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Biochemistry |
Department of Bacteriology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan1
Department of Molecular Medicine, Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan2
Hokkaido Institute of Public Health, N19, W12, Sapporo 060-0819, Japan3
Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196, Yasaka, Abashiri 099-2422, Japan4
Author for correspondence: Keiji Oguma. Tel: +81 86 235 7162. Fax: +81 86 235 7162. e-mail: kuma{at}med.okayama-u.ac.jp
The 16S toxin and one subcomponent of haemagglutinin (HA), designated HA1, were purified from a type D culture of Clostridium botulinum by a newly established procedure, and their HA activities as well as that of purified type C 16S toxin were characterized. SDS-PAGE analysis indicated that the free HA1 forms a polymer with a molecular mass of approximately 200 kDa. Type C and D 16S toxins agglutinated human erythrocytes in the same manner. Their HA titres were dramatically reduced by employing erythrocytes that had been previously treated with neuraminidase, papain or proteinase K, and were inhibited by the addition of N-acetylneuraminic acid to the reaction mixtures. In a direct-binding test to glycolipids such as SPG (NeuAc
2-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-Cer) and GM3 (NeuAc2-3Galß1-4Glcß1-Cer), and glycoproteins such as glycophorin A and/or B prepared from the erythrocytes, both toxins bound to sialylglycolipids and sialoglycoproteins, but bound to neither neutral glycolipids nor asialoglycoproteins. On the basis of these results, it was concluded that type C and D 16S toxins bind to erythrocytes through N-acetylneuraminic acid. HA1 showed no haemagglutination activity, although it did bind to sialylglycolipids. We therefore speculate that binding to glycoproteins rather than to glycolipids may be important in causing haemagglutination by type C and D 16S toxins.
Keywords: Clostridium botulinum, haemagglutinin, glycolipid, glycoprotein
Abbreviations: CBB, Coomassie brilliant blue; HA, haemagglutinin; MLD, minimal lethal dose
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