Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage.
Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.
Vaidyanathan
V V
VV
Department of Biochemistry, Medizinische Hochschule Hannover, Germany.
Yoshino
K
K
Jahnz
M
M
Dörries
C
C
Bade
S
S
Nauenburg
S
S
Niemann
H
H
Binz
T
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D007536:Isomerism; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25
D000596:Amino Acids; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; C494208:SNAP25 protein, human; C107017:Snap23 protein, mouse; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096324:botulinum toxin type C; C096326:botulinum toxin type E