Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton

Gangliosides have been implicated in exerting multiple physiological functions, and it is important to understand how their distribution is regulated in the cell membrane. By using freeze-fracture immunolabeling electron microscopy, we showed that GM1 and GM3 make independent clusters that are significantly reduced by cholesterol depletion. In the present study, we examined the effects of actin depolymerization/polymerization and Src-family kinase inhibition on the GM1 and GM3 clusters. Both GM1 and GM3 clustering was reduced when the actin cytoskeleton was perturbed by latrunculin A or jasplakinolide, but the decrease was less significant than that induced by cholesterol depletion. On the other hand, inhibition of Src-family kinases decreased GM3 clustering more drastically than did cholesterol depletion, whereas its effect on GM1 clustering was less significant. GM1 and GM3 were segregated from each other in unperturbed cells, but co-clustering increased significantly after actin depolymerization. Our results indicate that the GM1 and GM3 clusters in the cell membrane are regulated in different ways and that segregation of the two gangliosides depends on the intact actin cytoskeleton.     

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.     

Cholesterol alters the interaction of glycosphingolipid GM3 with alpha5beta1 integrin and increases integrin-mediated cell adhesion to fibronectin

Integrins bind to their ligand in the extracellular matrix (ECM), such as fibronectin (FN), through a specific interaction between the amino acid motifs in the ligand, and binding sites in the extracellular domains of the integrin molecule generated jointly by its alpha and beta subunits. It has been proposed that membrane cholesterol and glycosphingolipids (GSLs) can regulate integrin-ECM interactions and it has been demonstrated that increased membrane cholesterol leads to increased cell adhesion to FN. Here, we have shown that a specific glycosphingolipid GM3 binds directly to alpha5beta1 integrin and an increase in membrane cholesterol results in the redistribution of GM3-associated alpha5beta1 integrin molecules specifically on the surface that is in contact with the substratum. Our results suggest that GM3-associated alpha5beta1 integrins bind less avidly to FN than GM3-free integrins and that cholesterol and GM3 play an interdependent role in the distribution of alpha5beta1integrin molecules in the membrane and regulation of cell adhesion.     

Ganglioside GM3 inhibits hepatoma cell motility via down‐regulating activity of EGFR and PI3K/AKT signaling pathway

Two related sublines derived from murine ascites hepatoma cell lines Hca‐F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3‐suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. J. Cell. Biochem. 114: 1616–1624, 2013. © 2013 Wiley Periodicals, Inc.     

Cytokine Stimulation Promotes Increased Glucose Uptake Via Translocation at the Plasma Membrane of GLUT1 in HEK293 Cells

Interleukin‐3 (IL‐3) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) are two of the best‐characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL‐3 and GM‐CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL‐3 and GM‐CSF are present in several non‐hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL‐3 and GM‐CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL‐3 and GM‐CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3‐kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non‐insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non‐hematopoietic cells. J. Cell. Biochem. 110: 1471–1480, 2010. © 2010 Wiley‐Liss, Inc.     

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4–5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56^lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56^lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56^lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56^lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56^lck, we analyzed this association in U937, a CD4+and p56^lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56^lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.     

Glycosphingolipid Domains on Cell Plasma Membrane

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.     

Lyso-GM3, its dimer,and multimer: their synthesis,and their effect on epidermal growth factor-induced receptor tyrosine kinase

Glycosphingolipids, particularly gangliosides, are known to modulate growth factor receptor tyrosine kinase. A well-documented example is the inhibitory effect of GM3 on kinase associated with epidermal growth factor receptor (EGFR) in human epidermoid carcinoma A431 cells. Lyso-GM3 was detected as a minor component in A431 cells, and may function as an auxiliary factor in GM3-dependent inhibition of EGFR. We studied the inhibitory effect of chemically synthesized GM3, lyso-GM3, and its derivatives, on EGFR function, based on their interaction in membrane microdomain, with the following major findings: (1) GM3, EGFR, and caveolin coexist, but tetraspanins CD9 and CD82 are essentially absent, within the same low-density membrane fraction, separated by sucrose density gradient ultracentrifugation. (2) Strong interaction between EGFR and GM3 was indicated by increasing binding of EGFR to GM3-coated polystyrene beads, in a GM3 dose-dependent manner. Confocal microscopy results suggested that three components in the microdomain (GM3, EGFR, and caveolin) are closely associated. (3) Lyso-GM3 or lyso-GM3 dimer strongly inhibited EGFR kinase activity, in a dose-dependent manner, while lyso-GM3 trimer and tetramer did not. >50 μM lyso-GM3 was cytolytic, while >50 μM lyso-GM3 dimer was not cytolytic, yet inhibited EGFR kinase strongly. Thus, lyso-GM3 and its dimer exert an auxiliary effect on GM3-induced inhibition of EGFR kinase and cell growth, and lyso-GM3 dimer may be a good candidate for pharmacological inhibitor of epidermal tumor growth.     

Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as “glycosignaling domains.” In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Binding affinity of GM3 lactone for influenza virus

We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.     

Association of Cellular Prion Protein with Gangliosides in Plasma Membrane Microdomains of Neural and Lymphocytic Cells

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrP^c presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrP^c and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrP^c-enriched microdomains. In lymphoblastoid T cells PrP^c molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrP^c was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrP^c-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrP^c immunoprecipitates. The physical association of PrP^c with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrP^c-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.     

Antigenicity of the carbohydrate moiety of ganglioside GM3 having 3-O-acetyl ceramide

To elucidate the effect of a modification of ceramide on antigenicity of the carbohydrate of ganglioside, the reactivity of O-acetyl GM3 having 3-O-acetyl ceramide, which has been characterized as a gliomarelated ganglioside, with monoclonal antibody M2590 was examined in comparison to that of non-acetylated GM3, by means of quantitative enzyme-linked immunosorbent assay, TLC-immunostaining and liposome immune lysis assay. In all these assay systems, O-acetyl GM3 showed less activity than GM3 as follows: GM3 was detected till 0.1 nmol in TLC-immunostaining, whereas O-acetyl GM3 could not be detected even at 0.25 nmol; the GM3 reaction was approximately twofold that of O-acetyl GM3 at each diluted point in the enzyme-linked immunosorbent assay; and 20% of the liposomes containing GM3 were lysed at 6 mol%, while liposomes containing O-acetyl GM3 did not lyse at that concentration. The lesser antigenicity of the sugar moiety of O-acetyl GM3 could be ascribed to the presence of an acetyl group in the ceramide at the 3-position of sphingosine.     

Activation of ganglioside GM3 biosynthesis in human monocyte/macrophages during culturing <Emphasis Type="Italic">in vitro</Emphasis>

We found that GM3 levels in human peripheral blood monocytes and cultured monocyte-derived macrophages were 0.37 and 2.7 μg per million cells, respectively. GM3 synthase of monocytes and to a greater extent of monocyte-derived macrophages was shown to be able to sialylate endogenous substrate, lactosylceramide (LacCer), to form GM3. With exogenously added LacCer, GM3 synthase activity was 57.1 and 563 pmol/h per mg protein in monocytes and monocyte-derived macrophages, respectively. The revealed changes in ganglioside GM3 biosynthesis are specific as the activity of some other sialyltransferases under these conditions was not altered. Human anti-GM3 synthase antibody detected in monocytes a main protein with molecular weight of 60 kD and minor proteins with molecular masses of 52 and 64 kD. In monocyte-derived macrophages the amounts of 60 kD protein and especially 64 kD protein sharply rose. Thus, the increase in ganglioside GM3 levels, GM3 synthase activity, and the enzyme amounts during culturing of monocyte/macrophages may be one of the mechanisms of in vivo increased ganglioside GM3 levels in arterial atherosclerotic lesions. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 7, pp. 948–954.     

Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells

Abstract: Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12-amino acid sequence located in the NH₂-terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca²⁺-independent and not disassociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The association of prosaposin-GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22-mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide-induced neurite outgrowth, as well as prosaptide-enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein-mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.     

神经节苷脂类抑制BT325细胞系的生长

应用自提的神经节苷脂GM3, 牛脑神经节苷脂(bovine brain gangliosides, BBG)加入低血清培养的人多形成胶质细胞瘤BT325细胞中观察其对该细胞系的影响; 以及GM3、BBG对表皮生长因子(EGF)刺激该细胞系生长的拮抗作用. 结果表明GM3、BBG均能抑制BT325细胞生长,GM3的抑制作用远高于BBG, 其抑制率为60.28% (P＜0.01), BBG为19.33％,在含不同浓度的EGF培养液中分别加入GM3、BBG均可拮抗EGF对BT325生长的刺激作用, GM3的拮抗作用远高于BBG.     

神经节苷脂GM_3对人白血病J6-2细胞PKC转位及DAG含量的影响

应用SDS PAGE及Western印迹技术检测神经节苷脂GM3 处理前后人白血病J6 2细胞不同类型PKC在细胞内的转位情况 ,同时利用高效薄板层析技术观察了细胞内DAG含量的变化 ,从而探讨GM3 抑制PKC活性的机制 .实验发现 ,GM3 处理后胞液PKCα明显增加 ,而颗粒结合PKCα则相对减少 ;GM3 对其它亚型PKC在细胞内分布无显著影响 .同时还发现 ,GM3 处理后细胞内DAG含量降低 (P <0 0 5 ) .结果表明 ,GM3 抑制PKCα由胞浆向质膜转位 ,对其它亚型PKC在细胞内转位无影响 .提示GM3 抑制的PKC亚型可能是PKCα .同时GM3 降低细胞内DAG含量 ,这可能与GM3抑制PKCα活性机制有关     

Development of Antibody to Human GM3 Synthase and Immunodetection of the Enzyme in Human Tissues

Polyclonal antibody was raised to a cloned fragment of human GM3 synthase. Affinity purified R27C1 antibody to the tagged recombinant protein inhibited GM3 synthase activity in human liver and HL-60 cells in a dose-dependent manner. However, the R27C1 antibody did not affect liver sialyltransferase activity towards asialofetuin. We are the first to measure GM3 synthase activity in human liver (194 +/- 60 pmol NeuAc/h per mg protein), which was about 10-fold lower than in phorbol myristate acetate-stimulated HL-60 cells (1353 +/- 573 pmol NeuAc/h per mg protein). On immunoblotting the R27C1 antibody recognized a common protein band in a number of human tissues (liver, brain, atherosclerotic aortic intima, HL-60 cells) with molecular mass of about 60 kD, which is similar to that of the purified GM3 synthase from rat liver. In human liver and aortic intima, the 60-kD band was almost a single band, which makes possible the use of the R27C1 antibody for immunohistochemical studies in these tissues.     

Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells

Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cells in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.