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Biochemistry. 1997 Mar 18;36(11):3061-7.

Importance of two adjacent C-terminal sequences of SNAP-25 in exocytosis from intact and permeabilized chromaffin cells revealed by inhibition with botulinum neurotoxins A and E.

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1
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, U.K.

Abstract

Types A and E botulinum neurotoxin (BoNT) are Zn2+-requiring endoproteases which cleave nine and twenty-six residues, respectively, from the C-terminus of synaptosomal-associated protein of Mr = 25 kDa (SNAP-25). Involvement of SNAP-25 in the exocytosis of large dense-core vesicles in bovine adrenochromaffin cells was examined by measuring cleavage of SNAP-25 in relation to the levels of Ca2+-evoked catecholamine release from cells exposed to BoNT/A or /E, either before or after permeabilization. The dose-dependency of inhibition of exocytosis correlated closely with the extents of SNAP-25 cleavage in cells permeabilized and then treated with BoNT/E. In intact cells exposed to 66 nM BoNT/A, virtually all of the SNAP-25 was truncated, accompanied by a near-complete inhibition of exocytosis; however, after their permeabilization a significant level of secretion was recorded upon Ca2+-stimulation. Importantly, this BoNT/A-resistant release from the permeabilized cells was dramatically lowered by subsequently adding BoNT/E, which further truncated the SNAP-25 fragment (lacking the C-terminal nine residues) that had been produced earlier by BoNT/A. Moreover, anti-SNAP-25 IgG decreased the BoNT/A-insensitive exocytosis. When permeabilized cells were exposed to either neurotoxin, both blocked MgATP-dependent secretion but only BoNT/E attenuated the energy-independent phase. These distinct inhibitory effects of the two neurotoxins demonstrate that residues 197-205 at the C-terminus of SNAP-25 are absolutely essential for exocytosis from intact cells whereas even after their removal a significant proportion of the exocytotic response can be elicited from permeabilized cells, but this is reliant on amino acids 180-196. Moreover, the latter but not residues 197-205 are implicated in a late, MgATP-independent step of exocytosis, which is blocked by BoNT/E but nonsusceptible to BoNT/A.

PMID:
9115981
DOI:
10.1021/bi9622478
[Indexed for MEDLINE]
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