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.
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Research Article|
March 25 1999
SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion
Carmen GONELLE-GISPERT;
Carmen GONELLE-GISPERT
*Laboratoires de Recherche Louis Jeantet, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland
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Philippe A. HALBAN;
Philippe A. HALBAN
*Laboratoires de Recherche Louis Jeantet, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland
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Heiner NIEMANN;
Heiner NIEMANN
†Institut für Biochemie, Medizinische Hochschule Hannover, OE 4310, 30623 Hannover, Germany
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Michael PALMER;
Michael PALMER
‡Institut für medizinische Mikrobiologie, Johannes Gutenberg Universität, Hochhaus am Augustusplatz, 55101 Mainz, Germany
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Stefan CATSICAS;
Stefan CATSICAS
§Institut de Biologie Cellulaire et de Morphologie, 9 rue du Bugnon, 1005 Lausanne, Switzerland
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Karin SADOUL
Karin SADOUL
1
*Laboratoires de Recherche Louis Jeantet, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland
1To whom correspondence should be addressed (e-mail karin.sadoul@medecine.unige.ch).
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Publisher: Portland Press Ltd
Received:
October 20 1998
Revision Received:
January 04 1999
Accepted:
January 26 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 339 (1): 159–165.
Article history
Received:
October 20 1998
Revision Received:
January 04 1999
Accepted:
January 26 1999
Citation
Carmen GONELLE-GISPERT, Philippe A. HALBAN, Heiner NIEMANN, Michael PALMER, Stefan CATSICAS, Karin SADOUL; SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion. Biochem J 1 April 1999; 339 (1): 159–165. doi: https://doi.org/10.1042/bj3390159
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