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+ -dependent endoproteases that potently block neurosecretion. Syntaxin is presently thought to be the sole substrate for BoNT/C1, and synaptosomal-associated protein of Mr = 25 000 (SNAP-25) is selectively proteolyzed by types A and E. In this study, the effects of C1 on Ca2+ -regulated exocytosis of dense core granules from adreno-chromaffin cells were examined together with its underlying molecular action. Intact chromaffin cells were exposed to the toxin, and catecholamine release therefrom was then measured in conjunction with the monitoring of syntaxin cleavage by Western blotting. A good correlation was obtained between degradation of syntaxin 1A/B and reduction in Ca2+- or Ba2+-dependent secretion. 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. Notably, the cleaved SNAP-25 product was similar in size to that produced 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 treatment of chromaffin cells abolished Ca2+ -evoked secretion following digitonin permeabilization, compared with partial inhibition by BoNT/A, suggesting the importance of syntaxin for catecholamine release. Unexpectedly, C1 failed to proteolyze a soluble recombinant SNAP-25, even though it served as an efficient substrate for BoNT/A. These interesting observations suggest that C1 can only efficiently cleave SNAP-25 in intact cells, possibly due to the existence therein of a unique conformation and/or the participation of accessory factors.
Foran
P
P
Department of Biochemistry, Imperial College, London, UK.
Lawrence
G W
GW
Shone
C C
CC
Foster
K A
KA
Dolly
J O
JO
D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000633:toxicity*; D002118:Calcium / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability; D002838:Chromaffin System / Q000187:drug effects* / Q000378:metabolism*; D004072:Digitonin; D010450:Endopeptidases / Q000378:metabolism; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009498:Neurotoxins / Q000633:toxicity*; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25
D002395:Catecholamines; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D010450:Endopeptidases; D001905:Botulinum Toxins; D004072:Digitonin; D002118:Calcium