title: In vitro reconstitution of the Clostridium botulinum type D progenitor toxin.
abstract: Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kouguchi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Sagane
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sunagawa
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohyama
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification*; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism; D006388:Hemagglutinins / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D006388:Hemagglutinins; D011498:Protein Precursors; C096325:botulinum toxin type D; D010450:Endopeptidases; D014357:Trypsin; D001905:Botulinum Toxins