Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain

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.

The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity

“…In contrast, the protein-protein interaction of scAA-A with its non-glycosylated protein receptor SV2C was reduced about 10-fold ( Figure 4A), although the SV2C binding site was allocated to a β-strand in H CC A [15,45] present in scAA-A. Similar findings were obtained for rA∆H CN [17]. However, the SV2C site is in close proximity to the H CN interface and in the absence of H CN A as in scAA-A the translocation domain H N might shield access to the SV2 site in H CC A (Figure 1).…”

“…Although an isolated BoNT/A H CN (EGFP-H CN /A) binds to the plasma membrane of neurons in discrete spots identified as sphingomyelin-enriched membrane microdomains without being internalized [50], a directed mutagenesis analysis of a site suggested for interaction with PIP did not confirm this hypothesis [17]. In the latter study, also a BoNT/A H CN deletion mutant was generated, replacing the segment I874-Q1091 by two glycines (=rA∆H CN ) and subsequently characterized in detail.…”

“…Their results are in accordance with the 500-fold reduction of biological activity of our deletion mutants scAA-A and scAAL12A ( Figure 3B). Their rA∆H CN lacked high-affinity binding to rat cultured cerebellar granule neurons at 4 • C as well as subsequent internalization [17]. It remains unclear whether the rA∆H CN maintained its ability to bind to complex polysialo-gangliosides.…”

“…Only the H CN of BoNT/A harbors part of the N-glycan binding site of its protein receptor SV2 [15,16]. Accordingly, a mutant form of recombinant BoNT/A lacking H CN (rA∆H CN ) showed a 33,000-fold reduced lethality compared to wild-type BoNT/A, presumably due to impaired SV2C binding [17]. However, any additional function of H CN A and the role of the 25 kDa H CN domain in all other serotypes still remains elusive.…”

The structure of the tetanus toxin reveals pH‐mediated domain dynamics

“…In TeNT, H C interacts extensively with the two other domains via its N-terminal subdomain (H CN ). It was recently demonstrated that H CN of BoNT/A contributes to glycan binding of SV2C [45] and is essential for toxicity [46], in addition to some evidence that it binds…”