Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25. Identification of the minimal essential C-terminal residues.
Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1-197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1-206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1-201, 1-202, 1-203 afforded a significant return of exocytosis, unlike shorter forms 1-197, -198, -199, or -200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism.
O'Sullivan
G A
GA
Department of Biochemistry, Imperial College London SW7 2AY, United Kingdom.
Mohammed
N
N
Foran
P G
PG
Lawrence
G W
GW
Oliver Dolly
J
J
D000818:Animals; D001905:Botulinum Toxins / Q000506:poisoning*; D016466:CHO Cells; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects* / Q000378:metabolism; D006224:Cricetinae; D005089:Exocytosis / Q000187:drug effects*; D008565:Membrane Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050825:Synaptosomal-Associated Protein 25; D001905:Botulinum Toxins