RESUMEN
In this paper, we describe the effects of the expression of GM3 synthase at high levels in human ovarian carcinoma cells. Overexpression of GM3 synthase in A2780 cells consistently resulted in elevated ganglioside (GM3, GM2 and GD1a) levels. GM3 synthase overexpressing cells had a growth rate similar to wild-type cells, but showed a strongly reduced in vitro cell motility accompanied by reduced levels of the epithelial-mesenchymal transition marker alpha smooth muscle actin. A similar reduction in cell motility was observed upon treatment with exogenous GM3, GM2, and GM1, but not with GD1a. A photolabeling experiment using radioactive and photoactivable GM3 highlighted several proteins directly interacting with GM3. Among those, caveolin-1 was identified as a GM3-interacting protein in GM3 synthase overexpressing cells. Remarkably, caveolin-1 was markedly upregulated in GM3 synthase overexpressing cells. In addition, the motility of low GM3 synthase expressing cells was also reduced in the presence of a Src kinase inhibitor; on the other hand, higher levels of the inactive form of c-Src were detected in GM3 synthase overexpressing cells, associated with a ganglioside- and caveolin-rich detergent insoluble fraction.
Asunto(s)
Carcinoma/enzimología , Caveolina 1/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/enzimología , Sialiltransferasas/biosíntesis , Actinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Cartilla de ADN/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Esfingolípidos/química , Familia-src Quinasas/metabolismoRESUMEN
Niemann-Pick disease (NPD) type A is a neurodegenerative disorder caused by sphingomyelin (SM) accumulation in lysosomes relying on reduced or absent acid sphingomyelinase (ASM) activity. NPD-A patients develop progressive neurodegeneration including cerebral and cerebellar atrophy, relevant Purkinje cell and myelin deficiency with death within 3 years. ASM'knock-out' (ASMKO) mice, an animal model of NPD-A, develop a phenotype largely mimicking that of NPD-A. The mechanisms underlying myelin formation are poorly documented in ASMKO mice. In this study we determined the content of four myelin-specific proteins, myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin associated glycoprotein (MAG) and proteolipid protein (PLP), and that of myelin-enriched sphingolipids in the brains of ASMKO and wild-type mice in early stages of post-natal (pn) life. Protein and mRNA analysis revealed that in ASMKO mice beginning from 4 post-natal weeks (wk-pn), the expression levels of MAG, CNP, and MBP were below those observed in wild-type mice and the same applied to PLP at 10 wk-pn. Moreover, at 4 wk-pn the expression of SOX10, one of the transcription factors involved in oligodendrocyte development and maintenance was lower in ASMKO mice. Lipid analysis showed that SM and the gangliosides GM3 and GM2 accumulated in the brains of ASMKO mice, as opposed to galactocerebroside and galactosulfocerebroside that, in parallel with the mRNAs of UDP-galactose ceramide galactosyltransferase and galactose-3-O-sulfotransferase 1, the two transferases involved in their synthesis, decreased. Myelin lipid analysis showed a progressive sphingomyelin accumulation in ASMKO mice; noteworthy, of the two sphingomyelin species known to be resolved by TLC, only that with the lower Rf accumulated. The immunohistochemical analysis showed that the reduced expression of myelin specific proteins in ASMKO mice at 10 wk-pn was not restricted to the Purkinje layer of the cerebellar cortex but involved the cerebral cortex as well. In conclusion, reduced oligodendrocyte metabolic activity is likely to be the chief cause of myelin deficiency in ASMKO mice, thus shedding light on the molecular dysfunctions underlying neurodegeneration in NPD-A.
Asunto(s)
Encéfalo/metabolismo , Proteínas de la Mielina/metabolismo , Enfermedad de Niemann-Pick Tipo A/metabolismo , Factores de Transcripción SOXE/deficiencia , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/deficiencia , Animales , Encéfalo/enzimología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Proteínas de la Mielina/genética , Enfermedad de Niemann-Pick Tipo A/genética , Factores de Transcripción SOXE/biosíntesis , Factores de Transcripción SOXE/genética , Esfingolípidos/genética , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
Thin layer chromatography is the easiest way to analyze the total glycosphingolipid mixtures extracted, and, in some cases, partially purified from tissues and cultured cells. Several solvent systems have been introduced to separate the complex mixtures as a function of their composition, presence of contaminants and, in some cases, of their quantity. In addition, colorimetric, enzymatic, immunological and radiochemical detection procedures are available for their recognition. The method does not allow to determine the chemical structure of separated molecules, but gives a very economical and very accessible first information on their possible structure on the basis of their chromatographic mobility in comparison with standards, and of their reactivity to the staining procedures. In this paper we show how to perform mono and two-dimensional thin layer chromatography of the total lipid mixture extracted from mouse brains and, in a few cases, from cells in culture. Table 1 shows the structures of reported lipids.
Asunto(s)
Cromatografía en Capa Delgada/métodos , Gangliósidos/análisis , Animales , Secuencia de Carbohidratos , Línea Celular Tumoral , Ceramidas/análisis , Ceramidas/química , Gangliósidos/química , Humanos , Ratones , Datos de Secuencia Molecular , Ratas , Solventes , Sulfoglicoesfingolípidos/análisisRESUMEN
The cholesterol, sphingolipid, and glycerophospholipid content of total brain, of detergent-resistant membranes prepared from the total brain, and of cerebellar granule cells differentiated in culture from wild type (WT) and acid sphingomyelinase knockout (ASMKO) were studied. Brains derived from 7-month-old ASMKO animals showed a fivefold higher level of sphingomyelin and a significant increase in ganglioside content, mainly because of monosialogangliosides GM3 and GM2 accumulation, while the cholesterol and glycerophospholipid content was unchanged with respect to WT animals. An increase in sphingomyelin, but not in gangliosides, was also detected in cultured cerebellar granule neurons from ASMKO mice, indicating that ganglioside accumulation is not a direct consequence of the enzyme defect. When a detergent-resistant membrane fraction was prepared from ASMKO brains, we observed that a higher detergent-to-protein ratio was needed than in WT animals. This likely reflects a reduced fluidity in restricted membrane areas because of a higher enrichment in sphingolipids in the case of ASMKO brain.
Asunto(s)
Encéfalo/metabolismo , Metabolismo de los Lípidos/genética , Neuronas/metabolismo , Esfingomielina Fosfodiesterasa/deficiencia , Secuencias de Aminoácidos/genética , Animales , Animales Recién Nacidos , Encéfalo/anatomía & histología , Células Cultivadas , Cerebelo/citología , Detergentes/farmacología , Gangliósidos/metabolismo , Lípidos de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Esfingosina/metabolismo , Tritio/metabolismoRESUMEN
Sphingolipids are polar membrane lipids present as minor components in eukaryotic cell membranes. Sphingolipids are highly enriched in nervous cells, where they exert important biological functions. They deeply affect the structural and geometrical properties and the lateral order of cellular membranes, modulate the function of several membrane-associated proteins, and give rise to important intra- and extracellular lipid mediators. Sphingolipid metabolism is regulated along the differentiation and development of the nervous system, and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system: sphingolipids in the nervous system participate to several signaling pathways controlling neuronal survival, migration, and differentiation, responsiveness to trophic factors, synaptic stability and synaptic transmission, and neuron-glia interactions, including the formation and stability of central and peripheral myelin. In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal sphingolipid patterns and altered membrane organization that participate to several events related to the pathogenesis of these diseases. The most impressive consequence of this deregulation is represented by anomalous sphingolipid-protein interactions that are at least, in part, responsible for the misfolding events that cause the fibrillogenic and amyloidogenic processing of disease-specific protein isoforms, such as amyloid beta peptide in Alzheimer's disease, huntingtin in Huntington's disease, alpha-synuclein in Parkinson's disease, and prions in transmissible encephalopathies. Targeting sphingolipid metabolism represents today an underexploited but realistic opportunity to design novel therapeutic strategies for the intervention in these diseases.
Asunto(s)
Membrana Celular/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Esfingolípidos/metabolismo , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Membrana Celular/química , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Vaina de Mielina/química , Vaina de Mielina/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades por Prión/patología , Enfermedades por Prión/fisiopatología , Esfingolipidosis/patología , Esfingolipidosis/fisiopatología , Esfingolípidos/químicaRESUMEN
Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of beta-galactosidase and beta-glucosidase. To determine the presence of beta-galactosidase and beta-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.