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• W2175864900 abstract "Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli (EHEC), which cause human infections with an often fatal outcome. Vero cell lines, derived from African green monkey kidney, represent the gold standard for determining the cytotoxic effects of Stxs. Despite their global use, knowledge about the exact structures of the Stx receptor glycosphingolipids (GSLs) and their assembly in lipid rafts is poor. Here we present a comprehensive structural analysis of Stx receptor GSLs and their distribution to detergent-resistant membranes (DRMs), which were prepared from Vero-B4 cells and used as lipid raft equivalents. We identified globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) as the GSL receptors for Stx1a, Stx2a, and Stx2e subtypes using TLC overlay detection combined with MS. The uncommon Stx receptor, globopentaosylceramide (Gb5Cer, Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), which was specifically recognized (in addition to Gb3Cer and Gb4Cer) by Stx2e, was fully structurally characterized. Lipoforms of Stx receptor GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:0/C24:1 or C16:0 fatty acid. Moreover, co-occurrence with lipid raft markers, SM and cholesterol, in DRMs suggested GSL association with membrane microdomains. This study provides the basis for further exploring the functional impact of lipid raft-associated Stx receptors for toxin-mediated injury of Vero-B4 cells. Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli (EHEC), which cause human infections with an often fatal outcome. Vero cell lines, derived from African green monkey kidney, represent the gold standard for determining the cytotoxic effects of Stxs. Despite their global use, knowledge about the exact structures of the Stx receptor glycosphingolipids (GSLs) and their assembly in lipid rafts is poor. Here we present a comprehensive structural analysis of Stx receptor GSLs and their distribution to detergent-resistant membranes (DRMs), which were prepared from Vero-B4 cells and used as lipid raft equivalents. We identified globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) as the GSL receptors for Stx1a, Stx2a, and Stx2e subtypes using TLC overlay detection combined with MS. The uncommon Stx receptor, globopentaosylceramide (Gb5Cer, Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), which was specifically recognized (in addition to Gb3Cer and Gb4Cer) by Stx2e, was fully structurally characterized. Lipoforms of Stx receptor GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:0/C24:1 or C16:0 fatty acid. Moreover, co-occurrence with lipid raft markers, SM and cholesterol, in DRMs suggested GSL association with membrane microdomains. This study provides the basis for further exploring the functional impact of lipid raft-associated Stx receptors for toxin-mediated injury of Vero-B4 cells. Derived from the kidney of an African green monkey (Cercopithecus aethiops) in the 1960s, Vero cells are one of the most common mammalian continuous cell lines used in research (1.Ammermann N.C. Beier-Sexton M. Azad A.F. Growth and maintenance of Vero cell lines.Curr. Protoc. Microbiol. 2008; Appendix 4 (Appendix 4E)Google Scholar). They are the most widely accepted cells for the production of cell culture-derived human influenza vaccines (2.Nicolson C. Major D. Wood J.M. Robertson J.S. Generation of influenza vaccine viruses on Vero cells by reverse genetics: an H5N1 candidate vaccine strain produced under a quality system.Vaccine. 2005; 23: 2943-2952Crossref PubMed Scopus (150) Google Scholar, 3.Barrett P.N. Portsmouth D. Ehrlich H.J. Developing cell culture-derived pandemic vaccines.Curr. Opin. Mol. Ther. 2010; 12: 21-30PubMed Google Scholar, 4.Feng S.Z. Jiao P.R. Qi W.B. Fan H.Y. Liao M. Development and strategies of cell-culture technology for influenza vaccine.Appl. Microbiol. Biotechnol. 2011; 89: 893-902Crossref PubMed Scopus (30) Google Scholar, 5.Montomoli E. Khadang B. Piccirella S. Trombetta C. Mennitto E. Manini I. Stanzani V. Lapini G. Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production.Expert Rev. 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Specifically, Vero cells represent the gold standard for determining cytotoxic effects of bacterial exotoxins such as the Shiga toxins (Stxs) from Escherichia coli, starting from the very early investigations on Stx from Shigella dysenteriae type 1 and verotoxin (also referred to as Shiga-like toxin) from E. coli until recent studies on the E. coli O104:H4 outbreak strain isolated in 2011 in Germany employing verocytotoxicity assays (11.Konowalchuk J. Speirs J.I. Stavric S. Vero response to a cytotoxin of Escherichia coli..Infect. Immun. 1977; 18: 775-779Crossref PubMed Google Scholar, 12.Scotland S.M. Smith H.R. Rowe B. Two distinct toxins active on Vero cells from Escherichia coli O157.Lancet. 1985; 2: 885-886Abstract PubMed Scopus (153) Google Scholar, 13.Yutsudo T. Honda T. Miwatani T. Takeda Y. Characterization of purified Shiga toxin from Shigella dysenteriae 1.Microbiol. Immunol. 1986; 30: 1115-1127Crossref PubMed Scopus (27) Google Scholar, 14.Lingwood C.A. Law H. 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Comparative genomic analysis of two novel sporadic Shiga toxin-producing Escherichia coli O104:H4 strains isolated 2011 in Germany.PLoS One. 2015; 10: e0122074Crossref PubMed Scopus (14) Google Scholar). Stxs are produced by enterohemorrhagic E. coli (EHEC), which represent a certain subgroup of Stx-producing E. coli being responsible for global causes of diarrhea. The significant risk of developing two potentially life-threatening extraintestinal complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes EHEC infections a public health problem of serious concern (25.Karmali M.A. Infection by verocytotoxin-producing Escherichia coli..Clin. Microbiol. Rev. 1989; 2: 15-38Crossref PubMed Scopus (1115) Google Scholar, 26.Bielaszewska M. Mellmann A. Zhang W. Köck R. Fruth A. Bauwens A. Peters G. Karch H. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study.Lancet Infect. 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Shiga toxin/verocytotoxin-producing Escherichia coli infections: practical clinical perspectives.Microbiol. Spectr. 2014; 2doi:10.1128/microbiolspec.EHEC-0025-2014Crossref PubMed Scopus (43) Google Scholar, 29.Terajima J. Iyoda S. Ohnishi M. Watanabe H. Shiga toxin (verotoxin)-producing Escherichia coli in Japan.Microbiol. Spectr. 2014; 2Crossref PubMed Scopus (33) Google Scholar) owing to their cytotoxic capacity toward Vero cells, preferentially target microvascular endothelial cells of the human kidneys and the brain (30.Motto D. Endothelial cells and thrombotic microangiopathy.Semin. Nephrol. 2012; 32: 208-214Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 31.Trachtman H. Austin C. Lewinski M. Stahl R.A. Renal and neurological involvement in typical Shiga toxin-associated HUS.Nat. Rev. Nephrol. 2012; 8: 658-669Crossref PubMed Scopus (155) Google Scholar, 32.Bauwens A. Betz J. Meisen I. Kemper B. Karch H. Müthing J. Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature.Cell. Mol. Life Sci. 2013; 70: 425-457Crossref PubMed Scopus (63) Google Scholar) that follow gastrointestinal infection and culminate as renal insufficiency and an often fatal outcome (26.Bielaszewska M. Mellmann A. Zhang W. Köck R. Fruth A. Bauwens A. Peters G. Karch H. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study.Lancet Infect. Dis. 2011; 11: 671-676Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 31.Trachtman H. Austin C. Lewinski M. Stahl R.A. Renal and neurological involvement in typical Shiga toxin-associated HUS.Nat. Rev. Nephrol. 2012; 8: 658-669Crossref PubMed Scopus (155) Google Scholar, 33.Zoja C. Buelli S. Morigi M. Shiga toxin-associated hemolytic uremic syndrome: pathophsyiology of endothelial dysfunction.Pediatr. Nephrol. 2010; 25: 2231-2240Crossref PubMed Scopus (149) Google Scholar). Stxs are composed of a single 30 kDa A subunit and a pentamer of noncovalently attached identical 7 kDa B subunits (34.Bergan J. Dyve Lingelem A.B. Simm R. Skotland T. Sandvig K. Shiga toxins.Toxicon. 2012; 60: 1085-1107Crossref PubMed Scopus (150) Google Scholar). The B pentamer binds to cell surface-exposed glycosphingolipids (GSLs) of the globo-series and is therefore dependent on these lipids for transport into the cells (35.Sandvig K. Bergan J. Kavaliauskiene S. Skotland T. Lipid requirements for entry of protein toxins into cells.Prog. Lipid Res. 2014; 54: 1-13Crossref PubMed Scopus (64) Google Scholar). After retrograde routing of the holotoxin to the endoplasmic reticulum and cleavage of the A subunit, the A1 fragment halts eukaryotic protein biosynthesis by inactivating ribosomes leading to cell death (36.Johannes L. Römer W. Shiga toxins–from cell biology to biomedical applications.Nat. Rev. Microbiol. 2010; 8: 105-116Crossref PubMed Scopus (373) Google Scholar, 37.Sandvig K. Bergan J. Dyve A.B. Skotland T. Torgersen M.L. Endocytosis and retrograde transport of Shiga toxin.Toxicon. 2010; 56: 1181-1185Crossref PubMed Scopus (102) Google Scholar). The Stx subtypes, Stx1a, Stx2a, and Stx2e [for nomenclature of Stx subtypes refer to Scheutz et al. (38.Scheutz F. Teel L.D. Beutin L. Piérard D. Buvens G. Karch H. Mellmann A. Caprioli A. Tozzoli R. Morabito S. et al.Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature.J. Clin. Microbiol. 2012; 50: 2951-2963Crossref PubMed Scopus (556) Google Scholar)], which have so far been investigated in more detail, can be distinguished with regard to their exact binding specificity (39.Müthing J. Meisen I. Zhang W. Bielaszewska M. Mormann M. Bauerfeind R. Schmidt M.A. Friedrich A.W. Karch H. Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.Glycobiology. 2012; 22: 849-862Crossref PubMed Scopus (50) Google Scholar). Variants of the Stx1a subtype have been shown to exhibit preferential and moderate binding toward globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer), respectively. Stx2a variants prefer Gb3Cer and exhibit only marginal binding toward Gb4Cer, while Stx2e binds, in addition to Gb3Cer and a preference for Gb4Cer, also to the Forssman GSL, which represents a pentahexosylceramide with GalNAcα3GalNAcβ3Galα4Galβ4Glcβ1Cer structure (39.Müthing J. Meisen I. Zhang W. Bielaszewska M. Mormann M. Bauerfeind R. Schmidt M.A. Friedrich A.W. Karch H. Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.Glycobiology. 2012; 22: 849-862Crossref PubMed Scopus (50) Google Scholar), indicating promiscuous binding of Stx2e to globo-series-related GSLs with elongated Gb3Cer/Gb4Cer core structures. GSLs belong to the structurally very diverse group of sphingolipids (40.Levery S.B. Glycosphingolipid structural analysis and glycosphingolipidomics.Methods Enzymol. 2005; 405: 300-369Crossref PubMed Scopus (129) Google Scholar, 41.Müthing J. Distler U. Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry.Mass Spectrom. Rev. 2010; 29: 425-479Crossref PubMed Scopus (67) Google Scholar, 42.Merrill Jr, A.H. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics.Chem. 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Rev. 2011; 111: 6387-6422Crossref PubMed Scopus (505) Google Scholar) and they play important roles in the formation of the nervous system, where they are especially abundant (45.Yu R.K. Nakatani Y. Yanagisawa M. The role of glycosphingolipid metabolism in the developing brain.J. Lipid Res. 2009; 50: S440-S445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 46.Yu R.K. Tsai Y.T. Ariga T. Functional roles of gangliosides in neurodevelopment: an overview of recent advances.Neurochem. Res. 2012; 37: 1230-1244Crossref PubMed Scopus (140) Google Scholar, 47.Schengrund C.L. Gangliosides: glycosphingolipids essential for normal neural development and function.Trends Biochem. Sci. 2015; 40: 397-406Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). “Sphingolipidomic” analysis is now becoming feasible, at least for ceramides and other bioactive sphingolipid backbones, using MS (48.Zheng W. Kollmeyer J. Symolon H. Momin A. Munter E. Wang E. Kelly S. Allegood J.C. Liu Y. Peng Q. et al.Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.Biochim. Biophys. Acta. 2006; 1758: 1864-1884Crossref PubMed Scopus (443) Google Scholar), including novel MALDI MS imaging techniques (49.Soltwisch J. Kettling H. Vens-Cappell S. Wiegelmann M. Müthing J. Dreisewerd K. Mass spectrometry imaging with laser-induced postionization.Science. 2015; 348: 211-215Crossref PubMed Scopus (199) Google Scholar). GSLs are believed to be involved in many cellular events such as trafficking, signaling, and cellular interaction (50.Jennemann R. Gröne H.J. Cell-specific in vivo functions of glycosphingolipids: lessons from genetic deletions of enzymes involved in glycosphingolipid synthesis.Prog. Lipid Res. 2013; 52: 231-248Crossref PubMed Scopus (52) Google Scholar), as well as inflammation (51.Furukawa K. Ohmi Y. Kondo Y. Ohkawa Y. Tajima O. 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Cholesterol, sphingolipids, and glycolipids: what do we know about their role in raft-like membranes.Chem. Phys. Lipids. 2014; 184: 82-104Crossref PubMed Scopus (138) Google Scholar). Although the understanding of the structure of lipid rafts in living cells is quite limited, they are suggested to be involved in a great variety of cellular functions and biological events (53.Lingwood D. Simons K. Lipid rafts as a membrane-organizing principle.Science. 2010; 327: 46-50Crossref PubMed Scopus (3202) Google Scholar, 54.Simons K. Gerl M.J. Revitalizing membrane rafts: new tools and insights.Nat. Rev. Mol. Cell Biol. 2010; 11: 688-699Crossref PubMed Scopus (988) Google Scholar, 55.Sonnino S. Prinetti A. Membrane domains and the “lipid raft” concept.Curr. Med. Chem. 2013; 20: 4-21PubMed Google Scholar). 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Here we present the first comprehensive investigation of Vero cells regarding identification and structural characterization of globo-series Stx receptor GSLs for various Stx subtypes, their molecular assembly with phospholipids, and cholesterol in membrane microdomains using DRMs and Stx-mediated cytotoxicity. This study might be helpful to further our understanding of Vero cell sensitivity to various Stx subtypes and the functional role of lipid rafts in kidney epithelial cells. Vero-B4 cells were obtained from the Leibniz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; DSMZ number ACC 33). The cell line, which was established from the kidney of a normal adult African green monkey (Cercopithecus aethiops) in Japan (1.Ammermann N.C. Beier-Sexton M. Azad A.F. Growth and maintenance of Vero cell lines.Curr. Protoc. Microbiol. 2008; Appendix 4 (Appendix 4E)Google Scholar), was grown in MEM culture medium with nonessential amino acids and 1 mM sodium pyruvate (Lonza, Verviers, Belgium), MEM vitamin mix (Lonza, Wakersville, MD) plus 5% FCS (PAA, Pasching, Austria) in a humidified atmosphere at 37°C with 5% CO2. The cells grow as adherent-elongated fibroblast-like cells in monolayers. Cultures were routinely passaged every 2 to 3 days using 0.25% trypsin-EDTA (Invitrogen, Karlsruhe, Germany; catalog number 25200) before cells became confluent. Appropriate cell production for the isolation of preparative amounts of GSLs from total cells (see Purification of neutral GSLs from Vero-B4 cells below) and for the preparation of sucrose density gradient fractions (see Preparation of sucrose density gradient fractions: DRMs and nonDRM fractions below) was performed in 175 cm2 tissue culture flasks (Greiner Bio-One, Frickenhausen, Germany), as previously described for endothelial cells (61.Bauwens A. Bielaszewska M. Kemper B. Langehanenberg P. von Bally G. Reichelt R. Mulac D. Humpf H-U. Friedrich A.W. Kim K.S. et al.Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells.Thromb. Haemost. 2011; 105: 515-528Crossref PubMed Scopus (81) Google Scholar, 62.Betz J. Bielaszewska M. Thies A. Humpf H.U. Dreisewerd K. Karch H. Kim K.S. Friedrich A.W. Müthing J. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.J. Lipid Res. 2011; 52: 618-634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 63.Storck W. Meisen I. Gianmoena K. Pläger I. Kouzel I.U. Bielaszewska M. Haier J. Mormann M. Humpf H.U. Karch H. et al.Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation.Biol. Chem. 2012; 393: 785-799Crossref PubMed Scopus (18) Google Scholar). Indirect determination of cell viability was performed using the crystal violet assay, as previously described (61.Bauwens A. Bielaszewska M. Kemper B. Langehanenberg P. von Bally G. Reichelt R. Mulac D. Humpf H-U. Friedrich A.W. Kim K.S. et al.Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells.Thromb. Haemost. 2011; 105: 515-528Crossref PubMed Scopus (81) Google Scholar, 62.Betz J. Bielaszewska M. Thies A. Humpf H.U. Dreisewerd K. Karch H. Kim K.S. Friedrich A.W. Müthing J. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.J. Lipid Res. 2011; 52: 618-634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 63.Storck W. Meisen I. Gianmoena K. Pläger I. Kouzel I.U. Bielaszewska M. Haier J. Mormann M. Humpf H.U. Karch H. et al.Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation.Biol. Chem. 2012; 393: 785-799Crossref PubMed Scopus (18) Google Scholar). Briefly, Vero-B4 cells were grown until subconfluence in tissue culture flasks (Greiner Bio-One), trypsinized and seeded in 100 μl volumes in 96-well tissue culture plates (Corning Inc., Corning, NY) (initial cell seeding density of 4 × 103 cells/well). One hundred microliters of Stx-containing supernatants from E. coli cultures (see Stx1a, Stx2a, Stx2e, anti-Stx, anti-GSL, and secondary antibodies below) were added in various dilutions in cell culture medium to cell culture plate wells (final volume of 200 μl) and incubated for 48 h (37°C, 5% CO2). Stx-free cell culture medium served as a control. Incubation was stopped by removal of the diluted supernatants. Remaining adherent cells were fixed with formalin, stained with crystal violet, and densitometrically quantified (61.Bauwens A. Bielaszewska M. Kemper B. Langehanenberg P. von Bally G. Reichelt R. Mulac D. Humpf H-U. Friedrich A.W. Kim K.S. et al.Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells.Thromb. Haemost. 2011; 105: 515-528Crossref PubMed Scopus (81) Google Scholar, 62.Betz J. Bielaszewska M. Thies A. Humpf H.U. Dreisewerd K. Karch H. Kim K.S. Friedrich A.W. Müthing J. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.J. Lipid Res. 2011; 52: 618-634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 63.Storck W. Meisen I. Gianmoena K. Pläger I. Kouzel I.U. Bielaszewska M. Haier J. Mormann M. Humpf H.U. Karch H. et al.Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation.Biol. Chem. 2012; 393: 785-799Crossref PubMed Scopus (18) Google Scholar). Results represent the mean ± SD of quadruplicate determinations and are portrayed as percentage values of untreated control cells. Neutral GSLs were isolated from total cells and purified, as previously described (62.Betz J. Bielaszewska M. Thies A. Humpf H.U. Dreisewerd K. Karch H. Kim K.S. Friedrich A.W. Müthing J. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.J. Lipid Res. 2011; 52: 618-634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 63.Storck W. Meisen I. Gianmoena K. Pläger I. Kouzel I.U. Bielaszewska M. Haier J. Mormann M. Humpf H.U. Karch H. et al.Shiga toxin glycosphingolipid" @default.
• W2175864900 created "2016-06-24" @default.
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• W2175864900 date "2015-12-01" @default.
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• W2175864900 title "Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment" @default.
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