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. 1995 Mar 2;128(6):1019–1028. doi: 10.1083/jcb.128.6.1019

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release

PMCID: PMC2120411  PMID: 7896868

Abstract

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP- 25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

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Selected References

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  1. Ahnert-Hilger G., Bader M. F., Bhakdi S., Gratzl M. Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: inhibitory effect of tetanus toxin on catecholamine secretion. J Neurochem. 1989 Jun;52(6):1751–1758. doi: 10.1111/j.1471-4159.1989.tb07253.x. [DOI] [PubMed] [Google Scholar]
  2. Ahnert-Hilger G., Weller U. Comparison of the intracellular effects of clostridial neurotoxins on exocytosis from streptolysin O-permeabilized rat pheochromocytoma (PC 12) and bovine adrenal chromaffin cells. Neuroscience. 1993 Mar;53(2):547–552. doi: 10.1016/0306-4522(93)90219-6. [DOI] [PubMed] [Google Scholar]
  3. Ahnert-Hilger G., Wiedenmann B. The amphicrine pancreatic cell line, AR42J, secretes GABA and amylase by separate regulated pathways. FEBS Lett. 1992 Dec 7;314(1):41–44. doi: 10.1016/0014-5793(92)81457-w. [DOI] [PubMed] [Google Scholar]
  4. Asfari M., Janjic D., Meda P., Li G., Halban P. A., Wollheim C. B. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992 Jan;130(1):167–178. doi: 10.1210/endo.130.1.1370150. [DOI] [PubMed] [Google Scholar]
  5. Ashton A. C., Dolly J. O. Microtubule-dissociating drugs and A23187 reveal differences in the inhibition of synaptosomal transmitter release by botulinum neurotoxins types A and B. J Neurochem. 1991 Mar;56(3):827–835. doi: 10.1111/j.1471-4159.1991.tb01998.x. [DOI] [PubMed] [Google Scholar]
  6. Baekkeskov S., Aanstoot H. J., Christgau S., Reetz A., Solimena M., Cascalho M., Folli F., Richter-Olesen H., De Camilli P., Camilli P. D. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature. 1990 Sep 13;347(6289):151–156. doi: 10.1038/347151a0. [DOI] [PubMed] [Google Scholar]
  7. Banerjee A., Martin T. F., DasGupta B. R. Nerve growth factor induces sensitivity to botulinum neurotoxin type A in norepinephrine-secreting PC12 cells. Neurosci Lett. 1993 Dec 24;164(1-2):93–96. doi: 10.1016/0304-3940(93)90865-i. [DOI] [PubMed] [Google Scholar]
  8. Baumert M., Maycox P. R., Navone F., De Camilli P., Jahn R. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J. 1989 Feb;8(2):379–384. doi: 10.1002/j.1460-2075.1989.tb03388.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bennett M. K., Calakos N., Scheller R. H. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 1992 Jul 10;257(5067):255–259. doi: 10.1126/science.1321498. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Bittner M. A., DasGupta B. R., Holz R. W. Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells. J Biol Chem. 1989 Jun 25;264(18):10354–10360. [PubMed] [Google Scholar]
  12. Black J. D., Dolly J. O. Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems. Neuroscience. 1987 Nov;23(2):767–779. doi: 10.1016/0306-4522(87)90094-7. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Blasi J., Chapman E. R., Yamasaki S., Binz T., Niemann H., Jahn R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 1993 Dec;12(12):4821–4828. doi: 10.1002/j.1460-2075.1993.tb06171.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Catsicas S., Larhammar D., Blomqvist A., Sanna P. P., Milner R. J., Wilson M. C. Expression of a conserved cell-type-specific protein in nerve terminals coincides with synaptogenesis. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):785–789. doi: 10.1073/pnas.88.3.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. DasGupta B. R., Tepp W. Protease activity of botulinum neurotoxin type E and its light chain: cleavage of actin. Biochem Biophys Res Commun. 1993 Jan 29;190(2):470–474. doi: 10.1006/bbrc.1993.1071. [DOI] [PubMed] [Google Scholar]
  17. Dayanithi G., Ahnert-Hilger G., Weller U., Nordmann J. J., Gratzl M. Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin. Neuroscience. 1990;39(3):711–715. doi: 10.1016/0306-4522(90)90254-2. [DOI] [PubMed] [Google Scholar]
  18. De Camilli P., Jahn R. Pathways to regulated exocytosis in neurons. Annu Rev Physiol. 1990;52:625–645. doi: 10.1146/annurev.ph.52.030190.003205. [DOI] [PubMed] [Google Scholar]
  19. Galli T., Chilcote T., Mundigl O., Binz T., Niemann H., De Camilli P. Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells. J Cell Biol. 1994 Jun;125(5):1015–1024. doi: 10.1083/jcb.125.5.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gansel M., Penner R., Dreyer F. Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals. Pflugers Arch. 1987 Aug;409(4-5):533–539. doi: 10.1007/BF00583812. [DOI] [PubMed] [Google Scholar]
  21. Geddes J. W., Hess E. J., Hart R. A., Kesslak J. P., Cotman C. W., Wilson M. C. Lesions of hippocampal circuitry define synaptosomal-associated protein-25 (SNAP-25) as a novel presynaptic marker. Neuroscience. 1990;38(2):515–525. doi: 10.1016/0306-4522(90)90047-8. [DOI] [PubMed] [Google Scholar]
  22. Gomperts B. D., Tatham P. E. Regulated exocytotic secretion from permeabilized cells. Methods Enzymol. 1992;219:178–189. doi: 10.1016/0076-6879(92)19020-7. [DOI] [PubMed] [Google Scholar]
  23. Hayashi T., McMahon H., Yamasaki S., Binz T., Hata Y., Südhof T. C., Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 1994 Nov 1;13(21):5051–5061. doi: 10.1002/j.1460-2075.1994.tb06834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Herbert V., Lau K. S., Gottlieb C. W., Bleicher S. J. Coated charcoal immunoassay of insulin. J Clin Endocrinol Metab. 1965 Oct;25(10):1375–1384. doi: 10.1210/jcem-25-10-1375. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Le Douarin N. M. On the origin of pancreatic endocrine cells. Cell. 1988 Apr 22;53(2):169–171. doi: 10.1016/0092-8674(88)90375-3. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Matteoli M., Haimann C., Torri-Tarelli F., Polak J. M., Ceccarelli B., De Camilli P. Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7366–7370. doi: 10.1073/pnas.85.19.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McInnes C., Dolly J. O. Ca2(+)-dependent noradrenaline release from permeabilised PC12 cells is blocked by botulinum neurotoxin A or its light chain. FEBS Lett. 1990 Feb 26;261(2):323–326. doi: 10.1016/0014-5793(90)80582-4. [DOI] [PubMed] [Google Scholar]
  30. Molgo J., Comella J. X., Angaut-Petit D., Pecot-Dechavassine M., Tabti N., Faille L., Mallart A., Thesleff S. Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions. J Physiol (Paris) 1990;84(2):152–166. [PubMed] [Google Scholar]
  31. Molgó J., Dasgupta B. R., Thesleff S. Characterization of the actions of botulinum neurotoxin type E at the rat neuromuscular junction. Acta Physiol Scand. 1989 Dec;137(4):497–501. doi: 10.1111/j.1748-1716.1989.tb08786.x. [DOI] [PubMed] [Google Scholar]
  32. Montecucco C., Schiavo G. Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci. 1993 Sep;18(9):324–327. doi: 10.1016/0968-0004(93)90065-u. [DOI] [PubMed] [Google Scholar]
  33. Navone F., Di Gioia G., Jahn R., Browning M., Greengard P., De Camilli P. Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals. J Cell Biol. 1989 Dec;109(6 Pt 2):3425–3433. doi: 10.1083/jcb.109.6.3425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Navone F., Jahn R., Di Gioia G., Stukenbrok H., Greengard P., De Camilli P. Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol. 1986 Dec;103(6 Pt 1):2511–2527. doi: 10.1083/jcb.103.6.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Niemann H., Blasi J., Jahn R. Clostridial neurotoxins: new tools for dissecting exocytosis. Trends Cell Biol. 1994 May;4(5):179–185. doi: 10.1016/0962-8924(94)90203-8. [DOI] [PubMed] [Google Scholar]
  36. Orci L. Macro- and micro-domains in the endocrine pancreas. Diabetes. 1982 Jun;31(6 Pt 1):538–565. doi: 10.2337/diab.31.6.538. [DOI] [PubMed] [Google Scholar]
  37. Orci L. The insulin factory: a tour of the plant surroundings and a visit to the assembly line. The Minkowski lecture 1973 revisited. Diabetologia. 1985 Aug;28(8):528–546. doi: 10.1007/BF00281987. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Oyler G. A., Higgins G. A., Hart R. A., Battenberg E., Billingsley M., Bloom F. E., Wilson M. C. The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations. J Cell Biol. 1989 Dec;109(6 Pt 1):3039–3052. doi: 10.1083/jcb.109.6.3039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Oyler G. A., Polli J. W., Higgins G. A., Wilson M. C., Billingsley M. L. Distribution and expression of SNAP-25 immunoreactivity in rat brain, rat PC-12 cells and human SMS-KCNR neuroblastoma cells. Brain Res Dev Brain Res. 1992 Feb 21;65(2):133–146. doi: 10.1016/0165-3806(92)90172-s. [DOI] [PubMed] [Google Scholar]
  41. Pevsner J., Hsu S. C., Braun J. E., Calakos N., Ting A. E., Bennett M. K., Scheller R. H. Specificity and regulation of a synaptic vesicle docking complex. Neuron. 1994 Aug;13(2):353–361. doi: 10.1016/0896-6273(94)90352-2. [DOI] [PubMed] [Google Scholar]
  42. Polak M., Scharfmann R., Seilheimer B., Eisenbarth G., Dressler D., Verma I. M., Potter H. Nerve growth factor induces neuron-like differentiation of an insulin-secreting pancreatic beta cell line. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5781–5785. doi: 10.1073/pnas.90.12.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Prentki M., Janjic D., Wollheim C. B. The regulation of extramitochondrial steady state free Ca2+ concentration by rat insulinoma mitochondria. J Biol Chem. 1983 Jun 25;258(12):7597–7602. [PubMed] [Google Scholar]
  45. Prentki M., Matschinsky F. M. Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev. 1987 Oct;67(4):1185–1248. doi: 10.1152/physrev.1987.67.4.1185. [DOI] [PubMed] [Google Scholar]
  46. Reetz A., Solimena M., Matteoli M., Folli F., Takei K., De Camilli P. GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J. 1991 May;10(5):1275–1284. doi: 10.1002/j.1460-2075.1991.tb08069.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Sanna P. P., Bloom F. E., Wilson M. C. Dibutyryl-cAMP induces SNAP-25 translocation into the neurites in PC12. Brain Res Dev Brain Res. 1991 Mar 18;59(1):104–108. doi: 10.1016/0165-3806(91)90035-h. [DOI] [PubMed] [Google Scholar]
  49. Schiavo G., Benfenati F., Poulain B., Rossetto O., Polverino de Laureto P., DasGupta B. R., Montecucco C. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature. 1992 Oct 29;359(6398):832–835. doi: 10.1038/359832a0. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Schiavo G., Rossetto O., Santucci A., DasGupta B. R., Montecucco C. Botulinum neurotoxins are zinc proteins. J Biol Chem. 1992 Nov 25;267(33):23479–23483. [PubMed] [Google Scholar]
  52. Schiavo G., Santucci A., Dasgupta B. R., Mehta P. P., Jontes J., Benfenati F., Wilson M. C., Montecucco C. Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett. 1993 Nov 29;335(1):99–103. doi: 10.1016/0014-5793(93)80448-4. [DOI] [PubMed] [Google Scholar]
  53. Schiavo G., Shone C. C., Rossetto O., Alexander F. C., Montecucco C. Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin. J Biol Chem. 1993 Jun 5;268(16):11516–11519. [PubMed] [Google Scholar]
  54. Stecher B., Gratzl M., Ahnert-Hilger G. Reductive chain separation of botulinum A toxin--a prerequisite to its inhibitory action on exocytosis in chromaffin cells. FEBS Lett. 1989 May 8;248(1-2):23–27. doi: 10.1016/0014-5793(89)80424-7. [DOI] [PubMed] [Google Scholar]
  55. Stecher B., Weller U., Habermann E., Gratzl M., Ahnert-Hilger G. The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells. FEBS Lett. 1989 Sep 25;255(2):391–394. doi: 10.1016/0014-5793(89)81129-9. [DOI] [PubMed] [Google Scholar]
  56. Steinhardt R. A., Bi G., Alderton J. M. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science. 1994 Jan 21;263(5145):390–393. doi: 10.1126/science.7904084. [DOI] [PubMed] [Google Scholar]
  57. Söllner T., Bennett M. K., Whiteheart S. W., Scheller R. H., Rothman J. E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 1993 Nov 5;75(3):409–418. doi: 10.1016/0092-8674(93)90376-2. [DOI] [PubMed] [Google Scholar]
  58. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  59. Teitelman G., Lee J. K. Cell lineage analysis of pancreatic islet development: glucagon and insulin cells arise from catecholaminergic precursors present in the pancreatic duct. Dev Biol. 1987 Jun;121(2):454–466. doi: 10.1016/0012-1606(87)90182-5. [DOI] [PubMed] [Google Scholar]
  60. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Trimble W. S., Cowan D. M., Scheller R. H. VAMP-1: a synaptic vesicle-associated integral membrane protein. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4538–4542. doi: 10.1073/pnas.85.12.4538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wiedenmann B., Franke W. W., Kuhn C., Moll R., Gould V. E. Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci U S A. 1986 May;83(10):3500–3504. doi: 10.1073/pnas.83.10.3500. [DOI] [PMC free article] [PubMed] [Google Scholar]

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