Abbreviations used : BoNT, botulinal neurotoxin ; GST, glutathione S-transferase ; GT, glutathione ; L chain, light chain ; NSF, N-ethylmaleimide sensitive fusion protein ; PAGE, polyacrylamide gel electrophoresis ; SDS, sodium dodecyl sulfate ; SNAP, soluble NSF attachment protein ; SNAP-23, 23-kDa isoform of SNAP-25 ; SNAP-25, synaptosome-associated protein of 25 kDa ; hSNAP-23 and mSNAP-23, human and murine SNAP-23, respectively ; SNARE, soluble NSF attachment protein receptor ; VAMP, vesicle-associated membrane protein.
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
Version of Record online: 18 JAN 2002
Journal of Neurochemistry
Volume 72, Issue 1, pages 327–337, January 1999
How to Cite
Vaidyanathan, V. V., Yoshino, K.-i., Jahnz, M., Dörries, C., Bade, S., Nauenburg, S., Niemann, H. and Binz, T. (1999), Proteolysis of SNAP-25 Isoforms by Botulinum Neurotoxin Types A, C, and E. Journal of Neurochemistry, 72: 327–337. doi: 10.1046/j.1471-4159.1999.0720327.x
- Issue online: 18 JAN 2002
- Version of Record online: 18 JAN 2002
- Clostridial neurotoxins;
- Protease binding assay
Abstract : 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 × 10-7M) 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.