Ionizing radiations increase the activity of the cell surface glycohydrolases and the plasma membrane ceramide content.

We detected significant levels of ß-glucosidase, ß-galactosidase, sialidase Neu3 and sphingomyelinase activities associated with the plasma membrane of fibroblasts from normal and Niemann-Pick subjects and of cells from breast, ovary, colon and neuroblastoma tumors in culture. All of the cells subjected to ionizing radiations showed an increase of the activity of plasma membrane ß-glucosidase, ß-galactosidase and sialidase Neu3, in addition of the well known increase of activity of plasma membrane sphingomyelinase, under similar conditions. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of ß-glucosidase and ß-galactosidase was parallel to the increase of irradiation dose up to 60 Gy and continued with time, at least up to 72 h from irradiation. ß-glucosidase increased up to 17 times and ß-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20 Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of plasma membrane ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the plasma membranes and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells.

Cell surface associated glycohydrolases in normal and Gaucher disease fibroblasts.

Gaucher disease (GD) is the most common lysosomal disorder and is caused by an inherited autosomal recessive deficiency in ß-glucocerebrosidase. This enzyme, like other glycohydrolases involved in glycosphingolipid (GSL) metabolism, is present in both plasma membrane (PM) and intracellular fractions. We analyzed the activities of CBE-sensitive ß-glucosidase (GBA1) and AMP-DNM-sensitive ß-glucosidase (GBA2) in total cell lysates and PM of human fibroblast cell lines from control (normal) subjects and from patients with GD clinical types 1, 2, and 3. GBA1 activities in both total lysate and PM of GD fibroblasts were low, and their relative percentages were similar to those of control cells. In contrast, GBA2 activities were higher in GD cells than in control cells, and the degree of increase differed among the three GD types. The increase of GBA2 enzyme activity was correlated with increased expression of GBA2 protein as evaluated by QRT-PCR. Activities of ß-galactosidase and ß-hexosaminidase in PM were significantly higher for GD cells than for control cells and also showed significant differences among the three GD types, suggesting the occurrence of cross-talk among the enzymes involved in GSL metabolism. Our findings indicate that the profiles of glycohydrolase activities in PM may provide a valuable tool to refine the classification of GD into distinct clinical types.

Plasma membrane-associated glycohydrolases along differentiation of murine neural stem cells.

The activities of plasma membrane associated sialidase Neu3, total ß-glucosidase, CBE-sensitive ß-glucosidase, non-lysosomal ß-glucosyl ceramidase GBA2, ß-galactosidase, ß-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive ß-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive ß-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.

Plasma membrane-associated glycohydrolases activation by extracellular acidification due to proton exchangers.

In this paper, we show that the pH optimum for the plasma membrane (PM)-associated activity of four glycohydrolases (conduritol B epoxide sensitive ß-glucosidase, ß-glucosidase GBA2, ß-hexosaminidase and ß-galactosidase) measured on intact cells is acidic. Moreover, we show that drugs able to modify the efflux of protons across the PM, thus locally affecting the extracellular proton concentration close to the PM, are able to modulate the activities of these enzymes. These data strongly suggest that pH-dependent modulation of PM-associated glycohydrolases activities could be an effective way to locally modulate the cell surface glycoconjugate composition.

A glycosphingolipid/caveolin-1 signaling complex inhibits motility of human ovarian carcinoma cells.

The genetic (stable overexpression of sialyltransferase I, GM3 synthase) or pharmacological (selective pressure by N-(4-hydroxyphenyl)retinamide)) manipulation of A2780 human ovarian cancer cells allowed us to obtain clones characterized by higher GM3 synthase activity compared with wild-type cells. Clones with high GM3 synthase expression had elevated ganglioside levels, reduced in vitro cell motility, and enhanced expression of the membrane adaptor protein caveolin-1 with respect to wild-type cells. In high GM3 synthase-expressing clones, both depletion of gangliosides by treatment with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and silencing of caveolin-1 by siRNA were able to strongly increase in vitro cell motility. The motility of wild-type, low GM3 synthase-expressing cells was reduced in the presence of a Src inhibitor, and treatment of these cells with exogenous gangliosides, able to reduce their in vitro motility, inactivated c-Src kinase. Conversely, ganglioside depletion by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol treatment or caveolin-1 silencing in high GM3 synthase-expressing cells led to c-Src kinase activation. In high GM3 synthase-expressing cells, caveolin-1 was associated with sphingolipids, integrin receptor subunits, p130(CAS), and c-Src forming a Triton X-100-insoluble noncaveolar signaling complex. These data suggest a role for gangliosides in regulating tumor cell motility by affecting the function of a signaling complex organized by caveolin-1, responsible for Src inactivation downstream to integrin receptors, and imply that GM3 synthase is a key target for the regulation of cell motility in human ovarian carcinoma.

Cell surface sphingolipid glycohydrolases in neuronal differentiation and aging in culture.

Qualitative and quantitative changes in glycosphingolipids, together with changes in the expression of the corresponding glycosyltransferases, have been reported along neuronal differentiation and aging. Plasma membrane (PM) glycosphingolipid pattern and content are the result of a complex network of metabolic pathways, including those potentially involving the activity of PM glycohydrolases. We analyzed the total cell activities of sialyltransferase I, II and IV, sialidase, ß-galactosidase and ß-glucosidase, and the PM-associated activities of sialidase Neu3, ß-galactosidase, Conduritol B Epoxide-sensitive ß-glucosidase and ß-glucosidase GBA2 in rat cerebellar granule cells along differentiation and aging in culture. Sialyltransferase activities increased during cell differentiation, in agreement with the known increase of the total ganglioside content during neuronal maturation. The remodeling of ganglioside pattern could be because of the augmented activities of total sialidase and, within PM, to the action of the cell surface associated sialidase Neu3. Sialidase activities remained high during aging, in agreement with the known progressive ganglioside reduction in brain senescence. As PM ß-galactosidase and ß-glucosidase activities and parallely ceramide levels markedly increased along in vitro aging, PM ceramide production in neurons might be because of local catabolism of glycosphingolipids and not only to that of sphingomyelin, as already reported in human fibroblasts.

Secondary alterations of sphingolipid metabolism in lysosomal storage diseases.

In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal membrane sphingolipid patterns and altered plasma membrane organization. In this paper, we review the potential importance of these alterations to the pathogenesis of these diseases and focus the reader's attention on some secondary alterations of sphingolipid metabolism that have been sporadically reported in the literature. Moreover, we present a detailed analysis of the lipid composition of different central nervous system and extraneural tissues from the acid sphingomyelinase-deficient mouse, the animal model for Niemann-Pick disease type A, characterized by the accumulation of sphingomyelin. Our data show an unexpected, tissue specific selection of the accumulated molecular species of sphingomyelin, and an accumulation of GM3 and GM2 gangliosides in both neural and extraneural tissues, that cannot be solely explained by the lack of acid sphingomyelinase.

Remodeling of sphingolipids by plasma membrane associated enzymes.

The sphingolipid plasma membrane content and pattern is the result of several processes, among which the main, in term of quantity, are: neo-biosynthesis in endoplasmic reticulum and Golgi apparatus, membrane turnover with final catabolism in lysosomes and membrane shedding. In addition to this, past and recent data suggest that the head group of sphingolipids can be opportunely modified at the plasma membrane level, probably inside specific membrane lipid domains, by the action of enzymes involved in the sphingolipids metabolism, working directly at the cell surface. The number of membrane enzymes, hydrolases and transferases, acting on membrane sphingolipids is growing very rapidly. In this report we describe some properties of these enzymes.

Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids.

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.

Deregulated sphingolipid metabolism and membrane organization in neurodegenerative disorders.

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.

GM3 synthase overexpression results in reduced cell motility and in caveolin-1 upregulation in human ovarian carcinoma cells.

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.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells.

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Fine tuning of cell functions through remodeling of glycosphingolipids by plasma membrane-associated glycohydrolases.

The plasma membrane (PM) sphingolipid composition is the result of a series of well-known metabolic pathways comprising neobiosynthesis in the endoplasmic reticulum and in the Golgi apparatus followed by vesicular delivery to the plasma membrane, membrane turnover with final catabolism in lysosomes, and shedding of membrane components. In addition to this, the head group of PM sphingolipids can be opportunely modified by the action of PM associated hydrolases and transferases. The number of enzymes for glycosphingolipid metabolism that have been shown to be associated with the plasma membrane and the information on their properties are growing very rapidly. In this review, we will focus on the possible role and on the involvement of the plasma membrane-associated glycohydrolases in modulating cell functions.

Activity of plasma membrane beta-galactosidase and beta-glucosidase.

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.

Brain lipid composition in grey-lethal mutant mouse characterized by severe malignant osteopetrosis.

The grey-lethal mouse (gl/gl) mutant most closely resembles the severe human malignant autosomal recessive OSTM1-dependent form of osteopetrosis that it has been described to be associated with neurological abnormalities. For this reason, we have analyzed the brain lipid composition (gangliosides, neutral glycosphingolipids, phospholipids and cholesterol), from gl/gl mice at different ages of development and compared with wild type mice. Both cholesterol and glycerophospholipid content and pattern in the gl/gl and control mice were very similar. In contrast, significant differences were observed in the content of several sphingolipids. Higher amount of the monosialogangliosides GM2 and GM3, and lower content of sphingomyelin, sulfatide and galactosylceramide were observed in the gl/gl brain with respect to controls. The low content of sphingomyelin, sulfatide and galactosylceramide is consistent with the immunohistochemical results showing that in the grey-lethal brain significant depletion and disorganization of the myelinated fibres is present, thus supporting the hypothesis that loss of function of the OSTM1 causes neuronal impairment and myelin deficit.

Glycosphingolipid behaviour in complex membranes.

Glycosphingolipids, due to their tendency to form laterally separated liquid-ordered phases, possess a high potential for the creation of order in biological membranes. The formation of glycosphingolipid-rich domains within the membrane has profound consequences on the membrane organization at different levels, and on the conformational and biological properties of membrane-associated proteins and multimolecular protein complexes. In this review, we will discuss 1) how glycosphingolipids influence the lateral organization of biological membranes; 2) how glycosphingolipids influence the function of membrane-associated proteins.