Analysis of tryptophan-rich region in Clostridium septicum alpha-toxin involved with binding to glycosylphosphatidylinositol-anchored proteins

Authors

  • Masafumi Mukamoto,

    Corresponding author
    • Laboratory of Veterinary Epidemiology, Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
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  • Ryota Kimura,

    1. Laboratory of Veterinary Epidemiology, Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
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  • Mudenda B. Hang'ombe,

    1. Department of Paraclinical Studies, School of Veterinary Medicine, University of Zambia, Lusaka, Zambia
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  • Tomoko Kohda,

    1. Laboratory of Veterinary Epidemiology, Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
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  • Shunji Kozaki

    1. Laboratory of Veterinary Epidemiology, Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan
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Correspondence

Masafumi Mukamoto, Laboratory of Veterinary Epidemiology, Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Orai-kita, Izumisano, Osaka 598-8531, Japan. Tel: +81 72 4635690; fax: +81 72 4635691; email: mukamoto@vet.osakafu-u.ac.jp

Abstract

Clostridium septicum alpha-toxin has a unique tryptophan-rich region (302NGYSEWDWKWV312) that consists of 11 amino acid residues near the C-terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan-rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)-anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI-anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan-rich region plays an important role in the cytotoxic mechanism of alpha-toxin, especially in the binding to GPI-anchored proteins as cell receptors.

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