Maria Grazia Ciampa

The oxysterol–CXCR2 axis plays a key role in the recruitment of tumor-promoting neutrophils

Tumor-derived oxysterols recruit protumor neutrophils in an LXR-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression.

GM1 Ganglioside: Past Studies and Future Potential

Gangliosides (sialic acid-containing glycosphingolipids) are abundant in neurons of all animal species and play important roles in many cell physiological processes, including differentiation, memory control, cell signaling, neuronal protection, neuronal recovery, and apoptosis. Gangliosides also function as anchors or entry points for various toxins, bacteria, viruses, and autoantibodies. GM1, a ganglioside component of mammalian brains, is present mainly in neurons. GM1 is one of the best studied gangliosides, and our understanding of its properties is extensive. Simple and rapid procedures are available for preparation of GM1 as a natural compound on a large scale, or as a derivative containing an isotopic radionuclide or a specific probe. Great research interest in the properties of GM1 arose from the discovery in the early 1970s of its role as receptor for the bacterial toxin responsible for cholera pathogenesis.

Gangliosides as regulators of cell signaling: ganglioside‐protein interactions or ganglioside‐driven membrane organization?

It has been over 30 years now since the first pieces of information (obtained by physico-chemical studies on model membranes) suggested that the components of natural membranes are not randomly distributed but are, rather, hierarchically organized in ‘membrane domains’. The notion that lipid segregation occurs at the cell surface, leading to the dynamic separation of ‘lipid rafts’ (i.e. membrane domains whose properties are driven by the lipid segregation) capable of sorting or concentrating proteins belonging to specific cell signaling cassettes is now widely accepted, even if the physiological role of lipid rafts is still a matter of intense debate. The role of lipid segregation as the driving force for membrane organization in cells was originally suggested by subfractionation studies that highlighted the peculiar lipid and protein composition of lipid rafts (Brown and Rose 1992). Recently, it has been suggested that the membrane segregation of GM1 ganglioside can force the transbilayer distribution of cholesterol, adding a new dimension to lipiddriven membrane organization (Rondelli et al. 2012). On the other hand, the ability of glycosphingolipids (GSL) to modify some functional properties of several membraneassociated proteins (including enzymes, receptors and adhesion molecules) has been proved in many laboratories. Moreover, some GSL species are in the immediate surroundings of proteins necessary for cell signaling (Kabayama et al. 2007) in living cells. These constitute the basis of the concept that GSL can modulate the information transduction process across the membrane. The importance of the GSL oligosaccharide chain in this process has been emphasized by many studies, suggesting that specific GSL-protein interactions mediated by the GSL oligosaccharide chains are relevant in this context. However, it has been recently proven that lactosylceramide involved in the signal transduction process leading to the phosphorylation cascade necessary for neutrophil activation requires a specific structure of both the oligosaccharide and the ceramide moieties (Iwabuchi et al. 2008). Gangliosides, sialic acid-containing GSL, have often been reported to participate in cell signaling processes. The involvement of GD3 ganglioside, 1, in cell signaling has been suggested by several studies, thanks to the worldwide availability of an excellent experimental tool, the anti-GD3 monoclonal antibody R24. R24 is probably the best, and certainly the most widely used, anti-ganglioside antibody for ganglioside recognition and immunoprecipitation studies. GD3 is placed, wrongly or rightly, in the middle of many cellular processes, associating it, strangely enough, with the Greek goddess Hecate whose three faces are turned in as many directions (Malisan and Testi 2005).

Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[3H]LacCer-(N3), but not C18-[3H]LacCer-(N3), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[3H]LacCer-(N3) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[3H]LacCer-(N3) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.

Anti-GM1/GD1a complex antibodies in GBS sera specifically recognize the hybrid dimer GM1-GD1a

It is now emerging the new concept that the antibodies from some patients with Guillain-Barré syndrome (GBS) recognize an antigenic epitope formed by two different gangliosides, a ganglioside complex (GSC). We prepared the dimeric GM1-GD1a hybrid ganglioside derivative that contains two structurally different oligosaccharide chains to mimic the GSC. We use this compound to analyze sera from GBS patients by high-performance thin-layer chromatography immunostaining and enzyme-linked immunosorbent assay. We also synthesized the dimeric GM1-GM1 and GD1a-GD1a compounds that were used in control experiments together with natural gangliosides. The hybrid dimeric GM1-GD1a was specifically recognized by human sera from GBS patients that developed anti-oligosaccharide antibodies specific for grouped complex oligosaccharides, confirming the information that GBS patients developed antibodies against a GSC. High-resolution (1)H-(13)C heteronuclear single-quantum coherence-nuclear overhauser effect spectroscopy nuclear magnetic resonance experiments showed an interaction between the IV Gal-H1 of GM1 and the IV Gal-H2 of GD1a suggesting that the two oligosaccharide chains of the dimeric ganglioside form a single epitope recognized by a single-antibody domain. The availability of a method capable to prepare several hybrid gangliosides, and the availability of simple analytical approaches, opens new perspectives for the understanding and the therapy of several neuropathies.

Membrane lipid domains in the nervous system.

The structural properties of the lipids forming biological membranes determine a very high level of lateral organization within membranes. Lipid-driven membrane organization allows the segregation of membrane-associated components into membrane lipid domains, now worldwide known as lipid rafts, acting as dynamic platforms for signal transduction, protein processing and membrane turnover. Many processes necessary to the correct functions of nervous system occur in lipid rafts and are dependent on lipid raft organization. Thus, an altered lipid composition frequently occurring in neurodegenerative diseases leads to anomalous lipid raft organization and then to deregulated cell signaling.

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), [3H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([3H]SM-N3). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [3H]SM-N3. 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.

A lysosome–plasma membrane–sphingolipid axis linking lysosomal storage to cell growth arrest

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose‐loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids (i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp‐1 on the PM of sucrose‐loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose‐loaded fibroblasts. The inhibition of β‐ glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.—Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome‐plasma membrane‐sphingolipid axis linking lysosomal storage to cell growth arrest. FASEB J. 32, 5685–5702 (2018). www.fasebj.org

Altered expression of ganglioside GM3 molecular species and a potential regulatory role during myoblast differentiation

Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process.

Serum Antibodies to Glycans in Peripheral Neuropathies

In peripheral neuropathies, such as sensorimotor neuropathies, motor neuron diseases, or the Guillain-Barré syndrome, serum antibodies recognizing saccharide units, portion of oligosaccharides, or oligosaccharide chains, have been found. These antibodies are called anti-glycosphingolipid (GSL) or anti-ganglioside antibodies. However, the information on the aglycone carrying the hydrophilic oligosaccharide remains elusive. The absolute and unique association of GSL to the onset, development and symptomatology of the peripheral neuropathies could be misleading. Here, we report some thoughts on the matter.