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Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) and showed that the lethality of this mutant is reduced 3.3 × 104-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔHCN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with 125I-BoNT/A (Kd = 0.46 nM). At a higher concentration, rAΔHCN did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within HCN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCN makes additional contributions to productive internalization/translocation steps beyond binding to neurons.

Numerous naturally occurring toxins can perturb biological systems when they invade susceptible cells. Coupling of pertinent targeting ligands to the active domains of such proteins provides a strategy for directing these to particular cellular populations implicated in disease. A novel approach described herein involved fusion of one mutated immunoglobulin G (IgG) binding moiety of staphylococcal protein A to the SNARE protease and translocation domain of botulinum neurotoxin A (BoNT/A). This chimera could be monovalently coupled to IgG or via its Fc region to recombinant targeting ligands. The utility of the resulting conjugates is demonstrated by the delivery of a SNARE protease into a cell line expressing tropomyosin receptor kinase A (TrkA) through coupling to anti-TrkA IgG or a fusion of Fc and nerve-growth factor. Thus, this is a versitile and innovative technology for conjugating toxins to diverse ligands for retargeted cell delivery of potential therapeutics.

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