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 (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/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 rHC/A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/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 HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization.

Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A’s substrate and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A infiltrates neurons and retains its SNAP23 activity. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.One Sentence SummaryDirected evolution of the BoNT/A protease targets a new cellular protein, SNAP23, expanding its therapeutic potential.

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

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that also serves as an effective therapeutic for a variety of neuromuscular and glandular diseases and disorders. The observed pharmacological effect of BoNT/A is due to specific targeting and entry into motor nerve terminals within muscles followed by cleavage of the SNARE protein, SNAP-25, inducing a block in neurotransmission and temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells are mediated by its binding domain (HC/A), which binds to GT1b ganglioside and protein cell surface receptors. Previously, fibroblast growth factor receptor 3 (FGFR3) was identified as a BoNT/A receptor, in addition to synaptic vesicle protein (SV2). To further study BoNT/A interactions with FGFRs, an FCS & TIRF receptor dimerization assay was developed to measure dimerization of FGFRs in live cells, as FGFR dimerization can be considered an indirect measure for receptor-ligand binding interaction and downstream signaling. The ability of HC/A to facilitate dimerization of three FGFR subtypes (FGFR1-3) was assessed. Recombinant HC/A (rHC/A) was shown to dimerize FGFR subtypes in the rank order FGFR3c > FGFR2b > FGFR1c. With potencies (EC50 values) defined as the concentration of ligand required to dimerize 50% of the receptors, wild type rHC/A dimerized FGFR3c with an EC50 of 24 nM, similar to FGF9, a native FGFR3c ligand, which had an EC50 of 15 nM, while FGFR1c and FGFR2b required higher rHC/A concentrations (≥100 nM and 68 nM, respectively). Furthermore, addition of the GT1b ganglioside to the culture media resulted in increased dimerization, whereas a ganglioside mutant variant of HC/A (rHC/A W1266L;Y1267S) showed decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A (rHC/A T1145A;T1146A). These results support a model wherein BoNT/A interacts with FGFRs, gangliosides, and SV2 on the cell surface to facilitate cell uptake.

Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models

Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1MY) and E1191Q/S1199W (rBoNT/B1QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1MYin complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) Zephyr detects BoNT/A holotoxin at limits of detection (LOD) of 10.0 ± 2.5 ng/mL in assay buffer. Milk matrices (whole milk, 2% milk and non-fat milk) with BoNT/A holotoxin were detected at similar levels (7.4–7.9 ng/mL). BoNT/A complex was positive in carrot, orange, and apple juices at LODs of 32.5–75.0 ng/mL. The detection of BoNT/A complex in solid complex foods (ground beef, smoked salmon, green bean baby puree) ranged from 14.8 ng/mL to 62.5 ng/mL. Detection of BoNT/A complex in the viscous liquid egg matrix required dilution in assay buffer and gave a LOD of 171.9 ± 64.7 ng/mL. These results show that the CANARY® Zephyr assay can be a highly useful qualitative tool in environmental and food safety surveillance programs.

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death.  Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay (CANARY® Zephyr) detects BoNT/A in buffer and various food matrices rapidly in ≤ 40 min, in small volumes ≈ 50 μL, with minimal processing of samples, and is extremely portable (suitcase-sized equipment). BoNT/A was detected at limits of detection (LOD) < 0.075 ng ± 0.02 in assay buffer while milk matrices (non-fat, 2 %, whole milk) increased the LOD to < 0.175 – 0.314 ng. Limits of detection for the assay in complex foods were < 1 ng ± 0.0 (neutralized acidic juices-carrot, orange and apple); < 16.7 ng ± 7.7 (liquid egg); and varied from < 0. 39 – 3.125 ng for solid complex foods (ground beef, green bean baby puree, smoked salmon). These results show that the CANARY® Zephyr assay can be a highly useful tool in clinical, environmental, and food safety surveillance programs.