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Takao Taki - One of the best experts on this subject based on the ideXlab platform.
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Bio-recognition and functional lipidomics by Glycosphingolipid transfer technology.
Proceedings of the Japan Academy. Series B Physical and biological sciences, 2013Co-Authors: Takao TakiAbstract:Through Glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of Glycosphingolipids to peptides. The other is a physicochemical transfer of Glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of Glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-Glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original Glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen Glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring Glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual Glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for Glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced.(Communicated by Kunihiko SUZUKI, M.J.A.).
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A simple and quantitative purification of Glycosphingolipids and phospholipids by thin-layer chromatography blotting.
Analytical biochemistry, 1994Co-Authors: Takao Taki, Takeshi Kasama, Shizuo Handa, Dai IshikawaAbstract:A new and simple method for purifying Glycosphingolipids and phospholipids by using "TLC blotting" was established. Glycosphingolipids separated by two-dimensional thin-layer chromatography (TLC) were made visible with primuline reagent, and then bands were marked with a drawing colored pencil. The Glycosphingolipids that separated on the HPTLC plate were transferred by TLC blotting to a polyvinylidene difluoride membrane together with the color marks. The marked areas were excised after which their Glycosphingolipids were extracted and monitored by TLC. By this method, 20 Glycosphingolipids showing homogeneous bands on a HPTLC plate were isolated from the neutral Glycosphingolipid fraction of human meconium. Moreover, 10 kinds of acidic Glycosphingolipids were purified as homogeneous bands from the bovine acidic Glycosphingolipid fraction. The yields of Glycosphingolipids (13 different ones) ranged from 68 to 92%, the mean value being 82.3%. The Glycosphingolipids were confirmed to be purified as intact forms by mass spectrometric analysis and chromatographic mobilities on a HPTLC plate. The same procedure could also be used to purify phospholipids.
Susann Teneberg - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Glycosphingolipids from gastrointestinal stromal tumours.
Scientific reports, 2020Co-Authors: Licinia Santos, Chunsheng Jin, Taťána Gazárková, Anders Thornell, Olov Norlén, Karin Säljö, Susann TenebergAbstract:Gastrointestinal stromal tumours (GISTs) are the major nonepithelial neoplasms of the human gastrointestinal tract with a worldwide incidence between 11 and 15 per million cases annually. In this study the acid and non-acid Glycosphingolipids of three GISTs were characterized using a combination of thin-layer chromatography, chemical staining, binding of carbohydrate recognizing ligands, and mass spectrometry. In the non-acid Glycosphingolipid fractions of the tumors globotetraosylceramide, neolactotetraosylceramide, and Glycosphingolipids with terminal blood group A, B, H, Lex, Lea, Ley and Leb determinants were found. The relative amounts of these non-acid compounds were different in the three tumour samples. The acid Glycosphingolipid fractions had sulfatide, and the gangliosides GM3, GD3, GM1, Neu5Acα3neolactotetraosylceramide, GD1a, GT1b and GQ1b. In summary, we have characterized the Glycosphingolipids of GISTs and found that the pattern differs in tumours from different individuals. This detailed characterization of Glycosphingolipid composition of GISTs could contribute to recognition of new molecular targets for GIST treatment and sub-classification.
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Glycosphingolipids Recognized by Acinetobacter baumannii.
Microorganisms, 2020Co-Authors: Miralda Madar Johansson, Karin Säljö, Mehjar Azzouz, Beatrice Häggendal, Henri Malmi, Anton Zaviolov, Susann TenebergAbstract:Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential Glycosphingolipid receptors of A. baumannii were examined by binding of 35S-labeled bacteria to Glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid Glycosphingolipids of human and rabbit small intestine was found. The binding-active Glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Galβ4GlcNAcβ3Galβ4Glcβ1Cer) and lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer). Further binding assays using reference Glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAcβ3Galβ4Glcβ1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as “lactosylceramide-binding”. Finally, by isolation and characterization of Glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue.
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Glycosphingolipids of human embryonic stem cells
Glycoconjugate Journal, 2017Co-Authors: Michael E. Breimer, Karin Säljö, Angela Barone, Susann TenebergAbstract:The application of human stem cell technology offers theoretically a great potential to treat various human diseases. However, to achieve this goal a large number of scientific issues remain to be solved. Cell surface carbohydrate antigens are involved in a number of biomedical phenomena that are important in clinical applications of stem cells, such as cell differentiation and immune reactivity. Due to their cell surface localization, carbohydrate epitopes are ideally suited for characterization of human pluripotent stem cells. Amongst the most commonly used markers to identify human pluripotent stem cells are the globo-series Glycosphingolipids SSEA-3 and SSEA-4. However, our knowledge regarding human pluripotent stem cell Glycosphingolipid expression was until recently mainly based on immunological assays of intact cells due to the very limited amounts of cell material available. In recent years the knowledge regarding Glycosphingolipids in human embryonic stem cells has been extended by biochemical studies, which is the focus of this review. In addition, the distribution of the human pluripotent stem cell Glycosphingolipids in human tissues, and Glycosphingolipid changes during human stem cell differentiation, are discussed.
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Characterization of moose intestinal Glycosphingolipids
Glycoconjugate Journal, 2015Co-Authors: Miralda Madar Johansson, John Benktander, Angela Barone, Benjamin Dedic, Klara Lundholm, Filip Berner Branzell, Susann TenebergAbstract:As a part of a systematic investigation of the species-specific expression of Glycosphingolipids, acid and non-acid Glycosphingolipids were isolated from three small intestines and one large intestine of the moose ( Alces alces ). The Glycosphingolipids were characterized by binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays, and by mass spectrometry. The non-acid fractions were complex mixtures, and all had Glycosphingolipids belonging to the lacto- and neolactoseries (lactotriaosylceramide, lactotetraosylceramide, neolactotetraosylceramide, Galα3-Le^x hexaosylceramide, and lacto-neolactohexaosylceramide), globo-series (globotriaosylceramide and globotetraosylceramide), and isogloboseries (isoglobotriaosylceramide). Penta- and heptaglycosylceramides with terminal Galili determinants were also characterized. Furthermore, Glycosphingolipids with terminal blood group O determinants (H triaosylceramide, H type 2 pentaosylceramide, H type 1 penta- and heptaosylceramide) were characterized in two of the moose small intestines, and in the one large intestine, while the third small intestine had Glycosphingolipids with terminal blood group A determinants (A tetraosylceramide, A type 1 hexa- and octaosylceramide, A dodecaosylceramide). The acid Glycosphingolipid fractions of moose small and large intestine contained sulfatide, and the gangliosides GM3, GD3, GD1a, GD1b, and also NeuGc and NeuAc variants of the Sd^a ganglioside and the sialyl-globopenta/SSEA-4 ganglioside. In humans, the NeuAc-globopenta/SSEA-4 ganglioside is a marker of embryonic and adult stem cells, and is also expressed in several human cancers. This is the first time sialyl-globopentaosylceramide/SSEA-4 has been characterized in a fully differentiated normal tissue, and also the first time NeuGc-globopentaosylceramide has been characterized.
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The repertoire of Glycosphingolipids recognized by Vibrio cholerae.
PloS one, 2013Co-Authors: John Benktander, Michael Lebens, Jonas Ångström, Hasse Karlsson, Omid Teymournejad, Sara K. Lindén, Susann TenebergAbstract:The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of Glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid Glycosphingolipids were thereby detected. After isolation of binding-active Glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct Glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto Glycosphingolipids with the GlcNAcβ3Galβ4GlcNAc sequence as the minimal binding epitope. Secondly, Glycosphingolipids with a terminal Galα3Galα3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto Glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing Glycosphingolipids represents two separate binding specificities.
Johannes M F G Aerts - One of the best experts on this subject based on the ideXlab platform.
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Lyso-Glycosphingolipids: presence and consequences
Essays in biochemistry, 2020Co-Authors: Marco Van Eijk, Maria J. Ferraz, Rolf G. Boot, Johannes M F G AertsAbstract:Lyso-Glycosphingolipids are generated in excess in Glycosphingolipid storage disorders. In the course of these pathologies glycosylated sphingolipid species accumulate within lysosomes due to flaws in the respective lipid degrading machinery. Deacylation of accumulating Glycosphingolipids drives the formation of lyso-Glycosphingolipids. In lysosomal storage diseases such as Gaucher Disease, Fabry Disease, Krabbe disease, GM1 -and GM2 gangliosidosis, Niemann Pick type C and Metachromatic leukodystrophy massive intra-lysosomal Glycosphingolipid accumulation occurs. The lysosomal enzyme acid ceramidase generates the deacylated lyso-Glycosphingolipid species. This review discusses how the various lyso-Glycosphingolipids are synthesized, how they may contribute to abnormal immunity in Glycosphingolipid storing lysosomal diseases and what therapeutic opportunities exist.
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Glycosphingolipids and insulin resistance.
Advances in experimental medicine and biology, 2011Co-Authors: Johannes M F G Aerts, Marco Van Eijk, Rolf G. Boot, Johanna E. M. Groener, Nora Bijl, Elisa Lombardo, Florence Bietrix, Nick Dekker, Albert K. Groen, Roelof OttenhoffAbstract:Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for Glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of Glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of Glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow Glycosphingolipids act as critical regulators, Importantly, since reductions in Glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.
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Glycosphingolipids—Nature, Function, and Pharmacological Modulation
Angewandte Chemie (International ed. in English), 2009Co-Authors: Tom Wennekes, Rolf G. Boot, Richard J. B. H. N. Van Den Berg, Gijsbert A. Van Der Marel, Herman S. Overkleeft, Johannes M F G AertsAbstract:The discovery of the Glycosphingolipids is generally attributed to Johan L. W. Thudichum, who in 1884 published on the chemical composition of the brain. In his studies he isolated several compounds from ethanolic brain extracts which he coined cerebrosides. He subjected one of these, phrenosin (now known as galactosylceramide), to acid hydrolysis, and this produced three distinct components. One he identified as a fatty acid and another proved to be an isomer of D-glucose, which is now known as D-galactose. The third component, with an "alkaloidal nature", presented "many enigmas" to Thudichum, and therefore he named it sphingosine, after the mythological riddle of the Sphinx. Today, sphingolipids and their glycosidated derivatives are the subjects of intense study aimed at elucidating their role in the structural integrity of the cell membrane, their participation in recognition and signaling events, and in particular their involvement in pathological processes that are at the basis of human disease (for example, sphingolipidoses and diabetes type 2). This Review details some of the recent findings on the biosynthesis, function, and degradation of Glycosphingolipids in man, with a focus on the Glycosphingolipid glucosylceramide. Special attention is paid to the clinical relevance of compounds directed at interfering with the factors responsible for Glycosphingolipid metabolism.
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Glycosphingolipids and insulin resistance.
Progress in lipid research, 2009Co-Authors: Mirjam Langeveld, Johannes M F G AertsAbstract:Obesity is associated with an increased risk for insulin resistance, a state characterized by impaired responsiveness of liver, muscle and adipose tissue to insulin. One class of lipids involved in the development of insulin resistance are the (glyco)sphingolipids. Ceramide, the most simple sphingolipid, directly inhibits phosphorylation of the insulin signaling mediator Akt/Protein Kinase B. More complex Glycosphingolipids, so-called gangliosides, block phosphorylation of the insulin receptor and down-stream signaling, possibly by exclusion of the insulin receptor from specific membrane domains. Pharmacological inhibition of Glycosphingolipid synthesis is found to markedly improve insulin sensitivity in rodent models of insulin resistance. Partial Glycosphingolipid reduction is well tolerated and may thus offer an attractive new treatment modality for obesity-induced insulin resistance and type II diabetes.
Ronald L. Schnaar - One of the best experts on this subject based on the ideXlab platform.
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Thin-layer chromatography of Glycosphingolipids.
Methods in enzymology, 1994Co-Authors: Ronald L. Schnaar, Leila K. NeedhamAbstract:Publisher Summary Glycosphingolipids are well suited for analysis by thin-layer chromatography (TLC), which is useful for monitoring purification, for qualitative and quantitative determination of expression in normal and pathological tissues, for partial structural analysis, and for detecting biological activities, including immunoreactivity and binding activity toward toxins, viruses, bacteria, and eukaryotic cells. Although quantitative TLC can be used to determine the concentration of resolved Glycosphingolipid species, it is useful to estimate the total concentration of Glycosphingolipid in a sample prior to or in association with TLC. Glycosphingolipid samples must first be freed of major contaminating lipids, proteins, and low molecular weight contaminants. The most widely used TLC developing solvents for Glycosphingolipids are mixtures of chloroform, methanol, and water (or aqueous salts), because they form a single phase at a range of hydrophobicities well suited for Glycosphingolipid resolution on silica gel TLC plates. Reversible stains are useful for preparative TLC. To improve recovery, the plate should not be allowed to dry thoroughly at any time after running. The simplest but least sensitive reversible staining method is to spray the TLC plate heavily with distilled water.
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Isolation of Glycosphingolipids.
Methods in enzymology, 1994Co-Authors: Ronald L. SchnaarAbstract:Publisher Summary Glycosphingolipids are classified primarily on the basis of their carbohydrate portion, which may vary from a single monosaccharide to large branched structures composed of >20 monosaccharide units. Techniques for Glycosphingolipid purification have largely focused on separations based on saccharide differences, resulting in purification to oligosaccharide homogeneity. There is additional variation in the ceramide, which is composed of any of several long-chain bases (of which sphingosine is the most common in mammalian tissues), each of which is further substituted, through amide linkage, with any of a variety of fatty acids. Glycosphingolipid purification involves three steps—(1) extraction of lipids from a biological source, using organic solvents, (2) bulk separation from major lipid and nonlipid contaminants, and (3) chromatographic resolution of individual species. Isolation of Glycosphingolipids requires repeated removal of solvents. For small samples this is most conveniently achieved under a stream of nitrogen in a heated block or water bath at ∼45°, using a commercial apparatus for multiple small samples. For large numbers of small samples containing volatile salts, which require vacuum for efficient removal, a SpeedVac concentrator is recommended.
Daniel J. Sillence - One of the best experts on this subject based on the ideXlab platform.
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Glycosphingolipid storage leads to the enhanced degradation of the B cell receptor in Sandhoff disease mice
Journal of Inherited Metabolic Disease, 2010Co-Authors: Danielle Te Vruchte, Aruna Jeans, Frances M. Platt, Daniel J. SillenceAbstract:Glycosphingolipid storage diseases are a group of inherited metabolic diseases in which Glycosphingolipids accumulate due to their impaired lysosomal breakdown. Splenic B cells isolated from NPC1, Sandhoff, GM1-gangliosidosis and Fabry disease mouse models showed large (20- to 30-fold) increases in disease specific Glycosphingolipids and up to a 4-fold increase in cholesterol. The magnitude of Glycosphingolipid storage was in the order NPC1 > Sandhoff ∼ GM1 gangliosidosis > Fabry. Except for Fabry disease, Glycosphingolipid storage led to an increase in the lysosomal compartment and altered Glycosphingolipid trafficking. In order to investigate the consequences of storage on B cell function, the levels of surface expression of B cell IgM receptor and its associated components were quantitated in Sandhoff B cells, since they are all raft-associated on activation. Both the B cell receptor, CD21 and CD19 had decreased cell surface expression. In contrast, CD40 and MHC II, surface receptors that do not associate with lipid rafts, were unchanged. Using a pulse chase biotinylation procedure, surface B cell receptors on a Sandhoff lymphoblast cell line were found to have a significantly decreased half-life. Increased co-localization of fluorescently conjugated cholera toxin and lysosomes was also observed in Sandhoff B cells. Glycosphingolipid storage leads to the enhanced formation of lysosomal lipid rafts, altered endocytic trafficking and increased degradation of the B cell receptor.
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New Insights into Glycosphingolipid Functions—Storage, Lipid Rafts, and Translocators
International review of cytology, 2007Co-Authors: Daniel J. SillenceAbstract:Glycosphingolipids are key components of eukaryotic cellular membranes. Through their propensity to form lipid rafts, they are important in membrane transport and signaling. At the cell surface, they are required for caveolar-mediated endocytosis, a process required for the action of many Glycosphingolipid-binding toxins. Glycosphingolipids also exist intracellularly, on both leaflets of organelle membranes. It is expected that dissecting the mechanisms of cell pathology seen in the Glycosphingolipid storage diseases, where lysosomal Glycosphingolipid degradation is defective, will reveal their functions. Disrupted cation gradients in Mucolipidosis type IV disease are interlinked with Glycosphingolipid storage, defective rab 7 function, and the activation of autophagy. Relationships between drug translocators and Glycosphingolipid synthesis are also discussed. Mass spectrometry of cell lines defective in drug transporters reveal clear differences in Glycosphingolipid mass and fatty acid composition. The potential roles of Glycosphingolipids in lipid raft formation, endocytosis, and cationic gradients are discussed.
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role of multiple drug resistance protein 1 in neutral but not acidic Glycosphingolipid biosynthesis
Journal of Biological Chemistry, 2004Co-Authors: Maria Fabiana De Rosa, Daniel J. Sillence, Cameron Ackerley, Clifford A LingwoodAbstract:Abstract Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246–6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral Glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid Glycosphingolipids are synthesized from distinct precursor Glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral Glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of Glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral Glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid Glycosphingolipid synthesis.