic acid and the derivative used for the subsequent glycosidic bond formation. The obtained glycosyl phytoceramide was further glycosylated with the sialyl galactose residue to afford a fully protected GM3 derivative, which was converted into the desired, final compound by using conventional deprotection procedures.     

Interaction of N-linked glycans, having multivalent GlcNAc termini, with GM3 ganglioside

GM3 ganglioside interacts specifically with complex-type N-linked glycans having multivalent GlcNAc termini, as shown for (1) and (2) below. (1) Oligosaccharides (OS) isolated from ConA-non-binding N-linked glycans of ovalbumin, whose structures were identified as penta-antennary complex-type with bisecting GlcNAc, having five or six GlcNAc termini (OS B1, B2), or bi-antennary complex-type having two GlcNAc termini (OS I). OS I is a structure not previously described. (2) Multi-antennary complex-type N-linked OS isolated from fetuin, treated by sialidase followed by β-galactosidase, having three or four GlcNAc termini exposed. These OS, conjugated to phosphatidylethanolamine (PE), showed clear interaction with ³H-labeled liposomes containing GM3, when various doses of OS-PE conjugate were adhered by drying to multi-well polystyrene plates. Interaction was clearly observed only with liposomes containing GM3, but not LacCer, Gb4, or GalNAcα1-3Gb4 (Forssman antigen). GM3 interaction with PE conjugate of OS B1 or B2 was stronger than that with PE conjugate of OS I. GM3 interacted clearly with PE conjugate of N-linked OS from desialylated and degalactosylated fetuin, but not native fetuin. No binding was observed to cellobiose-PE conjugate, or to OS-PE conjugate lacking GlcNAc terminus. Thus, GM3, but not other GSL liposomes, interacts with various N-linked OS having multiple GlcNAc termini, in general. These findings suggest that the concept of carbohydrate-to-carbohydrate interaction can be extended to interaction of specific types of N-linked glycans with specific GSLs. Natural occurrence of such interaction to define cell biological phenomena is under investigation. All solvent ratios are by volume. An erratum to this article can be found at     

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.     

Endogenously produced ganglioside GM3 endows etoposide and doxorubicin resistance by up-regulating Bcl-2 expression in 3LL Lewis lung carcinoma cells

The ganglioside patterns have been shown to dramatically change during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. To investigate the significance of the ganglioside GM3 in cancer malignancy, we established GM3-reconstituted cells by transfecting the cDNA of GM3 synthase into a GM3-deficient subclone of the 3LL Lewis lung carcinoma cell line (Uemura, S. (2003) Glycobiology, 13, 207-216). The GM3-reconstituted cells were resistant to apoptosis induced by etoposide and doxorubicin. There were no changes in the expression levels of topoisomerase IIalpha or P-glycoprotein, or in the uptake of doxorubicin between the GM3-reconstituted cells and the mock-transfected cells. To understand the mechanism of the etoposide-resistant phenotype acquired in the GM3-reconstituted cells, we investigated their apoptotic signaling. Although no difference was observed in the phosphorylation of p53 at serine-15-residue site by etoposide between the GM3-reconstituted cells and mock-transfected cells, the activation of both caspase-3 and caspase-9 was specifically inhibited in the former. We found that the anti-apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2) was increased in the GM3-reconstituted cells. Moreover, wild-type 3LL Lewis lung carcinoma cells, which have an abundance of GM3, exhibited no DNA fragmentation following etoposide treatment and expressed higher levels of the Bcl-2 protein compared with the J5 subclone. Thus, these results support the conclusion that endogenously produced GM3 is involved in malignant phenotypes, including anticancer drug resistance through up-regulating the Bcl-2 protein in this lung cancer cell line.     

Study on systematizing the synthesis of the a-series ganglioside glycans GT1a, GD1a, and GM1 using the newly developed N-Troc-protected GM3 and GalN intermediates

A first systematic synthesis of the glycan parts of the a-series gangliosides (GT1a, GD1a, and GM1) utilizing the newly developed N-Troc-protected GM3 and galactosaminyl building blocks is described. The key processes, including the assembly of the GM2 sequence and its conversion into the 3-hydroxy acceptor, were facilitated mainly by the high degree of participation and chemoselective cleavability of the Troc group in the galactosaminyl unit. Furthermore, the novel GM2 acceptor served as a good coupling partner during glycosylation with galactosyl, sialyl galactosyl, and disialyl galactosyl donors, successfully producing the GM1, GD1a, and GT1a glycans.     

TNFalpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3

Membrane microdomains (lipid rafts) are now recognized as criticalfor proper compartmentalization of insulin signaling, but theirrole in the pathogenesis of insulin resistance has not beeninvestigated. Detergent-resistant membrane microdomains (DRMs),isolated in the low-density fractions, are highly enriched incholesterol, glycosphingolipids and various signaling molecules.Tumor necrosis factor alpha (TNF) induces insulin resistancein type 2 diabetes, but its mechanism of action is not fullyunderstood. In other studies we have found a selective increasein the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytestreated with TNF, suggesting a specific function for GM3. Inthe DRMs from TNF-treated 3T3-L1 adipocytes, GM3 levels weredoubled compared with results in normal adipocytes. Additionally,insulin receptor (IR) accumulations in the DRMs were diminished,whereas caveolin and flotillin levels were unchanged. Furthermore,insulin-dependent IR internalization and intracellular movementof the IR substrate 1(IRS-1) were both greatly suppressed inthe treated cells, leading to an uncoupling of IR–IRS-1signaling. GM3 depletion was able to counteract the TNF-inducedinhibitions of IR internalization and accumulation into DRMs.Together, these findings provide compelling evidence that ininsulin resistance the insulin metabolic signaling defect canbe attributed to a loss of IRs in the microdomains due to anaccumulation of GM3.     

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.     

First total synthesis of alpha-(2-->3)/alpha-(2-->6)-disialyl lactotetraosyl ceramide and disialyl Lewis A ganglioside as cancer-associated carbohydrate antigens

The first total synthesis of alpha-(2-->3)/alpha-(2-->6)-disialyl lactotetraosyl (DSLc4) ceramide and alpha-(2-->3)/alpha-(2-->6)-disialyl Lewis A (DSLe(a)) ganglioside as cancer-associated antigens is described. The suitably protected lactotriose (Lc3) derivatives were successively glycosylated with sialic acid, sialyl-alpha-(2-->3)-D-galactose and/or L-fucose donors in a regio- and stereo-selective manner, to give the protected type I hexa- and hepta-saccharides, respectively, which were then converted to the target gangliosides by the introduction of ceramide and subsequent complete deprotection.     

Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes

Novel mono-O-acetylated GM3s, one containing 9-O-acetylN-glycolyl neuraminic acid and another containing 6-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of theO-acetyl residue was identified by the downfield shift of the methylene protons at C-9 ofN-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6-O-Ac GM3, theO-acetylated GM3 mixture was desialylated withArthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1. Abbreviations: Ac, acetyl; Gc, glycolyl; NeuGc,N-Gc neuraminic acid; GM3 (Gc), GM3 containing NeuGc (II³NeuGc-LacCer); 4-O-Ac GM3 (Gc), GM3 containing 4-O-Ac NeuGc; 9-O-Ac GM3 (Gc), GM3 containing 9-O-Ac NeuGc; 6-O-Ac GM3 (Gc), GM3 containing 6-O-Ac Gal; 1D-NMR, one-dimensional nuclear magnetic resonance spectrometry; 2D-COSY, two-dimensional chemical shift-correlated spectrometry; FAB-MS, fast atom bombardment-mass spectrometry; GLC, gas-layer chromatography; GC-MS, gas chromatography-mass spectrometry; TLC, thin-layer chromatography; Ggl, ganglioside; Cer, ceramide; CMH, monohexosylceramide; LacCer, lactosylceramide; 6-O-Ac LacCer, LacCer containing 6-O-Ac Gal; Me₂SO-d₆,²H₆-dimethylsufloxide; CMW, chloroform-methanol-water; Nomenclature and abbreviations of glycosphingolipids follow the system of Svennerholm (J Neurochem [1963]10: 613–23) and those recommended by the IUPAC-IUB Nomenclature Commission (Lipids [1977]12: 455–68).     

Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.     

Ganglioside GM3 modulates conformation of reconstituted Ca~(2 ) -ATPase

Using steady-state fluorescence and nanosecond time-resolved fluorescence techniques, the Ca 2 -ATPase conformational changes induced by ganglioside GM3 were studied with different quenchers. The results showed that GM3 could significantly increase the lifetime of intrinsic fluorescence of Ca2 -ATPase reconstituted into proteoliposomes, and could also weaken the intrinsic fluorescence quenching by KI or hypocrellin B, HB. Further-more, by using quenching kinetic analysis of the time-resolved fluorescence, in the presence of GM3, the quenching constant (Ksv) and quenching efficiency were significantly lowered. The obtained results suggest that the oligosaccha-ride chain and the ceramide moieties of the GM3 molecule could interact with its counterparts of the Ca2 -ATPase re-spectively, thus change the conformation of the hydrophobic domain of the enzyme, making the tryptophan residues in different regions shift towards the hydrophilic-hydrophobic interface, and hence shorten the distance between the hy     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

Accelerated release of exosome-associated GM1 ganglioside (GM1) by endocytic pathway abnormality: another putative pathway for GM1-induced amyloid fibril formation

Exosomes are extracellularly released small vesicles that are derived from multivesicular bodies formed via the endocytic pathway. We treated pheochromocytoma PC12 cells with chloroquine, an acidotropic agent, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment increased the level of GM1 ganglioside in cell media only when the cells were exposed to KCl for depolarization, which is known to enhance exosome release from neurons. In the sucrose-density-gradient fractionation of cell media, GM1 ganglioside was exclusively recovered with Alix, a specific marker of exosomes, in the fractions with the density corrresponding to that of exosomes. Notably, amyloid-β assembly was markedly accelerated when incubated with the exosome fraction prepared from the culture media of PC12 cells treated with chloroquine and KCl. Furthermore, amyloid-β assembly was significantly suppressed by the co-incubation with an antibody specific to GM1-bound amyloid-β, an endogenous seed for amyloid formation of Alzheimer's disease. Together with our previous finding that chloroquine treatment induces the accumulation of GM1 ganglioside in early endosomes, results of this study suggest that endocytic pathway abnormality accelerates the release of exosome-associated GM1 ganglioside following its accumulation in early endosomes. Furthermore, this study also suggests that extracellular amyloid fibril formation is induced by not only GM1 gangliosides accumulated on the surface of the cells but also those released in association with exosomes.     

GM1-induced activation of phosphatidylinositol 3-kinase: involvement of Trk receptors

The ganglioside GM1 promotes neuronal growth, differentiation, survival, phenotypic expression, and function restoration, by apparently interacting with neurotrophic factors and/or their receptors. In brain, GM1 activates the Trk receptors for neurotrophins and the Raf/MEK/ERK cascade in situ and in vivo . We have expanded these studies and explored whether GM1 recruits the phosphatidylinositol 3 (PI3)-kinase pathway in brain also. Incubating striatal slices with GM1 increased the activity of PI3-kinase in phosphotyrosine immunoprecipitates in a time- and concentration-dependent manner, and the response was blocked by the PI3-kinase inhibitors wortmannin and LY294002. PI3-kinase activation following GM1 was rapid and short lasting with an EC₅₀ of 5 μmol/L. There was a temporally parallel activation of the downstream PI3-kinase target Akt, which was prevented by PI3-kinase inhibition. PI3-kinase activity was found increased in Trk and Gab1 immunoprecipitates, and co-immunoprecipitation studies demonstrated the association of Trk and Gab1 after GM1 treatment. Enhanced PI3-kinase activity associated with Trk or Gab1 immunoprecipitates was blocked by the Trk inhibitor K252a. GM1 did not appear to transactivate Trk and did not alter the efflux of neurotrophins in striatal slices. Our findings suggest that GM1 induces activation of PI3-kinase that is, in part, mediated through Trk and Gab1.     

Effects of exogenous ganglioside GM1 on different stages of cell spreading studied by directly quantifying spreading rate

Abstract

We developed novel methods to directly quantify cell spreading rate. By comparing our methods with traditional methods, we found that the enhancement effects of fetal calf serum (FCS) or the inhibitory effects of exogenous ganglioside GM1 occurred at different stages of cell spreading. GM1 mainly influenced the early and late stages of cell spreading of HUVECs. In the presence of 0.5% FCS, GM1 significantly impaired the area-based spreading rates (127.4 ± 35.7 μm²/h and 22.2 ± 3.8 μm²/h, respectively) on the early (0–0.5 h) and late (12–24 h) stages of cell spreading compared with the controls (238.1 ± 11.7 μm²/h and 35.4 ± 19.5 μm²/h, respectively), which was confirmed by the data on the GM1-induced changes in average length of actin filaments during cell spreading. The real-time observation and quantification of cold-induced de-spreading of GM1-free or GM1-treated HUVECs further confirmed that GM1 can influence cell de-spreading process having inhibitory (0–10 min) or enhancement (10–20 min or 40–50 min) effects on different stages. The methods can be recruited for investigating effects of other reagents on different stages of cell spreading.     

GM1 ganglioside modulates prostaglandin E1 stimulated adenylyl cyclase in neuro-2A cells

This study demonstrates modulation by GM1 ganglioside of prostaglandin E1 (PGE1)-induced cAMP formation in Neuro-2a neuroblastoma cells. Pretreatment of the cells with neuraminidase, an enzyme that increases cell surface GM1, resulted in significant elevation of PGE1-induced cAMP formation, as did preincubation of the cells with nmolar concentrations of GM1. Pretreatment with brain ganglioside mixture lacking GM1 had no effect. Cholera toxin B subunit, a specific GM1-binding ligand, inhibited adenylyl cyclase. When the concentration of exogenous GM1 in which the cells were preincubated was increased from nmolar to molar levels there was a dose-responsive fall off in cAMP elevation, attributed to progressive inhibition of adenylyl cyclase by increasing GM1. These results are interpreted as indicating modulation of this PGE1 receptor in Neuro-2a cells by plasma membrane-localized GM1 in a structure-specific manner.Abbreviations PGE1 prostaglandin E1 - Ctx B B subunit of cholera toxin - BBG bovine brain ganglioside mixture - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - IBMX 3-isobutyl-1-methylxanthine - N'ase neuraminidase - D-PBS Dulbecco's phosphate-buffered saline