Abstract
Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B’s therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.
Highlights
Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin
By combining rational design and saturation mutagenesis, here we identified a series of point mutations in Botulinum neurotoxins (BoNTs)/B that increased its binding affinity to h-Syt II
Binding of WT HC of BoNT/B (HCB) to Syt I can be detected only in the presence of co-receptor gangliosides in pull-down assays, as Syt I has a lower binding affinity toward BoNT/B compared to Syt II15, 30. We found that both E1191M and E1191M/S1199Y mutants bound to h-Syt I in the absence of gangliosides in pull-down assays (Fig. 2c), suggesting that these mutants may enhance binding to h-Syt I
Summary
It has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B’s therapeutic efficacy and reduce adverse effects. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A–G)[1,2,3]. They are composed of a light chain (LC, ~50 kDa) and a heavy chain (HC, ~100 kDa), connected via a disulfide bond. Human motor neurons might be less sensitive to BoNT/B compared to mouse motor neurons, which is consistent with a long-standing clinical observation that BoNT/B has to be used at ~60–100-fold higher doses than BoNT/A to achieve the same level of effects in patients[32, 33]
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