Abstract:BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even tho… Show more
“…Compared with substrate recognition by other serotypes of BoNT, LC/D possesses unique features of substrate recognition (14,15,20,21 , and Pro 154 ) of LC/D. Mutation of each residue to alanine or asparagine had no effect or only a minor effect on substrate hydrolysis, whereas triple mutations to alanine resulted in a much stronger reduction of substrate hydrolysis, highlighting the complementary effects of the three residues forming this pocket.…”
Section: Discussionmentioning
confidence: 98%
“…The P1Ј site of VAMP-2 was shown to be important for LC/D substrate recognition, as seen in other serotypes of BoNT (14,15,20,21). Mutation of the VAMP-2 P1Ј residue (L60A) reduced LC/D substrate hydrolysis by ϳ25-fold.…”
Section: Dual Recognition Of the Vamp-2 P1 Site By The S1 Pocket Of Lc/dmentioning
confidence: 88%
“…A two-region substrate recognition model has been demonstrated in LC/A, LC/B, LC/E, LC/F, and the tetanus neurotoxin LC (14,15,20,21). The significance of the SNARE motif was not consistently proven in these toxins.…”
Background:The mechanism of botulinum neurotoxin D light chain (LC/D) substrate recognition is not well defined. Results: A dual recognition strategy employed by LC/D was revealed, in which one site of VAMP-2 was recognized by two independent, functionally similar LC/D sites that were complementary to each other. Conclusion: LC/D utilizes a unique mechanism for substrate recognition. Significance: This study provides insights for LC/D engineering and antitoxin development.
“…Compared with substrate recognition by other serotypes of BoNT, LC/D possesses unique features of substrate recognition (14,15,20,21 , and Pro 154 ) of LC/D. Mutation of each residue to alanine or asparagine had no effect or only a minor effect on substrate hydrolysis, whereas triple mutations to alanine resulted in a much stronger reduction of substrate hydrolysis, highlighting the complementary effects of the three residues forming this pocket.…”
Section: Discussionmentioning
confidence: 98%
“…The P1Ј site of VAMP-2 was shown to be important for LC/D substrate recognition, as seen in other serotypes of BoNT (14,15,20,21). Mutation of the VAMP-2 P1Ј residue (L60A) reduced LC/D substrate hydrolysis by ϳ25-fold.…”
Section: Dual Recognition Of the Vamp-2 P1 Site By The S1 Pocket Of Lc/dmentioning
confidence: 88%
“…A two-region substrate recognition model has been demonstrated in LC/A, LC/B, LC/E, LC/F, and the tetanus neurotoxin LC (14,15,20,21). The significance of the SNARE motif was not consistently proven in these toxins.…”
Background:The mechanism of botulinum neurotoxin D light chain (LC/D) substrate recognition is not well defined. Results: A dual recognition strategy employed by LC/D was revealed, in which one site of VAMP-2 was recognized by two independent, functionally similar LC/D sites that were complementary to each other. Conclusion: LC/D utilizes a unique mechanism for substrate recognition. Significance: This study provides insights for LC/D engineering and antitoxin development.
“…Of these, subtype F7 was composed exclusively of BoNT F genes from Clostridium baratii strains [21]. Further studies showed that BoNT F exhibits a strict requirement for residues for substrate recognition that distinguishes BoNT F from other zinc endoproteases, including BoNTs A and B [22,23].…”
The first two cases in France of botulism due to Clostridium baratii type F were identified in November 2014, in the same family. Both cases required prolonged respiratory assistance. One of the cases had extremely high toxin serum levels and remained paralysed for two weeks. Investigations strongly supported the hypothesis of a common exposure during a family meal with high level contamination of the source. However, all analyses of leftover food remained negative.
“…Plasmids for the expression of LC/T (1-436) and VAMP2 (1-97) and subsequent protein expression and purification were performed as previously described Chen, Wan). Site directed mutagenesis of pLC/T and pVAMP2 were performed using QuickChange (Stratagene) protocols as previously described .…”
Section: Plasmid Construction For Protein Expressionmentioning
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