Characterization of clostridium botulinum neurotoxin serotype A (BoNT/A) and fibroblast growth factor receptor interactions using a novel receptor dimerization assay

Abstract

FGFR2b required higher rH C /A concentrations (≥100 nM and 68 nM, respectively). Furthermore, 3 43 addition of the GT1b ganglioside to the culture media resulted in increased dimerization, whereas 44 a ganglioside mutant variant of H C /A (rH C /A W1266L;Y1267S) showed decreased dimerization. 45 Interestingly, reduced dimerization was also observed with an SV2 mutant variant of H C/ A (rH C /A 46 T1145A;T1146A). These results support a model wherein BoNT/A interacts with FGFRs, 47 48 49 Current word count: 299 words 50 PLOS one Abstract length: 300 words 51 52 53 54 55 56 57 58 59 60 4 61 65 specificity and high potency of BoNT/A has allowed its use in the treatment of a large number of 66 medical and aesthetic conditions [3, 5-7], relying on injection of picomolar (pM) concentrations 67 of the toxin. Though BoNT/A has been the subject of extensive study, greater understanding of 68 the complex mechanism associated with BoNT/A's neuronal specificity and cellular entry could 69 lead to further therapeutic applications. 70 BoNT/A is a single-chain protein activated by proteolytic cleavage to form a 150 kDa di-71 chain molecule. The di-chain is comprised of a light chain (L C /A) which encodes a Zn 2+ dependent 72 endopeptidase (~50 kDa), linked by a single disulfide bond and non-covalent interactions to a 73~100 kDa heavy chain (HC) containing the receptor binding and translocation domains [8]. The 74 50 kDa receptor binding domain, H C /A, is located at the C-terminal half of the HC and mediates 75 specific binding and internalization of the toxin into neurons. Following internalization, the 76 translocation domain (H N ) of BoNT/A residing at the N-terminal half of the HC facilitates the 77 translocation of L C /A from the endocytic vesicle into the cytosol. Once in the cytosol, L C /A 78 enzymatically cleaves the soluble N-ethylmaleimide-sensitive factor attachment protein receptor 79 (SNARE), synaptosomal-associated protein 25 (SNAP-25) [9, 10], which is essential for mediating 80 vesicular fusion and exocytosis. Cleavage of SNAP-25 leads to inhibition of 81 neurotransmitter/neuropeptide release, including acetylcholine, from neuronal cells and is

OnabotulinumtoxinA effects on trigeminal nociceptors

“…The botulinum neurotoxin type A (BoNT/A) complex is a 900 kDa protein complex composed of the 150 kDa neurotoxin protein, which is made up of an ∼100 kDa heavy chain (H C /A) and an ∼50 kDa light chain (L C /A), that is associated with several non-toxic neurotoxin-associated proteins (1). As part of BoNT/A’s classical MoA, the receptor binding domain of the H C /A first binds to polysialoganglioside (PSG), synaptic vesicle protein 2 (SV2), and potentially fibroblast growth factor receptor 3 (FGFR3) receptors on peripheral nerve terminals before being endocytosed (2,3). Once endosome-bound, the L C /A dissociates from the H C /A and translocates through the vesicular membrane into the cytosol where it enzymatically cleaves synaptosomal-associated protein-25 kDa (SNAP25), preventing soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis of small synaptic vesicles containing acetylcholine.…”