Cleavage of SNAP-25 by botulinum toxin type A requires receptor-mediated endocytosis, pH-dependent translocation, and zinc

Previously we reported that SNAP-25, synaptobrevin II, and syntaxin I, the intracellular substrates of botulinum toxin originally identified in nontar get tissues, were present in a recognized mammalian target tissue, the mous e hemidiaphragm. Furthermore, we reported that SNAP-25, syntaxin I, and syn aptobrevin II were cleaved by incubation of the intact hemidiaphragm in bot ulinum serotypes A, C, and D, respectively. The objective of the current st udy was to use the mouse phrenic nerve-hemidiaphragm preparation and botuli num serotype A to investigate 1) the relationship of substrate cleavage to toxin-induced paralysis, and 2) the relevance of substrate cleavage to the mechanism of toxin action. Immunoblot examination of tissues paralyzed by b otulinum toxin type A (10(-8) M) revealed less than or equal to 10% loss of SNAP-25 immunoreactivity at 1 h postparalysis, and greater than or equal t o 75% loss at 5 h postparalysis. Triticum vulgaris lectin, an agent that co mpetitively antagonizes toxin binding, antagonized toxin-induced paralysis as well as SNAP-25 cleavage. Methylamine hydrochloride, an agent that preve nts pH-dependent translocation, also antagonized toxin-induced paralysis an d SNAP-25 cleavage. Furthermore, zinc chelation antagonized toxin-induced p aralysis and SNAP-25 cleavage. These results demonstrate that cleavage of S NAP-25 by botulinum serotype A fulfills the requirements of the multistep m odel of botulinum toxin action that includes receptor-mediated endocytosis, pH-dependent translocation, and zinc-dependent proteolysis. Furthermore, t he minimal amount of SNAP-25 cleavage at 1 h postparalysis suggests that in activation of only a small but functionally important pool of SNAP-25 is ne cessary for paralysis.