title: Four molecules of the 33 kDa haemagglutinin component of the Clostridium botulinum serotype C and D toxin complexes are required to aggregate erythrocytes.
abstract: Normally, large-sized botulinum toxin complexes (L-TC) of serotype C and D are composed of a single neurotoxin, a single non-toxic non-haemagglutinin, two HA-70 molecules, four HA-33 molecules and four HA-17 molecules that assemble to form a 650 kDa L-TC. The 540 and 610 kDa TC species (designated here as L-TC2 and L-TC3, respectively) were purified in addition to the 650 kDa L-TC from the culture supernatants of serotype D strains (D-4947 and D-CB16) and serotype C strains (C-6814 and C-Yoichi). The 650 kDa L-TC from D-4947, D-CB16 and C-6814 showed haemagglutination and erythrocyte-binding activity, but their L-TC2 and L-TC3 species had only binding activity. In contrast, every TC species from C-Yoichi having the C-terminally truncated variant of HA-33 exhibited neither haemagglutination activity nor erythrocyte-binding activity. Four strain-specific HA-33/HA-17 complexes were isolated from the 650 kDa L-TC of each strain. The 650 kDa HA-hybrid L-TCs were reconstituted by various combinations of isolated HA-33/HA-17 complexes and haemagglutination-negative L-TC2 or L-TC3 from each strain. HA-hybrid 650 kDa L-TC, including at least one HA-33/HA-17 complex derived from C-Yoichi, lost haemagglutination activity, leading to the conclusion that the binding of four HA-33 molecules is required for haemagglutination activity of botulinum L-TC. The results of the modelling approach indicated that the structure of a variant C-Yoichi HA-33 molecule reveals clear deformation of the beta-trefoil domain responsible for the carbohydrate recognition site.
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Mutoh
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Hasegawa
authors.affiliation: Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku 156-8502, Japan.
authors.forename: Yozo
authors.initials: Y
authors.lastname: Nakazawa
authors.affiliation: Hokkaido Institute of Public Health, N19, W12, Kita-Ku, Sapporo 060-0819, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kouguchi
authors.affiliation: The Sars International Centre for Marine Molecular Biology, Thormøhlensgt 55, N-5008 Bergen, Norway.
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Sagane
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Niwa
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohyama
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003014:Clostridium botulinum / Q000737:chemistry*; D004912:Erythrocytes / Q000378:metabolism*; D006384:Hemagglutination / Q000502:physiology; D006386:Hemagglutination Tests; D006388:Hemagglutinins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D006388:Hemagglutinins; C096325:botulinum toxin type D; D001905:Botulinum Toxins; C096324:botulinum toxin type C