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SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion

Gonelle-Gispert, C.
Niemann, H.
Palmer, M.
Catsicas, S.
Published in Biochemical journal. 1999, vol. 339 ( Pt 1), p. 159-165
Abstract The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187-T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.
Keywords AnimalsBase SequenceBotulinum Toxins/pharmacologyCell LineDNA PrimersHumansInsulin/ secretionIslets of Langerhans/cytology/drug effects/ secretionMembrane ProteinsMutagenesis, Site-DirectedNerve Tissue Proteins/genetics/ metabolismProtein Isoforms/genetics/ metabolismRNA, Messenger/genetics/metabolismRatsSynaptosomal-Associated Protein 25
PMID: 10085240
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GONELLE-GISPERT, C. et al. SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion. In: Biochemical journal, 1999, vol. 339 ( Pt 1), p. 159-165. doi: 10.1042/bj3390159 https://archive-ouverte.unige.ch/unige:8761

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Deposited on : 2010-07-12

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