title: Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain.
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.
authors.affiliation: Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Università di Padova, Viale Ugo Bassi 58/B, 35131 Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Pirazzini
authors.forename: Tina
authors.initials: T
authors.lastname: Henke
authors.forename: Ornella
authors.initials: O
authors.lastname: Rossetto
authors.forename: Stefan
authors.initials: S
authors.lastname: Mahrhold
authors.forename: Nadja
authors.initials: N
authors.lastname: Krez
authors.forename: Andreas
authors.initials: A
authors.lastname: Rummel
authors.forename: Cesare
authors.initials: C
authors.lastname: Montecucco
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001905:Botulinum Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D019274:Botulinum Toxins, Type A; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009154:Mutation; D009474:Neurons / Q000187:drug effects; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D021381:Protein Transport; D011522:Protons
chemical_list: D009498:Neurotoxins; D011522:Protons; C096323:rimabotulinumtoxinB; D001224:Aspartic Acid; D018698:Glutamic Acid; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A
keywords: BafA1; Bafilomycin A1; BoNT/X; Botulinum neurotoxin; C-terminal domain of the H(C)-fragment; C-terminal half of HC; CGNs; FBS; H(C); H(CC); H(CN); H(N); HC; LC; MEM; MPN; N-terminal domain of the H(C)-fragment; N-terminal half of HC; PBS; PC12; Phrenic nerve hemidiaphragm toxicity test; SNAP-25; SNAREs; SV2; TM; Translocation; VAMP-2; WT; a pheochromocytoma cell line; bafilomycin A1; botulinum neurotoxin/serotype X; cerebellar granule neurons; foetal bovine serum; heavy chain; light chain; mice phrenic nerve; minimum essential medium; pH sensor; phosphate buffered saline; soluble N-ethyl maleimide sensitive factor attachment protein receptors; synaptic vesicle (glyco-)protein 2; synaptosomal-associated protein of 25kDa; triple mutant; vesicle associated membrane protein 2; wild-type