title: Specificity of botulinum protease for human VAMP family proteins.
abstract: The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT.
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka 598-8531, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ida
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Mori
authors.forename: Tomoko
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kohda
authors.forename: Masafumi
authors.initials: M
authors.lastname: Mukamoto
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.forename: Shunji
authors.initials: S
authors.lastname: Kozaki
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
mesh_terms: D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D003014:Clostridium botulinum / Q000201:enzymology*; D006801:Humans; D059748:Proteolysis; D050683:R-SNARE Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D050683:R-SNARE Proteins; C096323:rimabotulinumtoxinB; C096325:botulinum toxin type D; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096321:botulinum toxin type F