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. 1999 Apr 1;339(Pt 1):159–165.

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

C Gonelle-Gispert 1, P A Halban 1, H Niemann 1, M Palmer 1, S Catsicas 1, K Sadoul 1
PMCID: PMC1220140  PMID: 10085240

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.

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Selected References

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  1. Bark I. C., Hahn K. M., Ryabinin A. E., Wilson M. C. Differential expression of SNAP-25 protein isoforms during divergent vesicle fusion events of neural development. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1510–1514. doi: 10.1073/pnas.92.5.1510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bark I. C. Structure of the chicken gene for SNAP-25 reveals duplicated exon encoding distinct isoforms of the protein. J Mol Biol. 1993 Sep 5;233(1):67–76. doi: 10.1006/jmbi.1993.1485. [DOI] [PubMed] [Google Scholar]
  3. Bark I. C., Wilson M. C. Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25. Gene. 1994 Feb 25;139(2):291–292. doi: 10.1016/0378-1119(94)90773-0. [DOI] [PubMed] [Google Scholar]
  4. Bark I. C., Wilson M. C. Regulated vesicular fusion in neurons: snapping together the details. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4621–4624. doi: 10.1073/pnas.91.11.4621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett M. K., García-Arrarás J. E., Elferink L. A., Peterson K., Fleming A. M., Hazuka C. D., Scheller R. H. The syntaxin family of vesicular transport receptors. Cell. 1993 Sep 10;74(5):863–873. doi: 10.1016/0092-8674(93)90466-4. [DOI] [PubMed] [Google Scholar]
  6. Binz T., Blasi J., Yamasaki S., Baumeister A., Link E., Südhof T. C., Jahn R., Niemann H. Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J Biol Chem. 1994 Jan 21;269(3):1617–1620. [PubMed] [Google Scholar]
  7. Blasi J., Binz T., Yamasaki S., Link E., Niemann H., Jahn R. Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins. J Physiol Paris. 1994;88(4):235–241. doi: 10.1016/0928-4257(94)90086-8. [DOI] [PubMed] [Google Scholar]
  8. Blasi J., Chapman E. R., Link E., Binz T., Yamasaki S., De Camilli P., Südhof T. C., Niemann H., Jahn R. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature. 1993 Sep 9;365(6442):160–163. doi: 10.1038/365160a0. [DOI] [PubMed] [Google Scholar]
  9. Bock J. B., Klumperman J., Davanger S., Scheller R. H. Syntaxin 6 functions in trans-Golgi network vesicle trafficking. Mol Biol Cell. 1997 Jul;8(7):1261–1271. doi: 10.1091/mbc.8.7.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bock J. B., Lin R. C., Scheller R. H. A new syntaxin family member implicated in targeting of intracellular transport vesicles. J Biol Chem. 1996 Jul 26;271(30):17961–17965. doi: 10.1074/jbc.271.30.17961. [DOI] [PubMed] [Google Scholar]
  11. Boschert U., O'Shaughnessy C., Dickinson R., Tessari M., Bendotti C., Catsicas S., Pich E. M. Developmental and plasticity-related differential expression of two SNAP-25 isoforms in the rat brain. J Comp Neurol. 1996 Apr 1;367(2):177–193. doi: 10.1002/(SICI)1096-9861(19960401)367:2<177::AID-CNE2>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  12. Boyd R. S., Duggan M. J., Shone C. C., Foster K. A. The effect of botulinum neurotoxins on the release of insulin from the insulinoma cell lines HIT-15 and RINm5F. J Biol Chem. 1995 Aug 4;270(31):18216–18218. doi: 10.1074/jbc.270.31.18216. [DOI] [PubMed] [Google Scholar]
  13. Catsicas S., Grenningloh G., Pich E. M. Nerve-terminal proteins: to fuse to learn. Trends Neurosci. 1994 Sep;17(9):368–373. doi: 10.1016/0166-2236(94)90045-0. [DOI] [PubMed] [Google Scholar]
  14. Elferink L. A., Trimble W. S., Scheller R. H. Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system. J Biol Chem. 1989 Jul 5;264(19):11061–11064. [PubMed] [Google Scholar]
  15. Gaisano H. Y., Ghai M., Malkus P. N., Sheu L., Bouquillon A., Bennett M. K., Trimble W. S. Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells. Mol Biol Cell. 1996 Dec;7(12):2019–2027. doi: 10.1091/mbc.7.12.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gutierrez L. M., Viniegra S., Rueda J., Ferrer-Montiel A. V., Canaves J. M., Montal M. A peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory vesicle docking in chromaffin cells. J Biol Chem. 1997 Jan 31;272(5):2634–2639. doi: 10.1074/jbc.272.5.2634. [DOI] [PubMed] [Google Scholar]
  17. Gutiérrez L. M., Cànaves J. M., Ferrer-Montiel A. V., Reig J. A., Montal M., Viniegra S. A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca(2+)-dependent exocytosis in chromaffin cells. FEBS Lett. 1995 Sep 18;372(1):39–43. doi: 10.1016/0014-5793(95)00944-5. [DOI] [PubMed] [Google Scholar]
  18. Hess D. T., Slater T. M., Wilson M. C., Skene J. H. The 25 kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS. J Neurosci. 1992 Dec;12(12):4634–4641. doi: 10.1523/JNEUROSCI.12-12-04634.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jagadish M. N., Fernandez C. S., Hewish D. R., Macaulay S. L., Gough K. H., Grusovin J., Verkuylen A., Cosgrove L., Alafaci A., Frenkel M. J. Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2. Biochem J. 1996 Aug 1;317(Pt 3):945–954. doi: 10.1042/bj3170945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kiraly-Borri C. E., Morgan A., Burgoyne R. D., Weller U., Wollheim C. B., Lang J. Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin. Biochem J. 1996 Feb 15;314(Pt 1):199–203. doi: 10.1042/bj3140199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Land J., Zhang H., Vaidyanathan V. V., Sadoul K., Niemann H., Wollheim C. B. Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal beta-cells. FEBS Lett. 1997 Dec 8;419(1):13–17. doi: 10.1016/s0014-5793(97)01411-7. [DOI] [PubMed] [Google Scholar]
  23. Lane S. R., Liu Y. Characterization of the palmitoylation domain of SNAP-25. J Neurochem. 1997 Nov;69(5):1864–1869. doi: 10.1046/j.1471-4159.1997.69051864.x. [DOI] [PubMed] [Google Scholar]
  24. Lang J., Nishimoto I., Okamoto T., Regazzi R., Kiraly C., Weller U., Wollheim C. B. Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits. EMBO J. 1995 Aug 1;14(15):3635–3644. doi: 10.1002/j.1460-2075.1995.tb00033.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li G., Hidaka H., Wollheim C. B. Inhibition of voltage-gated Ca2+ channels and insulin secretion in HIT cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca2+ channels. Mol Pharmacol. 1992 Sep;42(3):489–488. [PubMed] [Google Scholar]
  26. Linial M. SNARE proteins--why so many, why so few? J Neurochem. 1997 Nov;69(5):1781–1792. doi: 10.1046/j.1471-4159.1997.69051781.x. [DOI] [PubMed] [Google Scholar]
  27. Low S. H., Chapin S. J., Weimbs T., Kömüves L. G., Bennett M. K., Mostov K. E. Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol Biol Cell. 1996 Dec;7(12):2007–2018. doi: 10.1091/mbc.7.12.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martin F., Salinas E., Vazquez J., Soria B., Reig J. A. Inhibition of insulin release by synthetic peptides shows that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca(2+)- but not for guanosine 5'-[gamma-thio]triphosphate-induced secretion. Biochem J. 1996 Nov 15;320(Pt 1):201–205. doi: 10.1042/bj3200201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martín F., Moya F., Gutierrez L. M., Reig J. A., Soria B. Role of syntaxin in mouse pancreatic beta cells. Diabetologia. 1995 Jul;38(7):860–863. doi: 10.1007/s001250050364. [DOI] [PubMed] [Google Scholar]
  30. McMahon H. T., Ushkaryov Y. A., Edelmann L., Link E., Binz T., Niemann H., Jahn R., Südhof T. C. Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature. 1993 Jul 22;364(6435):346–349. doi: 10.1038/364346a0. [DOI] [PubMed] [Google Scholar]
  31. Nagamatsu S., Fujiwara T., Nakamichi Y., Watanabe T., Katahira H., Sawa H., Akagawa K. Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway. J Biol Chem. 1996 Jan 12;271(2):1160–1165. doi: 10.1074/jbc.271.2.1160. [DOI] [PubMed] [Google Scholar]
  32. Nagamatsu S., Nakamichi Y., Yamaguchi K., Sawa H., Akagawa K. Overexpressed syntaxin 1A/HPC-1 inhibits insulin secretion via a regulated pathway, but does not influence glucose metabolism and intracellular Ca2+ in insulinoma cell line beta TC3 cells. Biochem Biophys Res Commun. 1997 Feb 3;231(1):89–93. doi: 10.1006/bbrc.1997.6006. [DOI] [PubMed] [Google Scholar]
  33. Osen-Sand A., Catsicas M., Staple J. K., Jones K. A., Ayala G., Knowles J., Grenningloh G., Catsicas S. Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo. Nature. 1993 Jul 29;364(6436):445–448. doi: 10.1038/364445a0. [DOI] [PubMed] [Google Scholar]
  34. Osen-Sand A., Staple J. K., Naldi E., Schiavo G., Rossetto O., Petitpierre S., Malgaroli A., Montecucco C., Catsicas S. Common and distinct fusion proteins in axonal growth and transmitter release. J Comp Neurol. 1996 Apr 1;367(2):222–234. doi: 10.1002/(SICI)1096-9861(19960401)367:2<222::AID-CNE5>3.0.CO;2-7. [DOI] [PubMed] [Google Scholar]
  35. Oyler G. A., Polli J. W., Wilson M. C., Billingsley M. L. Developmental expression of the 25-kDa synaptosomal-associated protein (SNAP-25) in rat brain. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5247–5251. doi: 10.1073/pnas.88.12.5247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Praz G. A., Halban P. A., Wollheim C. B., Blondel B., Strauss A. J., Renold A. E. Regulation of immunoreactive-insulin release from a rat cell line (RINm5F). Biochem J. 1983 Feb 15;210(2):345–352. doi: 10.1042/bj2100345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rasmussen H., Zawalich K. C., Ganesan S., Calle R., Zawalich W. S. Physiology and pathophysiology of insulin secretion. Diabetes Care. 1990 Jun;13(6):655–666. doi: 10.2337/diacare.13.6.655. [DOI] [PubMed] [Google Scholar]
  38. Ravichandran V., Chawla A., Roche P. A. Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues. J Biol Chem. 1996 Jun 7;271(23):13300–13303. doi: 10.1074/jbc.271.23.13300. [DOI] [PubMed] [Google Scholar]
  39. Regazzi R., Sadoul K., Meda P., Kelly R. B., Halban P. A., Wollheim C. B. Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis. EMBO J. 1996 Dec 16;15(24):6951–6959. [PMC free article] [PubMed] [Google Scholar]
  40. Regazzi R., Wollheim C. B., Lang J., Theler J. M., Rossetto O., Montecucco C., Sadoul K., Weller U., Palmer M., Thorens B. VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion. EMBO J. 1995 Jun 15;14(12):2723–2730. doi: 10.1002/j.1460-2075.1995.tb07273.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Riddelle K. S., Green K. J., Jones J. C. Formation of hemidesmosomes in vitro by a transformed rat bladder cell line. J Cell Biol. 1991 Jan;112(1):159–168. doi: 10.1083/jcb.112.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rouiller D. G., Cirulli V., Halban P. A. Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet cell types. Exp Cell Res. 1990 Dec;191(2):305–312. doi: 10.1016/0014-4827(90)90019-7. [DOI] [PubMed] [Google Scholar]
  43. Sadoul K., Berger A., Niemann H., Weller U., Roche P. A., Klip A., Trimble W. S., Regazzi R., Catsicas S., Halban P. A. SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion. J Biol Chem. 1997 Dec 26;272(52):33023–33027. doi: 10.1074/jbc.272.52.33023. [DOI] [PubMed] [Google Scholar]
  44. Sadoul K., Lang J., Montecucco C., Weller U., Regazzi R., Catsicas S., Wollheim C. B., Halban P. A. SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. J Cell Biol. 1995 Mar;128(6):1019–1028. doi: 10.1083/jcb.128.6.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schiavo G., Rossetto O., Catsicas S., Polverino de Laureto P., DasGupta B. R., Benfenati F., Montecucco C. Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E. J Biol Chem. 1993 Nov 15;268(32):23784–23787. [PubMed] [Google Scholar]
  46. Tang B. L., Low D. Y., Lee S. S., Tan A. E., Hong W. Molecular cloning and localization of human syntaxin 16, a member of the syntaxin family of SNARE proteins. Biochem Biophys Res Commun. 1998 Jan 26;242(3):673–679. doi: 10.1006/bbrc.1997.8029. [DOI] [PubMed] [Google Scholar]
  47. Tang B. L., Low D. Y., Tan A. E., Hong W. Syntaxin 10: a member of the syntaxin family localized to the trans-Golgi network. Biochem Biophys Res Commun. 1998 Jan 14;242(2):345–350. doi: 10.1006/bbrc.1997.7966. [DOI] [PubMed] [Google Scholar]
  48. Tang B. L., Tan A. E., Lim L. K., Lee S. S., Low D. Y., Hong W. Syntaxin 12, a member of the syntaxin family localized to the endosome. J Biol Chem. 1998 Mar 20;273(12):6944–6950. doi: 10.1074/jbc.273.12.6944. [DOI] [PubMed] [Google Scholar]
  49. Veit M., Söllner T. H., Rothman J. E. Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25. FEBS Lett. 1996 Apr 29;385(1-2):119–123. doi: 10.1016/0014-5793(96)00362-6. [DOI] [PubMed] [Google Scholar]
  50. Wang H., Frelin L., Pevsner J. Human syntaxin 7: a Pep12p/Vps6p homologue implicated in vesicle trafficking to lysosomes. Gene. 1997 Oct 15;199(1-2):39–48. doi: 10.1016/s0378-1119(97)00343-0. [DOI] [PubMed] [Google Scholar]
  51. Weller U., Müller L., Messner M., Palmer M., Valeva A., Tranum-Jensen J., Agrawal P., Biermann C., Döbereiner A., Kehoe M. A. Expression of active streptolysin O in Escherichia coli as a maltose-binding-protein--streptolysin-O fusion protein. The N-terminal 70 amino acids are not required for hemolytic activity. Eur J Biochem. 1996 Feb 15;236(1):34–39. doi: 10.1111/j.1432-1033.1996.00034.x. [DOI] [PubMed] [Google Scholar]
  52. Wong S. H., Xu Y., Zhang T., Hong W. Syntaxin 7, a novel syntaxin member associated with the early endosomal compartment. J Biol Chem. 1998 Jan 2;273(1):375–380. doi: 10.1074/jbc.273.1.375. [DOI] [PubMed] [Google Scholar]

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