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 nontarget tissues, were present in a recognized mammalian target tissue, the mouse hemidiaphragm. Furthermore, we reported that SNAP-25, syntaxin I, and synaptobrevin II were cleaved by incubation of the intact hemidiaphragm in botulinum serotypes A, C, and D, respectively. The objective of the current study was to use the mouse phrenic nerve-hemidiaphragm preparation and botulinum 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 botulinum toxin type A (10(-8) M) revealed < or =10% loss of SNAP-25 immunoreactivity at 1 h postparalysis, and > or =75% loss at 5 h postparalysis. Triticum vulgaris lectin, an agent that competitively antagonizes toxin binding, antagonized toxin-induced paralysis as well as SNAP-25 cleavage. Methylamine hydrochloride, an agent that prevents pH-dependent translocation, also antagonized toxin-induced paralysis and SNAP-25 cleavage. Furthermore, zinc chelation antagonized toxin-induced paralysis and SNAP-25 cleavage. These results demonstrate that cleavage of SNAP-25 by botulinum serotype A fulfills the requirements of the multistep model of botulinum toxin action that includes receptor-mediated endocytosis, pH-dependent translocation, and zinc-dependent proteolysis. Furthermore, the minimal amount of SNAP-25 cleavage at 1 h postparalysis suggests that inactivation of only a small but functionally important pool of SNAP-25 is necessary for paralysis.
Kalandakanond
S
S
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, USA.
Coffield
J A
JA
D000818:Animals; D001692:Biological Transport; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000633:toxicity; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003964:Diaphragm / Q000187:drug effects / Q000378:metabolism; D004594:Electrophysiology; D004705:Endocytosis / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009465:Neuromuscular Agents / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000633:toxicity; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism; D010243:Paralysis / Q000139:chemically induced; D050825:Synaptosomal-Associated Protein 25; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D015032:Zinc / Q000502:physiology*
D002614:Chelating Agents; D008565:Membrane Proteins; D008744:Methylamines; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D014909:Wheat Germ Agglutinins; C027451:methylamine; D019274:Botulinum Toxins, Type A; D015032:Zinc