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

Samarani, Maura; Loberto, Nicoletta; Soldà, Giulia; Straniero, Letizia; Asselta, Rosanna; Duga, Stefano; Lunghi, Giulia; Zucca, Fabio A.; Mauri, Laura; Ciampa, Maria Grazia; Schiumarini, Domitilla; Bassi, Rajiv; Giussani, Paola; Chiricozzi, Elena; Prinetti, Alessandro; Aureli, Massimo; Sonnino, Sandro

doi:10.1096/fj.201701512rr

Cited by 35 publications

(29 citation statements)

References 71 publications

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“…In several cell lines of different origin, NLGase was found to be associated with the external leaflet of the PM where it catalyzes the in situ hydrolysis of glucosylceramide (GlcCer) to ceramide [ 27 , 28 , 29 ]. For this reason, we measured the enzymatic activity of NLGase directly at the cell surface of control and patient derived lymphoblastoid living cells.…”

Section: Resultsmentioning

confidence: 99%

“…Equivalent amounts of proteins associated with total cell lysates, determined by DC Protein Assay (Bio-Rad, Hercules, CA, USA), were separated on polyacrylamide gels and then transferred to PVDF (Polyvinylidene fluoride) membranes by electroblotting [ 29 ]. Blots were incubated with monoclonal rabbit anti-GCase (ab128879, Abcam, Cambridge, UK) or polyclonal rabbit anti-GAPDH (G9545, Sigma-Aldrich) primary antibodies at 4 °C overnight, followed by incubation with goat anti-rabbit HRP-conjugated (7074, Cell Signaling) secondary antibody and detection with a chemiluminescent kit (WESTAR ηC, Cyanagen, Bologna, Italy).…”

Section: Methodsmentioning

confidence: 99%

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Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia

Malekkou

Samarani

Drousiotou

et al. 2018

IJMS

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The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia

Malekkou

Samarani

Drousiotou

et al. 2018

IJMS

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“…; Samarani et al . ). Lipids were separated by Thin Layer Chromatography (TLC); then, GM1 ganglioside was specifically detected with HPR‐conjugated cholera toxin B (CT‐B) staining directly on TLC (Wu and Ledeen ; Valperta et al .…”

Section: Methodsmentioning

confidence: 97%

GM1 promotes TrkA‐mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA

Chiricozzi

Biase

Maggioni

et al. 2019

Journal of Neurochemistry

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Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA–GM1 oligosaccharide complex at the cell surface. To study the TrkA–GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross‐linked TrkA–GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA–GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent‐resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by ‘flopping’ down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1.

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“…Remarkably, this was accompanied by increased lysosomal biogenesis and lysosome fusion with the PM (Samarani et al . ). Obviously, increased lysosomal biogenesis in principle favors the traffic of lysosomal components to the cell surface with the above‐described mechanisms implying lysosomal fusion with the membrane.…”

Section: Sphingolipids and Lysosomal Storage Disorders (Lsds)mentioning

confidence: 97%

Sphingolipids and neuronal degeneration in lysosomal storage disorders

Grassi

Chiricozzi

Mauri

et al. 2018

Journal of Neurochemistry

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Ceramide, sphingomyelin, and glycosphingolipids (both neutral and acidic) are characterized by the presence in the lipid moiety of an aliphatic base known as sphingosine. Altogether, they are called sphingolipids and are particularly abundant in neuronal plasma membranes, where, via interactions with the other membrane lipids and membrane proteins, they play a specific role in modulating the cell signaling processes. The metabolic pathways determining the plasma membrane sphingolipid composition are thus the key point for functional changes of the cell properties. Unnatural changes of the neuronal properties are observed in sphingolipidoses, lysosomal storage diseases occurring when a lysosomal sphingolipid hydrolase is not working, leading to the accumulation of the substrate and to its distribution to all the cell membranes interacting with lysosomes. Moreover, secondary accumulation of sphingolipids is a common trait of other lysosomal storage diseases. Keywords: gangliosides, lysosome, membrane fusion, sphingolipidoses, sphingolipids, sphingomyelin.This article is part of the Special Issue "Lysosomal Storage Disorders".Sphingolipids are amphiphilic membrane lipids characterized by the presence of sphingosine, an amino-and-hydroxy alkylic long chain. After more than a century of studies, we now know their structure, their physico-chemical properties, their distribution and content in cells and tissues of many animal species, including humans . The final cellular site for sphingolipids is the plasma membrane, however, their complex metabolic pathway requires many intracellular processes, so that a not negligible portion of them is also found intracellularly (Sandhoff and Kolter 2003;Schulze et al. 2009;Kolter and Sandhoff 2010;Hannun and Obeid 2018). As amphiphilic compounds they contain a hydrophilic head group, for example a saccharide, an oligosaccharide, a phosphocholine, protruding into the cell aqueous environment, and a hydrophobic chain called ceramide, in which the sphingosine is linked with a fatty acid via an amidic bond, inserted into the outer layer of plasma membrane.Sphingolipids are typically asymmetric components of the plasma membranes enriched in the outer leaflet (van Meer and Hoetzl 2010;van Meer 2011;Fujimoto and Parmryd 2016), however, it cannot be excluded that a minor pool of them could be transiently inserted into the inner layer with the hydrophilic group facing the cytosol. The hydrophilic group, thanks to the presence of carbohydrates and/or ionic charges, attracts water and ions, forms hydrogen bonds and easily interacts with the group of membrane components protruding from the membrane itself. At the water-lipid interface, the sphingolipid ceramide amide linkage accepts hydrogen bonds on the carbonyl group and donates hydrogen bonds via the N-H group, allowing the formation of a double linkage, which works as a padlock within molecules (Fig. 1). The lipid moiety inserted into the lipid layer interacts with the lipid portion of the cholesterol in the membrane and with dip...

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

Cited by 35 publications

References 71 publications

Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia

Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia

GM1 promotes TrkA‐mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA

Sphingolipids and neuronal degeneration in lysosomal storage disorders

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