BOTULINUM NEUROTOXIN C1 CLEAVES BOTH SYNTAXIN AND SNAP-25 IN INTACT AND PERMEABILIZED CHROMAFFIN CELLS - CORRELATION WITH ITS BLOCKADE OF CATECHOLAMINE RELEASE

The seven types (A-G) of botulinum neurotoxin (BoNT) are Zn2+-dependen t endoproteases that potently block neurosecretion. Syntaxin is presen tly thought to be the sole substrate for BoNT/Cl, and synaptosomal-ass ociated protein of M(r) = 25 000 (SNAP-25) is selectively proteolyzed by types A and E. In this study, the effects of C1 on Ca2+-regulated e xocytosis of dense core granules fi om adreno-chromaffin cells were ex amined together with its underlying molecular action. Intact chromaffi n cells were exposed to the toxin, and catecholamine release therefrom was then measured in conjunction with the monitoring of syntaxin clea vage by Western blotting. A good correlation was obtained between degr adation of syntaxin 1A/B and reduction in Ca2+- or Ba2+-dependent secr etion. However, blotting with antibodies against a C-terminal peptide of SNAP-25 revealed the additional disappearance of immunoreactivity, with the same toxin concentration dependency as syntaxin breakdown. No tably, the cleaved SNAP-25 product was similar in size to that produce d by BoNT/A; however, contamination of BoNT/C1 by serotypes A or E was eliminated. Therefore, it is concluded that syntaxin 1A/B and SNAP-25 are cleaved in intact cells poisoned with only C1. Notably, C1 treatm ent of chromaffin cells abolished Ca2+-evoked secretion following digi tonin permeabilization, compared with partial inhibition by BoNT/A, su ggesting the importance of syntaxin for catecholamine release. Unexpec tedly, C1 failed to proteolyze a soluble recombinant SNAP-25, even tho ugh it served as an efficient substrate for BoNT/A. These interesting observations suggest that C1 can only efficiently cleave SNAP-25 in in tact cells, possibly due to the existence therein of a unique conforma tion and/or the participation of accessory factors.