Vanna Chigorno

The αβ T Cell Response to Self-Glycolipids Shows a Novel Mechanism of CD1b Loading and a Requirement for Complex Oligosaccharides

The structural basis for the T cell recognition of lipoglycans remains to be elucidated. We have described autoreactive T cells responsive to GM1 ganglioside presented by CD1b. We show that glycosphingolipids bind to CD1b on the cell surface at neutral pH and are recognized without internalization or processing. Furthermore, soluble GM-CD1b complexes stimulate specific T cells. Oligosaccharide groups containing five or more sugars are required to build a minimal epitope for TCR recognition. This suggests a mechanism for T cell recognition of glycosphingolipids in which much of the CD1b-bound ligand is exposed. Binding to CD1b is a highly reversible process and other ceramide-containing glycosphingolipids displace GM1. These nonantigenic compounds act as blockers and may prevent harmful autoreactivity in vivo.

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.

Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts

Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation. In this context, its production has been shown to occur via sphingomyelin hydrolysis or sphingosine acylation. Here, we show that in human fibroblasts, plasma membrane ceramide is also produced from ganglioside GM3 by detachment of sugar units. Membrane‐bound glycosylhydrolases have a role in this process. In fact, the production of ceramide from GM3 has been observed even under experimental conditions able to block endocytosis or lysosomal activity, and the overexpression of the plasma membrane ganglioside sialidase Neu3 corresponded to a higher production of ceramide in the plasma membrane. The increased activity of Neu3 was paralleled by an increase of GM3 synthase mRNA and GM3 synthase activity. Neu3‐overexpressing fibroblasts were characterized by a reduced proliferation rate and higher basal number of apoptotic cells in comparison with wild‐type cells. A similar behavior was observed when normal fibroblasts were treated with exogenous C2‐ceramide.—Valaperta, R., Chigorno, V., Basso, L., Prinetti, A., Bresciani, R., Preti, A., Miyagi, T., and Sonnino, S. Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts. FASEB J. 20, E450–E461 (2006)

Recognition by two-dimensional thin-layer chromatography and densitometric quantification of alkali-labile gangliosides from the brain of different animals.

A simple method for recognition and quantification of alkali-labile gangliosides is described. The method was worked out using authentic alkali-labile gangliosides in pure form (9-O-Ac-GT1b; 9-O-Ac-GQ 1b; lactone form of GD 1b) and applied to ganglioside mixtures from the brain of mouse, rat, rabbit, pig, and pigeon. The method consists of two-dimensional thin-layer chromatography on silica gel high-performance thin-layer chromatography plates employing the same solvent, chloroform/methanol/0.2% aqueous CaCl2, 50/40/10, for both runs. Prior to the second run the plate is exposed at room temperature for 5 h to ammonia vapors in order to split alkali-labile linkages. At the end of chromatography alkali-stable gangliosides appear lined along a diagonal starting from the origin; the spots corresponding to alkali-labile gangliosides lie out of the diagonal and can be individually detected and quantified on the basis of their sialic acid content. Up to 15 different spots, corresponding to as many alkali-labile gangliosides, can be recognized by this procedure.

Ganglioside molecular species containing C18- and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures

Gangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties. They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging. Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains. A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB). In fact, considering all the mammalian cell sphingolipids, gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons. C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging. Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span. In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties.

Densitometric quantification of brain gangliosides separated by two-dimensional thin layer chromatography

A procedure for accurate densitometric quantification of gangliosides separated by two-dimensional thin layer chromatography is reported. The procedure was set up employing 9 different pure gangliosides and was applied to the analysis of calf and pig brain gangliosides. Silica gel high performance thin layer plates, 10 x 10 cm. were two-dimensionally developed at 18-20 C with the following solvents: chloroform methanol 0.2% aqueous CaCl(2), 50/40/10 by volume, for the first run; n-propanol 17 M NH(4)OH/water, 6/2/1 by volume for the second run. Ganglioside spots were visualized by spraying with an Ehrlich reagent, which is specific for sialic acid, and heating at 120 C for 15 min. The spots were quantified by sequential scanning densitometry, linear responses being obtained for ganglioside amounts on the plate ranging from 0.1 to 6 nmol as bound sialic acid. The reproducibility of densitometric responses resulted to be acceptable since the standard deviation values were lower than +/- 15% of the mean values also for those ganglioside species contained in minor proportions. The ganglioside mixtures of calf and pig brain were resolved in about 20 spots. Of these 9 corresponded to gangliosides GM3, GM2, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b, which were identified with certainty and quantified. The identification of GM3 (carrying N-glycolylneuraminic acid), GD3, GD1a (carrying N-acetyl- and N-glycolyl-neuraminic acid) and GT1a was only tentative. All the other spots corresponded to unidentified gangliosides, some of them possibly new species.

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody

Rat cerebellar granule cells differentiated in culture were fed [1–3H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent‐insoluble sphingolipid‐enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1–3H]sphingosine‐fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c‐Src, Lyn and Fyn and in the GPI‐anchored neuronal adhesion molecule TAG‐1. The cell lysate and the sphingolipid‐enriched membrane fraction were subjected to immunoprecipitation with anti‐GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid‐enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid‐enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c‐Src, Lyn, Fyn and TAG‐1 were associated with the anti‐GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti‐GD3 antibody immunoprecipitation, and that the association of c‐Src, Fyn, Lyn, and TAG‐1 with the sphingolipid‐enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Association of gangliosides to fibroblasts in culture: A study performed with GM1 [14C]-labelled at the sialic acid acetyl group

The preparation of a GM1-ganglioside (GM1) [14C]-labelled in the sialic acid residue is reported. This can be obtained by re-N-acetylation in the presence of [1-14C]-acetic anhydride, of a GM1 derivative de-N-acetylated specifically on the sialic acid residue by alkaline hydrolysis of GM1 with tetramethylammonium hydroxide. The radiolabelled GM1 is utilized to investigate the binding properties and the mode of interaction of GM1 with cultured fibroblasts. Three different forms of association (one “serum-removable”, one “trypsin-removable” and one “trypsin-stable”) have been recognized to occur in a way that depended on cell culture conditions (presence or absence of fetal calf serum), ganglioside concentration (from, 5×10−9 M to 10−4 M) and incubation time (up to 24 h). Some metabolic modifications of GM1 during the period of high cell viability were also investigated.

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.