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FUNCTION Botulinum toxin causes flaccid paralysis by inhibiting neurotransmitter (acetylcholine) release from the presynaptic membranes of nerve terminals of the eukaryotic host skeletal and autonomic nervous system, with frequent heart or respiratory failure (PubMed:16252491, PubMed:8175689). Precursor of botulinum neurotoxin D for which a proteinaceous coreceptor is controversial. In double SV2A/SV2B knockout mice this toxin does not degrade its synaptobrevin target; introducing SV2A, SV2B or SV2C restores target cleavage (PubMed:21483489). Recognition of SV2 by this toxin does not occur via SV2 glycosylation or its large extracellular loop 4 (PubMed:21483489). Another group does not find a convincing interaction with SV2 (PubMed:21632541). Thus a protein receptor for this BoNT serotype has yet to be definitively proven. Recognizes at least 1 complex polysialylated ganglioside found on neural tissue. Electrical stimulation increases uptake of toxin in an ex vivo assay, presumably by transiently exposing a receptor usually found in eukaryotic target synaptic vesicles (PubMed:19650874, PubMed:21483489, PubMed:21632541). Upon synaptic vesicle recycling the toxin is taken up via the endocytic pathway; when the pH of the toxin-containing endosome drops a structural rearrangement occurs so that the N-terminus of the heavy chain (HC) forms pores that allows the light chain (LC) to translocate into the cytosol (By similarity). Once in the cytosol the disulfide bond linking the 2 subunits is reduced and LC cleaves its target protein on synaptic vesicles, preventing their fusion with the cytoplasmic membrane and thus neurotransmitter release (By similarity). Requires complex eukaryotic host polysialogangliosides for full neurotoxicity and for binding to neurons (PubMed:20704566, PubMed:21483489).FUNCTION Has proteolytic activity (PubMed:8175689, PubMed:8197120). After translocation into the eukaryotic host cytosol, inhibits neurotransmitter release by acting as a zinc endopeptidase that cleaves the '61-Lys-|-Leu-62' bond of synaptobrevin-1 (VAMP1), and the equivalent 'Lys-|-Leu' sites in VAMP2 and VAMP3 (PubMed:8175689). Cleaves the '49-Lys-|-Ile-50' bond of A.californica synaptobrevin (AC P35589) (PubMed:8197120). This chain probably has to be partially unfolded to translocate into the eukaryotic host cell cytosol (PubMed:15584922).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).CATALYTIC ACTIVITY Limited hydrolysis of proteins of the neuroexocytosis apparatus, synaptobrevins, SNAP25 or syntaxin. No detected action on small molecule substrates.COFACTOR Binds 1 zinc ion per subunit (PubMed:16519520, PubMed:26324071).ACTIVITY REGULATION Inhibited by dipicolinic acid, captopril, 1,10-phenanthroline and EDTA.BIOPHYSICOCHEMICAL PROPERTIES kcat is 0.59, 146.60 and 113.41 sec(-1) for over-expressed human VAMP1, VAMP2 and VAMP3 respectively.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).SUBCELLULAR LOCATION In animals that have ingested BoNT/D LC acts in the eukaryotic host cytosol (Probable).SUBCELLULAR LOCATION Upon incubation with hippocampal cell colocalizes with SV2C, probably in host synaptic vesicles (PubMed:21483489). Upon incubation with primary neurons HC colocalizes with synaptophysin probably in synaptic vesicles of presynaptic cells; a small portion also colocalizes with RAB5 and may be in synaptic vesicle protein sorting endosomes (PubMed:21632541). Probably integrates into the eukaryotic host synaptic vesicle membrane.DOMAIN Has protease activity (PubMed:8175689, PubMed:8197120).DOMAIN Has 3 functional domains; the translocation domain (TD) and the receptor-binding domain (RBD) which is further subdivided into N- and C-terminal domains (N-RBD and C-RBD, also called Hcn and Hcc) (PubMed:20704566, PubMed:20731382). The N-terminus of the TD wraps an extended belt around the perimeter of the LC which occludes the catalytic pocket (PubMed:26324071). The belt region may be a pseudosubstrate inhibitor which serves as an intramolecular chaperone for the LC prior to its translocation into the host cytosol (PubMed:17907800).BIOTECHNOLOGY Cargo proteins fused to the N-terminus of whole toxin are imported into neurons; stabilized cargo proteins are poorer substrates, suggesting partial unfolding of the cargo protein is necessary for import (PubMed:15584922).BIOTECHNOLOGY Can be used for targeted secretion inhibition in eukaryotic cells. A construct with a mammalian growth hormone-releasing factor ligand domain (GHRH) inserted between the LC and TD, when injected into rats, leads to decreased growth hormone production. The GHRH ligand domain binds to the GHRH receptor on somatotrophs where it is taken up into endosomes. There the TD inserts into the membrane, releasing LC which cleaves synaptobrevin and inhibits growth hormone exocytosis (PubMed:24029240, PubMed:26324071).MISCELLANEOUS There are seven antigenically distinct forms of botulinum neurotoxin: Types A, B, C, D, E, F, and G; new subtypes are quite frequent. Types C and D can undergo domain swapping to create hybrid types.MISCELLANEOUS Botulism poisoning is usually food-borne, either by ingesting toxin or bacterial-contaminated food, or less frequently by inhalation poisoning. In both cases the neurotoxin binds to the apical surface of epithelial cells in the gut or airway. Toxin undergoes receptor-mediated endocytosis (using a different receptor than on target nerve cells), transcytosis across the epithelial cells and release into the general circulation. Once in the general circulation it binds to its target cells.MISCELLANEOUS Botulinum type D neurotoxin is synthesized by D strains of C.botulinum which carry the appropriate bacteriophage.MISCELLANEOUS Botulinum type D South African (D-SA) neurotoxin is released from bacteria as a single chain and presumably cleaved by host proteases into the active dichain (PubMed:2668193). Botulinum type D-1873 neurotoxin is released from bacteria as an already-cleaved dichain (PubMed:16252491). Toxin from strain CB-16 phage d-16 phi is released as an alread-cleaved dichain (PubMed:8569530). Another toxin (possibly from phage CE-beta) is released from over-producing E.coli as a single chain (PubMed:15584922). Type D-4947 is released as a single chain (PubMed:17581814).MISCELLANEOUS This protein can also be encoded on a prophage.MISCELLANEOUS BoNT/D causes animal botulism; it is 1,000-fold less toxic in chickens than BoNT/C1 (PubMed:16115873, PubMed:16252491). Cattle botulism is often caused by type D (PubMed:17913314).SIMILARITY Belongs to the peptidase M27 family.CAUTION The existence of a proteinaceous coreceptor is unclear. In double SV2A/SV2B knockout mice this toxin does not degrade its VAMP target; introducing SV2A, SV2B or SV2C restores target cleavage (PubMed:21483489). However another group does not find a convincing interaction with SV2 (PubMed:21632541).
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