Nicoletta Loberto

Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNFα-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR–Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR β-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR–Cav1 complex by the interactions of IR with GM3 in microdomains.

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.

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 β peptide in Alzheimer’s disease, huntingtin in Huntington’s disease, α-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.

Ceramide and sphingomyelin species of fibroblasts and neurons in culture.

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

The membrane environment of endogenous cellular prion protein in primary rat cerebellar neurons

We studied the membrane environment of cellular prion protein in primary cultured rat cerebellar neurons differentiated in vitro. In these cells, about 45% of total cellular prion protein (corresponding to a 35‐fold enrichment) is associated with a low‐density, sphingolipid‐ and cholesterol‐enriched membrane fraction, that can be separated by flotation on sucrose gradient. Biotinylation experiments indicated that almost all prion protein recovered in this fraction was exposed at the cell surface. Prion protein was efficiently separated from this fraction by a monoclonal antibody immunoseparation procedure. Under conditions designed to preserve lipid‐mediated membrane organization, several proteins were found in the prion protein‐enriched membrane domains (i.e. the non‐receptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol‐anchored protein Thy‐1). The prion protein‐rich membrane domains contained, as well, about 50% of the sphingolipids, cholesterol and phosphatidylcholine present in the sphingolipid‐enriched membrane fraction. All main sphingolipids, including sphingomyelin, neutral glycosphingolipids and gangliosides, were similarly enriched in the prion protein‐rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted to be a complex entity, whose integrity requires a network of lipid‐mediated non‐covalent interactions.

Activity of plasma membrane β-galactosidase and β-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 β‐galactosidase and β‐glucosidase. To determine the presence of β‐galactosidase and β‐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.

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.

Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice

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.

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 β-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 β-glucosidase (GBA1) and AMP-DNM-sensitive β-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 β-galactosidase and β-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.

The adhesion protein TAG‐1 has a ganglioside environment in the sphingolipid‐enriched membrane domains of neuronal cells in culture

We studied the interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid‐enriched membrane domains by ganglioside photolabeling combined with cell surface biotin labeling. After cell photolabeling with radioactive photoactivable derivatives of GM3, GM1 and GD1b gangliosides, followed by cell surface biotin labeling, sphingolipid‐enriched domains were prepared and immunoprecipitated with streptavidin‐coupled beads, under experimental conditions preserving the integrity of the lipid domain. About 50% of the total radioactivity linked to proteins was associated with acylated tubulin, about 10% with a 135‐kDa protein present as a series of species with pI ranging from 6.5 to 8.0, about 5% with a protein of about 70u2003kDa and with pI near to 6.5. By immunoprecipitation with streptavidin‐coupled beads under conditions disrupting the integrity of the lipid domain, the 135u2003kDa protein was recovered in the immunoprecipitate, that did not contain tubulin. Thus, the 135u2003kDa protein has an exoplasmic domain, and it was then identified as the GPI‐anchored neural cell adhesion molecule TAG‐1. Remarkably, TAG‐1 was cross‐linked in a similar extent by the photoactivated ganglioside GM3, GM1 and GD1b. The three gangliosides bear different oligosaccharide chains, suggesting that the ganglioside/TAG−1 interaction is not specifically associated with the ganglioside oligosaccharide structure.