botA
Domain
Botulinum neurotoxin A light chain
Has protease activity (PubMed:7578132).
Botulinum neurotoxin A heavy chain
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). Upon trypsin digestion the isolated TD forms channels in bilayers when the cis side is acidic/oxidizing and the trans side is pH 7.0/reducing (PubMed:17666397, PubMed:19096517, PubMed:2446925). The RBD rotates 140 degrees around the TD in the presence of NTNHA (PubMed:22363010). The 3 major domains each serve as a chaperone for the other 2 to ensure they act only in the correct host cell context (PubMed:19096517). In BoNT/A structures the LC is separated from the RBD by the TD; the belt wraps around the perimeter of the LC, protecting Zn(2+) in the active site (PubMed:18032388, PubMed:19351593, PubMed:22363010). The belt region (449-545) may be a pseudosubstrate inhibitor which serves as an intramolecular chaperone for the LC prior to its translocation into the host cytosol (PubMed:17907800).
Function
Botulinum neurotoxin type A
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:15394302, PubMed:7578132). Precursor of botulinum neurotoxin A which has 2 coreceptors; complex polysialylated gangliosides found on neural tissue and specific membrane-anchored proteins of synaptic vesicles. Receptor proteins are exposed on host presynaptic cell membrane during neurotransmitter release, when the toxin heavy chain (HC) binds to them. 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 HC forms pores that allows the light chain (LC) to translocate into the cytosol (PubMed:17666397, PubMed:19096517). 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. Toxin activity requires polysialylated gangliosides; GT1b supports activity better than GD1a (PubMed:12089155). Binds to host peripheral neuronal presynaptic membranes via the synaptic vesicle glycoproteins SV2A, SV2B and SV2C (PubMed:16543415). It binds directly to the largest lumenal (intravesicular) loop of SV2A, SV2B and SV2C that is transiently exposed outside of cells during exocytosis; gangliosides enhance binding (PubMed:16543415, PubMed:16545378, PubMed:18815274). Recognizes an N-linked glycan on SV2 proteins (PubMed:18815274, PubMed:27294781). May also use FGFR3 as a receptor (PubMed:23696738). Toxin uptake into neural cells requires stimulation (incubation with K(+) to stimulate receptor exposure) to be internalized by receptor-mediated endocytosis (PubMed:16543415, PubMed:19650874, PubMed:21632541, PubMed:21832053). Subsequently the toxin colocalizes with its receptor in host cells (PubMed:16543415, PubMed:19650874). Toxin uptake can be blocked by the appropriate SV2 protein fragments in cell culture (PubMed:16543415).
Botulinum neurotoxin A light chain
Has proteolytic activity (PubMed:7578132). After translocation into the eukaryotic host cytosol LC hydrolyzes the '197-Gln-|-Arg-198' bond in SNAP25, blocking neurotransmitter release (PubMed:10694409, PubMed:11700044, PubMed:11827515, PubMed:19351593, PubMed:7578132, PubMed:8243676, PubMed:9886085). Recognizes the '146-Met--Gly-155' region of SNAP25, which confers substrate specificity (PubMed:15592454, PubMed:9886085). Hydrolyzes the '202-Thr-|-Arg-203' bond of mouse SNAP23, but not in human which has a different sequence (PubMed:9886085). Reduction of the interchain disulfide bond occurs in the host cytosol and probably prevents retrotranslocation into the synaptic vesicle (PubMed:17666397). Has slow (occurs over 4 weeks) autocatalytic cleavage, however it is not clear if this is physiologically relevant (PubMed:11565902).
Botulinum neurotoxin A heavy chain
Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of botulinum neurotoxin A light chain (LC) into host cytosol. Composed of 3 subdomains; the translocation domain (TD), and N-terminus and C-terminus of the receptor-binding domain (RBD) (PubMed:19096517). The RBD is responsible for binding to host epithelial cells and transcytosis across them; this uses different receptors than those on nerve cells (PubMed:21106906). RBD is also responsible for adherence of toxin to host nerve cell surface; HC alone prevents uptake of whole toxin by neural cells, and delays paralysis onset by 75% (PubMed:10413679, PubMed:6694738). Isolated RBD also delays paralysis onset (PubMed:21106906). The N-terminus of the RBD binds to phosphatidylinositol, which might play a role in membrane-binding (PubMed:19161982). Binds to host protein receptor synaptic vesicle glycoproteins SV2A, SV2B and SV2C via lumenal loop 4 (PubMed:16545378, PubMed:19650874, PubMed:24240280, PubMed:27294781, PubMed:27313224, PubMed:6370252). Binding can be inhibited by protein fragments from either the HC or SV2C (PubMed:24240280). Isolated HC significantly decreases uptake and toxicity of whole BoNT/A, but also interferes with uptake of BoNT/E and to a lesser extent BoNT/F (PubMed:19650874). The RBD recognizes the N-linked glycan on 'Asn-559' of SV2A, SV2B and SV2C; hydrogen-bonding occurs via 10 well-defined water molecules and stacking of hydrophobic residues (PubMed:27294781). Binds one host GT1b ganglioside, which serves as a coreceptor (PubMed:14731268, PubMed:18704164, PubMed:27958736). Modeling shows the HC can bind both coreceptors (a ganglioside and SV2 protein) simultaneously at different sites (PubMed:24240280). Crystals of the RBD with a GT1b analog can be grown at pH 5.5, indicating the toxin-ganglioside complex could be stable within the endosome (PubMed:18704164). Isolated RBD binds NTNHA (a bacterial protein that protects toxin) with high affinity at pH 6.0 but not at pH 7.5 (PubMed:22363010). The N-terminal belt (residues 449-545) wraps around the perimeter of the LC, probably protecting Zn(2+) in the active site; it is not required for channel formation by the TD domain but may serve to prevent premature LC dissociation from the translocation channel and to protect toxin prior to translocation (PubMed:17907800, PubMed:19351593, PubMed:22158863). The isolated TD forms transmembrane channels of about 15 Angstroms in the absence of a pH gradient; LC translocation requires a pH and redox gradient (pH 5.0/oxidizing in the cis compartment, pH 7.0/reducing in the trans compartment), LC does not unfold unless the cis pH is 6.0 or less (PubMed:17666397, PubMed:19096517, PubMed:2446925). Pores are presumably made by 1-2 toxin molecules (PubMed:23471747). While interaction with the RBD modulates the pH threshold for membrane insertion, the RBD is not essential for toxin degradation of SNAP25 in neural cells (PubMed:19096517).
Post-translational modifications
In a bacterial culture the precursor chain is initally cleaved on the amino side of Gly-445 and is processed more slowly between Lys-448 and Ala-449 to give the final mature heavy chain sequence.
Botulinum neurotoxin A light chain
Has slow autocatalytic activity, cleaves 250-Tyr-Tyr-251, 266-Phe-Gly-267, 419-Phe-Thr-420, 423-Phe-Glu-424, 430-Cys-Val-431, 432-Arg-Gly-433, 438-Lys-Thr-439, and probably 429-Leu-Cys-430 over a period of 4 weeks. Catalysis of the '197-Gln-|-Arg-198' bond in SNAP25 is estimated to be 10(5) more efficient than autocatalysis, leaving the physiological importance of autocatalysis in doubt (PubMed:11565902).
Botulinum neurotoxin A light chain
Ubiquitinated by host HECD2. Deubiquitination by host VCPIP1 prevents degradation by the proteasome.
Sequence Similarities
Belongs to the peptidase M27 family.
Cellular localization
- Botulinum neurotoxin type A
- Secreted
- Secreted
- Cell wall
- Host synapse
- Host presynaptic cell membrane
- Whole toxin may be released from the bacteria during cell wall exfoliation (PubMed:7592120). There are estimated to be 150-500 toxin molecules per um(2) of non-myelinated mouse hemidiaphragm nerve membrane (PubMed:6694738). In mouse hemidiaphragm binds only to nerve terminals, and not to muscle, blood vessels, connective tissue Schwann cells or myelin, toxin can be internalized by this preparation (PubMed:6694738).
- Botulinum neurotoxin A light chain
- Secreted
- Host cytoplasm
- Host cytosol
- Botulinum neurotoxin A heavy chain
- Secreted
- Host synapse
- Host presynaptic cell membrane
- Host cytoplasmic vesicle
- Host secretory vesicle
- Host synaptic vesicle membrane
- Multi-pass membrane protein
- Whole toxin may be released from the bacteria during cell wall exfoliation (PubMed:7592120). Colocalizes with its receptor SV2C (synaptic vesicle glycoprotein 2C) and VGAT (vesicular inhibitory amino acid transporter) in neurons (PubMed:24240280). In neurons HC colocalizes with synaptophysin or VAMP2 probably in synaptic vesicles, a portion also colocalizes with RAB5 and may be in synaptic vesicle protein sorting endosomes (PubMed:21632541, PubMed:21832053). Therefore there may be more than one uptake pathway at nerve terminals. Uptake of HC and whole toxin is slowed by dynamin inhibitors (PubMed:21832053). 1-2 molecules of HC are found in the host synaptic vesicle lumen, uptake and subsequent release of LC is very rapid (PubMed:21832053, PubMed:23471747).
Alternative names
atx, bonT, botA, Botulinum neurotoxin type A, BoNT/A, Bontoxilysin-A, Botulinum neurotoxin type A1, BOTOX