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Characterization of the interaction between subunits of the botulinum toxin complex produced by serotype D through tryptic susceptibility of the isolated components and complex forms

¹ Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan
² Hokkaido Institute of Public Health, N19, W12, Kita-Ku, Sapporo 060-0819, Japan
³ The Sars International Centre for Marine Molecular Biology, Thormøhlensgt 55, N-5008 Bergen, Norway
⁴ Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Japan
⁵ Department of Bacteriology, Okayama University, Medical School, 2-5-1 Shikata-Cho, Okayama 700-8558, Japan

Correspondence
Tohru Ohyama
t-oyama{at}bioindustry.nodai.ac.jp

The 650 kDa large toxin complex (L-TC) produced by Clostridium botulinum serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted in vitro by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

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