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.

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.