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

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 (FGFR13) in cells. rH(C)/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 approximate to 27 nM) > FGFR2b (EC50 approximate to 70 nM) > FGFR1c (EC50 approximate to 163 nM); rH(C)/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 approximate to 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.

Automated summary

The study demonstrates that recombinant H(C)/A can induce FGFR dimerization, potentially affecting the function of FGFRs directly or indirectly.