Abstract
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.
Highlights
They regain enzymically-active structures and separate from their HC after reduction of the inter-chain disulphide[17]
Note there was no significant difference in terms of yield (~5 mg/L culture) and purity between Recombinant BoNT/A devoid of the HCN (rA∆HCN) and Recombinantly-produced BoNT/A (rA)
The rA∆HCN SC was converted to a DC form with an expected Mr (~125 k); its constituent LC (~50 k) and HN-HCC (~75 k) were separated by SDS-PAGE only in the presence of reducing agent, confirming that the inter-chain disulphide had been formed in the expressed protein (Fig. 1b)
Summary
They regain enzymically-active structures and separate from their HC after reduction of the inter-chain disulphide[17]. The presence of a di-leucine motif in the LC of BoNT/A is responsible for it displaying the long-lasting duration of action in motor nerves[19], due to persistently truncating synaptosomal-associated protein of 25 k (SNAP-25) This substrate is susceptible to BoNT/E and /C1; the latter truncates syntaxin, whereas BoNT/B, /D, /F and /G cleave vesicle-associated membrane proteins (VAMPs) at distinct bonds [reviewed in ref. It is noted that BoNT/D uses SV2s as receptors and enters neurons independently of the status of glycosylation of SV223 This raises the possibility that HCN may serve additional roles e.g. binding to unknown receptors, aiding internalisation and facilitating the channel formation for translocation of protease. Recombinant BoNT/A devoid of the HCN (rA∆HCN) virtually fails to truncate intra-neuronal SNAP-25 This is due to a lack of high-affinity (KD = 0.46 nM) binding to neurons seen with 125I-radiolabelled BoNT/A. We have deduced that HCN is involved in binding to neurons and entry of BoNT/A leading to proteolytic inactivation of SNAP-25
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
