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 Arg(198) and Ala(199), depends on the presence of regions Asn(93) to Glu(145) and Ile(156) to Met(202), 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 (K-D = 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 Met(146)-Gln(197), and binding was optimal only with full-length SNAP-25, Proteolysis by BoNT/E required the presence of the domain Ile(156)-Asp(186). 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. Lys(185)Asp or Pro(182)Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.