Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain |
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| Authors: | Marco Pirazzini Tina Henke Ornella Rossetto Stefan Mahrhold Nadja Krez Andreas Rummel Cesare Montecucco Thomas Binz |
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| Affiliation: | 1. Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Università di Padova, Viale Ugo Bassi 58/B, 35131 Padova, Italy;2. Institut für Biochemie, OE 4310, Medizinische Hochschule Hannover, 30623 Hannover, Germany;3. Institut für Toxicologie, OE 5340, Medizinische Hochschule Hannover, 30623 Hannover, Germany |
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| Abstract: | Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion. |
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| Keywords: | BafA1, bafilomycin A1 BoNT/X, botulinum neurotoxin/serotype X CGNs, cerebellar granule neurons FBS, foetal bovine serum HC, heavy chain HC, C-terminal half of HC HCC, C-terminal domain of the HC-fragment HCN, N-terminal domain of the HC-fragment HN, N-terminal half of HC LC, light chain MEM, minimum essential medium MPN, mice phrenic nerve PBS, phosphate buffered saline PC12, a pheochromocytoma cell line SNAREs, soluble N-ethyl maleimide sensitive factor attachment protein receptors SNAP-25, synaptosomal-associated protein of 25 kDa SV2, synaptic vesicle (glyco-)protein 2 TM, triple mutant VAMP-2, vesicle associated membrane protein 2 WT, wild-type |
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