title: 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 rAΔHCNfailed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with125I-BoNT/A (Kd= 0.46 nM). At a higher concentration, rAΔHCNdid 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 HCNreduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCNmakes additional contributions to productive internalization/translocation steps beyond binding to neurons.
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Jiafu
authors.initials: J
authors.lastname: Wang
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Jianghui
authors.initials: J
authors.lastname: Meng
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Marc
authors.initials: M
authors.lastname: Nugent
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Minhong
authors.initials: M
authors.lastname: Tang
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: J Oliver
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000109:Acetylcholine / Q000737:chemistry; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D010243:Paralysis / Q000235:genetics* / Q000473:pathology; D061251:Primary Cell Culture; D011485:Protein Binding / Q000235:genetics; D000072417:Protein Domains / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011984:Sensory Receptor Cells / Q000187:drug effects*; D013572:Synaptic Vesicles / Q000737:chemistry / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A; D000109:Acetylcholine