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

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (H C /A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant H C /A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rH C /A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rH C /A dimerized FGFR subtypes in the rank order FGFR3c (EC 50 ≈ 27 nM) > FGFR2b (EC 50 ≈ 70 nM) > FGFR1c (EC 50 ≈ 163 nM); rH C /A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC 50 ≈ 18 nM). Mutating the ganglioside binding site in H C /A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of H C/ A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein H C /A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization. Botulinum neurotoxin type A (BoNT/A) is a 150 kDa metalloenzyme belonging to the family of neurotoxins produced by Clostridium botulinum. The toxin causes temporary muscle paralysis by inhibiting acetylcholine release at the neuromuscular junction 1-4. The neuronal specificity and high potency of BoNT/A has allowed its use in the treatment of a large number of medical and aesthetic conditions 3,5-7 , relying on injection of picomolar (pM) concentrations of the toxin. Though BoNT/A has been the subject of extensive study, greater understanding of the complex mechanism associated with BoNT/A's neuronal specificity and cellular entry could lead to further therapeutic applications. BoNT/A is a single-chain protein activated by proteolytic cleavage to form a 150 kDa di-chain molecule. The di-chain is composed of a light chain (L C /A), which encodes a Zn 2+-dependent endopeptidase (~ 50 kDa), linked by a single disulfide bond and non-covalent interactions to a ~ 100 kDa heavy chain (HC) containing the receptor binding and translocation domains 8. The 50 kDa receptor binding domain, H C /A, is located at the C-terminal half of the HC and mediates specific binding and internalization of the toxin into neurons. Following internalization, the translocation domain (H N) of BoNT/A, residing at the N-terminal half of the HC, facilitates the translocation of L C /A from the endocytic vesicle into the cytosol. Once in the cytosol, L C /A enzymatically cleaves the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptosomal-associated protein 25 (SNAP-25) 9,10 , which is essential for mediating vesicular fusion and exocytosis. Cleavage of SNAP-25 leads to inhibition of neurotransmitter/neuropeptide release, including acetylcholine, from neuronal cells and is responsible for BoNT/A's observed pharmacological effects on smooth and skeletal muscles and glands 1,11,12 .