Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey

Vertebrate lens tissues contain several species of acidic andneutral glycosphingolipids in relatively high amounts. However,the epithelia with capsule from dog and rhesus monkey lenseshad a simpler composition and lower content of glycosphingolipidsthan whole lenses. Gangliosides and neutral glycosphingolipidsin monolayer cultures of lens epithelial cells were also differentfrom those in whole lenses. Although -galactosyl (Gal1-3Ga1-R)or Lewis^x (Galß1-4[Fuc1-3]GlcNAc-R) epitopes werefound in glycosphingolipids from whole lenses, they were notdetected in those from monolayer cultures of dog and rhesusmonkey lens cells. In addition, significant changes in ganglio-seriesgangliosides were induced in monolayer cultures of both cells,where GM3 and GD3 were predominant. Immunofluorescence studyrevealed a characteristic distribution of cell surface gangliosidesin confluent monolayers. These findings suggest that glycosphingolipidsynthesis in lens epithelia is intrinsically different fromthat in cortical and nuclear fibres, and that the expressionof Lewis^x and -galactosyl epitopes in glycosphingolipids appearsto be associated with the differentiation of epithelial cellsto fibres. gangliosides glycosphingolipids lens epithelial cells Lewis^x rhesus monkey.     

Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

Characterization of acidic glycolipids in porcine plasma

Acidic glycosphingolipids including two sulfatides and five gangliosides were isolated from porcine plasma. They were characterized by NMR spectrometry as galactosylceramide-I³-sulfate and lactosylceramide-II³-sulfate, gangliosides GM3, GD3, GM1, GD1a and fucosyl GM1.     

Cortical distribution of gangliosides in Alzheimer's disease.

In Alzheimer's disease, all ganglio-series gangliosides (GM1, GD1a, GD1b and GT1b) were found to be decreased in temporal and frontal cortex, and nucleus basalis of Meynert. In addition, in Alzheimer's disease simple gangliosides (GM2, GM3) were elevated in frontal and parietal cortex, possibly correlating to accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.      

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.     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Glycosphingolipids from human T-lymphoma MOLT-4 cells

The glycosphingolipids of human lymphoma MOLT-4 cells were studied, using biochemical methods and specific antisera to gangliosides. The major neutral glycosphingolipids were found to be glucosyl- and lactosyl ceramides. GM3, GM2, GM1 and GD1a were identified as ganglioside components.     

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Neutral glycosphingolipids and gangliosides were isolated from3.7 x 10⁹ primary bovine aortic endothelial cells and structurallycharacterized by immunological and chemical methods. Glucosyl-and lactosylceramide were detected as the main neutral glycosphingolipids(28% and 40% of total orcinol stain, respectively); LcOse₃Cerand nLcOse₄Cer were expressed to somewhat minor amounts (16%and 10% of total orcinol stain, respectively), and nLcOse₆Ceroccurred only in trace quantities. No neutral glycosphingolipidsof the ganglio-series (GgOse₃Cer and GgOse₄Cer) and the globo-series(GbOse₄Cer and the Forssman antigen) have been detected; onlytraces of GbOse₃Cer were identified by TLC immunostaining. PositiveCD15 bands obtained by TLC overlay with anti-Galβ1–4(Fucl-3)GlcNAcβ1-Rantibody indicated the presence of lipid bound Lewis^x antigen,whereas the isomeric Lewis^a structure (Galβ1–3(Fuc1–4)GlcNAcβ1-R)was not detectable. G_M3 substituted with Neu5Gc and Neu5Ac ina 2:1 ratio was the major ganglioside comprising about 95% withinthe whole ganglioside fraction. G_M3-structures were furthercharacterized by FAB-MS and GC-MS of the native compounds andtheir permethylated derivatives. C₁₈-sphingosine was the onlylong chain base, whereas variation occurred due to C_24:0,24:1and C₁₆ fatty acids. Terminally 2–3 sialylated neolacto-seriesgangliosides with nL-cOse₄- and nLcOse₆Cer (<5% of totalresordnol stain) were found in almost equal quantities, whereasno 2–6 sialylated counterparts were detected. Fucosylatedgangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis^x,sialyl Lewis^a, and VIM-2 antigen) and sulfoglucuronyl-neolactoseries structures with HNK-1 epitope were not detectable inthe acidic glycosphingoiipid fraction by TLC immunostaining.Gangliotetraose-type gangliosides G_M1 and G_D1a (<1 % of totalresorcinol stain) as well as traces of G_D1b and G_T1b have beendistinctly identified by combined choleragenoid-TLC-immunostainingand previous neur-aminidase treatment.The expression of dominantglycosphingolipids lactosylceramide and G_M3(Neu5Gc) was provedby indirect immunofiuorescence microscopy of cell layers grownin chamber slides, each showing different plasma membrane andsubcellular distribution patterns. The results provide the basisfor investigation of the role of glycosphingolipids as cellsurface antigens of cellular interaction as well as receptorsfor blood components and mac-romolecules of the extracellularmatrix. gangliosides neutral glycosphingolipids antibodies Lewis^x antigen TLC immunostaining     

Gangliosides modulate sodium transport in cultured toad kidney epithelia

Cultured A6 epithelial cells from toad kidney form confluent monolayers with tight junctions separating the apical and basolateral membranes. These two membrane domains have distinct compositions and functions. Thus, sodium is actively transported across the epithelia from the apical to basolateral surface via amiloride-inhibitable sodium channels located in the apical membrane. Sodium transport is stimulated by vasopressin, cholera toxin, and 8-bromo-cAMP applied to the basolateral surface where the receptors, adenylate cyclase, and Na+/K+-ATPase are located. In a previous study (Spiegel, S., Blumenthal, R., Fishman, P.H., and Handler, J.S. (1985) Biochim. Biophys. Acta 821, 310-318), we demonstrated that exogenous gangliosides inserted into the apical membrane of A6 epithelia do not redistribute to the basolateral membrane. With the ability to vary selectively the ganglioside composition of the apical membrane, we examined the effects of gangliosides on sodium transport in A6 epithelia. When the apical surface of A6 epithelia were exposed to exogenous gangliosides, sodium transport in response to vasopressin, cholera toxin, and 8-bromo-cAMP was enhanced compared to epithelia not exposed to gangliosides. The effect was observed with bovine brain gangliosides, NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GD1a) and Gal beta-1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GM1), but not with the less complex ganglioside, Neu-Ac alpha 2----3Gal beta 1----4Glc beta 1----Cer (GM3). We examined A6 cells for endogenous gangliosides and found that, whereas GM3 was a major ganglioside, only trace amounts of GM1 and GD1a were present. Based on cell surface and metabolic labeling studies, these gangliosides were synthesized by the cells and were present on the apical as well as the basolateral surface. Bacterial sialidase, which hydrolyzes more complex gangliosides to GM1, was used to modify the endogenous gangliosides on the apical surface; after sialidase treatment, the epithelia were more responsive to vasopressin, cholera toxin, and 8-bromo-cAMP. Thus, gangliosides may be modulators of sodium channels present in the apical membrane of epithelial cells.     

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

Specific gangliosides increase rapidly in rat liver following partial hepatectomy

Rat liver gangliosides (sialic acid containing glycosphingolipids) were analyzed by HPTLC and HPLC following either partial hepatectomy or sham operation. Analysis of whole liver gangliosides by HPTLC demonstrated that within 6 h after partial (68%) hepatectomy, there was a significant increase in GM1 compared to both sham and control animals. By 48 h, GM1 was further increased and the polysialylgangliosides GD1a, GD1b and GT1b had also risen significantly, whereas changes in GM3 were negligible. Gangliosides associated with the plasma membrane were increased up to 3.5-fold in regenerating liver compared to sham-hepatectomized controls as assessed by HPLC. Although elevations in membrane gangliosides were associated with hepatocyte proliferation, they did not closely follow the growth curve. The time course of changes in ganglioside biosynthesis suggests differential upregulation of GM3 synthase and GD3 synthase in regenerating livers.     

Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers.

The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.     

Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin

Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).     

Gangliosides GD1a and GM3 induce interleukin-10 production by human T cells

Gangliosides are sialic acid-containing glycosphingolipids and exhibit various physiologic functions. Gangliosides GD1a and GM3 strongly induced interleukin-10 (IL-10) protein secretion and mRNA expression in T cells from normal human subjects while the other gangliosides were ineffective. IL-10 induction by both gangliosides was completely blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A, genistein, and tyrphostin AG 1288, but not by other signal transduction inhibitors. These results suggest that GD1a and GM3 may induce IL-10 production in T cells by regulating the PTK-dependent signaling pathway. These gangliosides may thus act as important immunoregulators via IL-10.     

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I)

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform–methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.