Renal distribution of ganglioside GM3 in rat models of types 1 and 2 diabetes

Ganglioside GM3 is particularly abundant in the kidney tissue and is thought to play an important role in the maintenance of the charge-selective filtration barrier of glomeruli. Altered expression of ganglioside GM3 was pathologically related with glomerular hypertrophy occurring in diabetic human and rat kidneys. Considering the role of GM3 ganglioside in kidney function, the aim of this study was to determine the difference in expression of GM3 ganglioside in glomeruli and tubules using immunofluorescence microscopy both in rat models of types 1 and 2 diabetes mellitus. Diabetes was induced with streptozotocin (55 mg/kg for type 1 diabetes and 35 mg/kg for type 2 diabetes) injection to male Sprague–Dawley rats which were fed with normal pellet diet (type 1 diabetes) or high-fat diet (type 2 diabetes). Rats were sacrificed 2 weeks after diabetes induction, frozen renal sections were stained with primary antibody GM3(Neu5Ac) and visualized by secondary antibody coupled with Texas red. In addition, renal gangliosides GM3 were analyzed by high-performance thin-layer chromatography followed by GM3 immunostaining. Immunofluorescent microscopy detected 1.7-fold higher GM3 expression in tubules and 1.25-fold higher GM3 in glomeruli of type 1 diabetes mellitus compared with control group. Type 2 diabetes mellitus rats showed slight GM3 increase in whole kidney, unchanged GM3 in glomeruli, but significant higher GM3 expression in tubules, compared with control animals. Taking into consideration increased tubular GM3 content in both types of diabetes, we could hypothesize the role of GM3 in early pathogenesis of diabetic nephropathy.     

GM2/GM3 controls the organizational status of CD82/Met microdomains: further studies in GM2/GM3 complexation

Glycoconjugate Journal - At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing...     

Secretary sialidase activity and GM3 ganglioside

The sialidase activities with GM3 ganglioside and sialyllactitol were demonstrated in the conditioned medium of human fibroblasts. pH versus activity profiles of conditioned medium with GM3 as substrate suggested the presence of two sialidases with optimal activities at pH 4.5 and pH 6.5. The GM3 sialidase activity at pH 6.5 was suppressed in the medium of contact-inhibited cells. This sialidase may function in the metabolism of cell surface GM3 since there was a selective loss of labeled sialic acid from GM3 at different times of incubation after pulse-labeling with a radioactive sialic acid precursor ([3H]N-acetyl-mannosamine) and a radioactive ceramide precursor ([14C]serine). In addition, a sialidase inhibitor, 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (NeuAc-2-en) resulted in a reversible growth inhibitory effect and the suppression of the sialidase activity in the medium. We have speculated that GM3 hydrolysis on the cell surface by the sialidase may be coordinated with the cell cycle and may be at its maximum during early in the G1 phase.     

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.     

Distribution of gangliosides, GM1 and GM3, in the rat oviduct

It is known that gangliosides, being ubiquitous membrane components, play important roles in cell-cell recognition, differentiation and transmembrane signalling. GM3, GM1 and GD1a were detected in the rat oviduct as major gangliosides by thin-layer chromatography (TLC) analysis. The total amounts of gangliosides from the oviducts at various times after hormone injection were not much changed. In order to identify their distribution and possible changes during ovulation, frozen sections of the rat oviducts were stained with specific monoclonal antibodies (MAbs) against the ganglio-series gangliosides. GM3 and GM1 were expressed in a different manner, but GD1a and other gangliosides were not immunohistochemically detected. In the ampullar region, GM3 was expressed in all the stroma and epithelial cells, but not GM1. GM1 was also not observed in epithelial cells. Staining by anti-GM1 monoclonal antibodies revealed long and minute thread-like structures in some of the stroma cells, whereas anti-GM3 monoclonal antibodies stained the entire cytoplasm, but not the nucleus, of all the stroma and epithelial cells. Other ganglio-series gangliosides, including GD1a, were not detected to some extent in the ampullar region by immunohistochemistry. Thus, these data suggest that GM3 and GM1 are oviduct-specific gangliosides.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Gangliosides GM3 and GD3 in human stomach and breast tumors

Gangliosides of human gastric and mammary tumours and of homologous normal tissues were studied by using biochemical methods and specific antisera. It was found that in most cases GM3, GD3 and GM1 are predominant gangliosides, whereas several polar components are minor ones. A comparison of the relative amount of ganglioside fractions revealed that in gastric tumours the per cent content of polar compounds is higher than in intact tissue; however, the absolute content of all gangliosides is markedly increased. A comparative study of the composition of mammary tumour and normal tissue gangliosides demonstrated two types of changes: i) the absolute content of all gangliosides in tumour tissue was increased and, ii) the increase in the content of total gangliosides was paralleled with the appearance of a new fraction (presumably GM4), the decrease of the GD3 content and the disappearance of polar gangliosides. A possible mechanism of this effect is discussed.     

A procedure for the preparation of GM3 ganglioside from GM1-lactone

A simple procedure is described for preparing GM3 ganglioside, from a few milligrams to grams, from GM1-lactone (Sonnino et al., (1985) Glycoconjugate J 2: 343-54) [1]. The synthesis was carried out under the following optimal conditions: 30 mM GM1-lactone in 0.25 M H2SO4 in DMSO, 30 min, 70 degrees C, nitrogen atmosphere, strong stirring. The yield of GM3 was 55%. The procedure applied to milligram amounts of GD1b-dilactone gave GD3 ganglioside.     

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with ¹³C-¹H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all ¹H and ¹³C chemical shifts is presented.     

Anti-GM3-lactam monoclonal antibodies of the IgG type recognize natural GM3-ganglioside lactone but not GM3-ganglioside

Immunization of mice with a synthetic GM₃-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM₃-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM₃-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM₃-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG₁, ) reacted with GM₃-ganglioside lactone. Binding of these two antibodies to the GM₃-lactam-BSA conjugate was inhibited by soluble glycosides of GM₂-, GM₃-, and GM₄-lactam and by GM₃- and GM₄-lactam, respectively, but not by Gb₃ or asialo-GM₁ and GM₂-saccharides. A third antibody (P3; IgG_2b, ) was inhibited by GM₂-, GM₃-, and GM₄-lactam, but did not recognize GM₃-ganglioside lactone.     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.     

The total synthesis of ganglioside GM3

Previous syntheses of ganglioside GM3 (NeuAc alpha3Gal beta4Glc beta1Cer) are reviewed, and both chemoenzymatic and chemical total synthetic approaches were investigated. In a chemoenzymatic approach, (2S,3R,4E)-5'-acetyl-alpha-neuraminyl-(2' --> 3')-beta-galactopyranosyl-(1' --> 4')-beta-glucopyranosyl-(1' <--> 1)-2-azido-4-octadecene-1,3-diol (azidoGM3) was readily prepared utilizing recombinant beta-Gal-(1' --> 3'/4')-GlcNAc alpha-(2' --> 3')-sialyltransferase enzyme, and was evaluated as a synthetic intermediate to ganglioside GM3. The chemical total synthesis of ganglioside GM3 was performed on one of the largest scales yet reported. The highlights of this synthesis include minimizing the steps necessary to prepare the lactosyl acceptor as a useful anomeric mixture, which was present in excess for the highly regioselective and fairly stereoselective sialylation with a known neuraminyl donor to give the protected GM3 trisaccharide. The synthetic methodology maximized convergence by a subsequent glycosidic coupling of the well-characterized GM3 trisaccharide trichloroacetimidate derivative with protected ceramide. The ganglioside GM3 was nearly homogeneous as the two glycosidic couplings utilized preparative HLPC purifications, and variations in the sphingosine base and fatty acyl group were under 0.1 and 0.2%, respectively.     

Change in the topographical distribution of GM3 during cell spreading and growth: immunostaining with monoclonal antibody against GM3

A monoclonal antibody, M2590, that recognizes hematoside (GM3) was used to analyze the immunostaining localization of GM3 of the surface of transformed and non-transformed hamster embryo fibroblasts and B16 melanoma cells. The reactivity of GM3 with the antibody changed markedly depending on the cell density. At the sparse density cells were clearly made visible by the antibody, but at the confluency the accessibility of the antibody to GM3 was greatly decreased. This density dependent change in the reactivity of GM3 was found for both normal and transformed cells. The staining pattern of GM3 was examined in relation to the actin fibers made visible with NBD-Phallacidin during cell spreading. When the cells were still round, the GM3 on microspikes or blebs was highly reactive with the antibody, and by the time cells showed circumferential staining of their actin fibers, GM3 had been distributed over the entire cell surface as punctuated spots. GM3 also was visible in substrate attachment materials (SAM). Trypsin treatment of SAM diminished the reactivity of GM3 with the antibody. The antibody did not inhibit cell attachment or spreading on a substratum coated with fibronectin or laminin.     

Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.     

GM1b and GM1b-GalNAc: major gangliosides of murine-derived macrophage-like WEHI-3 cells.

WEHI-3 cells, derived from a BALB/c mouse, are a myelomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas-liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%. The two major components were identified as (NeuAc)GM1b and (NeuAc)GM1b-GalNAc and the minor component as (NeuAc)GM1b-GalNAc-Gal. The presence of GM1b in this myelomonocytic cell line is consistent with its presence in other murine immune cells and tissues. GM1b-GalNAc and GM1b-GalNAc-Gal have been reported in T-lineage cells but not in resident or stimulated murine macrophages. Each of these monosialogangliosides belongs to the asialoGM1 synthetic pathway. Preliminary results indicate a disialo member of this pathway, GDlc, may also be present as a minor component. This ganglioside pathway, containing species which are not sialylated on the internal galactose, appears to be dominant in and may be characteristic of murine immune cells.     

GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells

The apical domain of epithelial cells is composed of distinct subdomains such as microvilli, primary cilia and a non-protruding region. Using the cholesterol-binding protein prominin-1 as a specific marker of plasma membrane protrusions we have previously proposed the co-existence of different cholesterol-based lipid microdomains (lipid rafts) within the apical domain [R?per, K., Corbeil, D. and Huttner, W.B. (2000), Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582-592]. To substantiate the hypothesis that the microvillar plasma membrane subdomains contain a distinct set of lipids compared to the planar portion we have investigated the distribution of prominin-1 and two raft-associated gangliosides GM(1) and GM(3) by fluorescence microscopy. GM(1) was found to co-localize with prominin-1 on microvilli whereas GM(3) was segregated from there suggesting its localization in the planar region. Regarding the primary cilium, overlapping fluorescent signals of GM(1) or GM(3) and prominin-1 were observed. Thus, our data demonstrate that specific ganglioside-enriched rafts are found in different apical subdomains and reveal that two plasma membrane protrusions with different structural bases (actin for the microvillus and tubulin for the cilium) are composed of distinct types of lipid.     

Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR-2

Angiogenesis is associated with growth, invasion, and metastasis of human solid tumors. Aberrant activation of endothelial cells and induction of microvascular permeability by a vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) signaling pathway is observed in pathological angiogenesis including tumor, wound healing, arthritis, psoriasis, diabetic retinopathy, and others. Here, we show that GM3 regulated the activity of various downstream signaling pathways and biological events through the inhibition of VEGF-stimulated VEGFR-2 activation in vascular endothelial cells in vitro. Furthermore, GM3 strongly blocked VEGF-induced neovascularization in vivo, in models including the chick chorioallantoic membrane and Matrigel plug assay. Interestingly, GM3 suppressed VEGF-induced VEGFR-2 activation by blocking its dimerization and also blocked the binding of VEGF to VEGFR-2 through a GM3-specific interaction with the extracellular domain of VEGFR-2, but not with VEGF. Primary tumor growth in mice was inhibited by subcutaneous injection of GM3. Immunohistochemical analyses showed GM3 inhibition of angiogenesis and tumor cell proliferation. GM3 also resulted in the suppression of VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 inhibits VEGFR-2-mediated changes in vascular endothelial cell function and angiogenesis, and might be of value in anti-angiogenic therapy.     

Compartmental Organization of the Synthesis of GM3, GD3, and GM2 in Golgi Membranes from Neural Retina Cells

The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [³H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[³H]Gal. Conversion of the synthesized [³H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [³H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.