Recombinant Clostridium botulinum D phage Botulinum neurotoxin type D (botD), partial

Function

Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of light chain (LC) into eukaryotic host cell cytosol. Composed of 3 subdomains; the translocation domain (TD), and N-terminus and C-terminus of the receptor-binding domain (RBD). The RBD is responsible for the adherence of the toxin to the eukaryotic target cell surface. The N-terminus of the TD wraps an extended belt around the perimeter of the LC, protecting Zn(2+) in the active site; it may also prevent premature LC dissociation from the translocation channel and protect toxin prior to translocation (PubMed:17907800). The TD inserts into synaptic vesicle membrane to allow translocation into the host cytosol (By similarity). The RBD binds eukaryotic host phosphatidylethanolamine, which may serve as toxin receptor (PubMed:16115873). Treatment of synaptosomes with proteinase K does not reduce HC binding, suggesting there is no protein receptor or it is protected from extracellular proteases (PubMed:16115873). HC significantly decreases uptake and toxicity of whole BoNT/D (PubMed:19650874, PubMed:21483489). HC also interferes with uptake of tetanus toxin (PubMed:19650874). Has 2 closely located carbohydrate-binding receptor sites and binds at least 1 GT1b ganglioside (PubMed:20704566). Bind gangliosides in the order GD2 > GT1b > GD1b (PubMed:21632541). Interacts with eukaryotic target protein SV2B (synaptic vesicle glycoprotein 2B) (PubMed:21483489). Expression of SV2A, SV2B or SV2C in mice knocked-out for the SV2 proteins restores entry of BoNT/D and cleavage of VAMP2, suggesting SV2 acts as its receptor (PubMed:21483489). Unlike BoNT/A and BoNT/E, toxin uptake is not mediated by large extracellular loop 4 of SV2 (PubMed:21483489). Another group finds very poor interaction with SV2 proteins, suggesting the possible protein receptor may not have been identified (PubMed:21632541)

Subcellular Location

Secreted

Subunit

Heterodimer; disulfide-linked heterodimer of a light chain (LC) and a heavy chain (HC) (PubMed:26324071). The LC has the proteolytic/pharmacological activity (PubMed:8175689, PubMed:8197120). The N- and C-termini of the HC mediate channel formation and eukaryotic host cell binding, respectively. Can also be purified in complex with a non-toxic component that is larger than the HC (PubMed:16252491). Single chain toxin from strain D-4947 copurifies with NTHNA, and in complexes that include NTNHA, HA-70, HA-33 and HA-17 (PubMed:11713244, PubMed:17581814). Dichain toxin from strain CB-16 phage d-16 phi copurifies with NTHNA, and in complexes that include NTNHA, HA-55, HA-33, HA-22 and HA-17 (PubMed:8569530). The stoichiometry of the whole complex has been modeled as one BoNT/D, one NTNHA, three HA-70, six HA-33 and three HA-17 (PubMed:17581814). HC interacts with eukaryotic protein synaptic vesicle glycoprotein 2B (SV2B), which may serve as its receptor (PubMed:21483489). Another group does not find a convincing interaction with SV2 (PubMed:21632541)

• Binz T. et al. Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D. — Nucleic Acids Res. (1990)
• Sunagawa H. et al. The complete amino acid sequence of the Clostridium botulinum type D neurotoxin, deduced by nucleotide sequence analysis of the encoding phage d-16 phi genome. — J. Vet. Med. Sci. (1992)
• Moriishi K. et al. Molecular diversity of neurotoxins from Clostridium botulinum type D strains. — Infect. Immun. (1989)
• Ohyama T. et al. Characterization of nontoxic-nonhemagglutinin component of the two types of progenitor toxin (M and L) produced by Clostridium botulinum type D CB-16. — Microbiol. Immunol. (1995)
• Kouguchi H. et al. In vitro reconstitution of the Clostridium botulinum type D progenitor toxin. — J. Biol. Chem. (2002)
• Hasegawa K. et al. A novel subunit structure of Clostridium botulinum serotype D toxin complex with three extended arms. — J. Biol. Chem. (2007)
• Yamasaki S. et al. Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. — J. Biol. Chem. (1994)
• Yamasaki S. et al. Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F. — Proc. Natl. Acad. Sci. U.S.A. (1994)