The role of the synaptic protein snap-25 in the potency of botulinum neurotoxin type A

Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137-142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca(2+). In this case, vesicular release is restored by increasing intracellular Ca(2+). These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca(2+).