title: Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes.
abstract: Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [Inoue, K. et al. (1999). Microbiology 145, 2533-2542]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.
authors.affiliation: PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan.|Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Fujinaga
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Inoue
authors.affiliation: Laboratory of Veterinary Immunology, Department of Veterinary Science, College of Agriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Watarai
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sakaguchi
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Arimitsu
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Jae-Chul
authors.initials: JC
authors.lastname: Lee
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Yingji
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Matsumura
authors.affiliation: PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan.|Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kabumoto
authors.affiliation: Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2422, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2422, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohyama
authors.affiliation: CREST, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan.|Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
authors.affiliation: Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Oguma
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003014:Clostridium botulinum; D004847:Epithelial Cells / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006168:Guinea Pigs; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C116382:16S toxin, Clostridium botulinum; D001427:Bacterial Toxins; D006388:Hemagglutinins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D001905:Botulinum Toxins; C096324:botulinum toxin type C