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Thin-Layer Chromatography in Structure and Recognition Studies of Shiga Toxin Glycosphingolipid Receptors

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Shiga Toxin-Producing E. coli

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2291))

Abstract

Glycosphingolipids (GSLs) consist of a ceramide (Cer) lipid anchor, which is typically composed of the long-chain aminoalcohol sphingosine (d18:1) and a fatty acid (mostly C16–C24) and a sugar moiety harboring to a great extent one to five monosaccharides. GSLs of the globo-series are well-recognized receptors of Shiga toxins (Stxs) released by Stx-producing Escherichia coli (STEC). Receptors for the Stx subtypes Stx1a and Stx2a are globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), whereby Gb3Cer represents their high-affinity and Gb4Cer their low-affinity receptor. In addition to Gb3Cer and Gb4Cer, Gb5Cer and Forssman GSL are further receptors of the Stx2e subtype rendering Stx2e unique among the various Stx subtypes. Thin-layer chromatography (TLC) is a convenient and ubiquitously employed method for analyzing GSL mixtures of unknown composition. In particular, TLC immunochemical overlay detection allows for sensitive identification of Stx-binding GSLs in complex mixtures directly on the TLC plate. For this purpose, specific anti-GSL antibodies or Stxs themselves in conjunction with anti-Stx antibodies can be used. The described protocols of antibody-mediated detection of TLC-separated globo-series GSLs and corresponding identification of Stx-binding globo-series GSLs will provide detailed advice for successful GSL analysis and particularly highlight the power of the TLC overlay technique.

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Acknowledgments

This chapter is dedicated to Prof. Dr. Elizabeth Hounsell, Emeritus Professor of Biological Chemistry at Birkbeck College London, who passed away on 21 February 2020 after a long and serious illness. Liz was a pioneer in the development and application of modern analytical techniques to structure determination of biologically important glycoconjugates. We lost an outstanding scientist and a very dear friend. She will be greatly missed, and we will always keep her in honorable memory. This work was supported by a grant from the German Research Foundation (DFG), grant number MU845/7-1 with reference number 404813761 (J.M.). We thank Dagmar Mense and Nikola Skutta for distinguished technical assistance. We are deeply indebted to Prof. em. Dr. Dr. h. c. Helge Karch for his intellectual leadership and long-lasting support of our scientific projects over so many years.

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  1. Institute for Hygiene, University of Münster, Münster, Germany

    Johanna Detzner, Gottfried Pohlentz & Johannes Müthing

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  1. Johanna Detzner
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  2. Gottfried Pohlentz
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  1. University of East Anglia, Norwich Medical School, Norwich, UK

    Stephanie Schüller

  2. National Reference Laboratory for E. coli and Shigellae, National Institute of Public Health, Prague, Czech Republic

    Martina Bielaszewska

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Detzner, J., Pohlentz, G., Müthing, J. (2021). Thin-Layer Chromatography in Structure and Recognition Studies of Shiga Toxin Glycosphingolipid Receptors. In: Schüller, S., Bielaszewska, M. (eds) Shiga Toxin-Producing E. coli . Methods in Molecular Biology, vol 2291. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1339-9_10

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