Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jan 1;361(Pt 1):67–76. doi: 10.1042/0264-6021:3610067

Processing of proSAAS in neuroendocrine cell lines.

Nino Mzhavia 1, Yimei Qian 1, Yun Feng 1, Fa-Yun Che 1, Lakshmi A Devi 1, Lloyd D Fricker 1
PMCID: PMC1222280  PMID: 11742530

Abstract

ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse-chase analysis using [(3)H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60-80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.

Full Text

The Full Text of this article is available as a PDF (271.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett D. L., Bailyes E. M., Nielsen E., Guest P. C., Rutherford N. G., Arden S. D., Hutton J. C. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J Biol Chem. 1992 Jul 25;267(21):15229–15236. [PubMed] [Google Scholar]
  2. Berman Y., Mzhavia N., Polonskaia A., Devi L. A. Impaired prohormone convertases in Cpe(fat)/Cpe(fat) mice. J Biol Chem. 2001 Jan 12;276(2):1466–1473. doi: 10.1074/jbc.M008499200. [DOI] [PubMed] [Google Scholar]
  3. Bloomquist B. T., Eipper B. A., Mains R. E. Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. Mol Endocrinol. 1991 Dec;5(12):2014–2024. doi: 10.1210/mend-5-12-2014. [DOI] [PubMed] [Google Scholar]
  4. Cameron A., Fortenberry Y., Lindberg I. The SAAS granin exhibits structural and functional homology to 7B2 and contains a highly potent hexapeptide inhibitor of PC1. FEBS Lett. 2000 May 12;473(2):135–138. doi: 10.1016/s0014-5793(00)01511-8. [DOI] [PubMed] [Google Scholar]
  5. Che F. Y., Yan L., Li H., Mzhavia N., Devi L. A., Fricker L. D. Identification of peptides from brain and pituitary of Cpe(fat)/Cpe(fat) mice. Proc Natl Acad Sci U S A. 2001 Jul 31;98(17):9971–9976. doi: 10.1073/pnas.161542198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Bie I., Marcinkiewicz M., Malide D., Lazure C., Nakayama K., Bendayan M., Seidah N. G. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments. J Cell Biol. 1996 Dec;135(5):1261–1275. doi: 10.1083/jcb.135.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feistner G. J., Højrup P., Evans C. J., Barofsky D. F., Faull K. F., Roepstorff P. Mass spectrometric charting of bovine posterior/intermediate pituitary peptides. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6013–6017. doi: 10.1073/pnas.86.16.6013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feng Y., Reznik S. E., Fricker L. D. Distribution of proSAAS-derived peptides in rat neuroendocrine tissues. Neuroscience. 2001;105(2):469–478. doi: 10.1016/s0306-4522(01)00200-7. [DOI] [PubMed] [Google Scholar]
  9. Flanagan T., Taylor L., Poulter L., Viveros O. H., Diliberto E. J., Jr A novel 1745-dalton pyroglutamyl peptide derived from chromogranin B is in the bovine adrenomedullary chromaffin vesicle. Cell Mol Neurobiol. 1990 Dec;10(4):507–523. doi: 10.1007/BF00712845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fricker L. D., Berman Y. L., Leiter E. H., Devi L. A. Carboxypeptidase E activity is deficient in mice with the fat mutation. Effect on peptide processing. J Biol Chem. 1996 Nov 29;271(48):30619–30624. doi: 10.1074/jbc.271.48.30619. [DOI] [PubMed] [Google Scholar]
  11. Fricker L. D., McKinzie A. A., Sun J., Curran E., Qian Y., Yan L., Patterson S. D., Courchesne P. L., Richards B., Levin N. Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing. J Neurosci. 2000 Jan 15;20(2):639–648. doi: 10.1523/JNEUROSCI.20-02-00639.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fricker L. D., Snyder S. H. Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3886–3890. doi: 10.1073/pnas.79.12.3886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gee P., Rhodes C. H., Fricker L. D., Angeletti R. H. Expression of neuropeptide processing enzymes and neurosecretory proteins in ependyma and choroid plexus epithelium. Brain Res. 1993 Jul 23;617(2):238–248. doi: 10.1016/0006-8993(93)91091-6. [DOI] [PubMed] [Google Scholar]
  14. Hsi K. L., Seidah N. G., De Serres G., Chrétien M. Isolation and NH2-terminal sequence of a novel porcine anterior pituitary polypeptide. Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60. FEBS Lett. 1982 Oct 18;147(2):261–266. doi: 10.1016/0014-5793(82)81055-7. [DOI] [PubMed] [Google Scholar]
  15. Jansen E., Ayoubi T. A., Meulemans S. M., Van de Ven W. J. Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements. J Biol Chem. 1995 Jun 23;270(25):15391–15397. doi: 10.1074/jbc.270.25.15391. [DOI] [PubMed] [Google Scholar]
  16. Kiefer M. C., Tucker J. E., Joh R., Landsberg K. E., Saltman D., Barr P. J. Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. DNA Cell Biol. 1991 Dec;10(10):757–769. doi: 10.1089/dna.1991.10.757. [DOI] [PubMed] [Google Scholar]
  17. Mains R. E., Berard C. A., Denault J. B., Zhou A., Johnson R. C., Leduc R. PACE4: a subtilisin-like endoprotease with unique properties. Biochem J. 1997 Feb 1;321(Pt 3):587–593. doi: 10.1042/bj3210587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mania-Farnell B. L., Botros I., Day R., Davis T. P. Differential modulation of prohormone convertase mRNA by second messenger activators in two cholecystokinin-producing cell lines. Peptides. 1996;17(1):47–54. doi: 10.1016/0196-9781(95)02044-6. [DOI] [PubMed] [Google Scholar]
  19. Martens G. J., Braks J. A., Eib D. W., Zhou Y., Lindberg I. The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5784–5787. doi: 10.1073/pnas.91.13.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Molloy S. S., Thomas L., VanSlyke J. K., Stenberg P. E., Thomas G. Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J. 1994 Jan 1;13(1):18–33. doi: 10.1002/j.1460-2075.1994.tb06231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mzhavia N., Berman Y., Che F. Y., Fricker L. D., Devi L. A. ProSAAS processing in mouse brain and pituitary. J Biol Chem. 2000 Nov 27;276(9):6207–6213. doi: 10.1074/jbc.M009067200. [DOI] [PubMed] [Google Scholar]
  22. Naggert J. K., Fricker L. D., Varlamov O., Nishina P. M., Rouille Y., Steiner D. F., Carroll R. J., Paigen B. J., Leiter E. H. Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat Genet. 1995 Jun;10(2):135–142. doi: 10.1038/ng0695-135. [DOI] [PubMed] [Google Scholar]
  23. Nakayama K. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J. 1997 Nov 1;327(Pt 3):625–635. doi: 10.1042/bj3270625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nielsen E., Welinder B. S., Madsen O. D. Chromogranin-B, a putative precursor of eight novel rat glucagonoma peptides through processing at mono-, di-, or tribasic residues. Endocrinology. 1991 Dec;129(6):3147–3156. doi: 10.1210/endo-129-6-3147. [DOI] [PubMed] [Google Scholar]
  25. Qian Y., Devi L. A., Mzhavia N., Munzer S., Seidah N. G., Fricker L. D. The C-terminal region of proSAAS is a potent inhibitor of prohormone convertase 1. J Biol Chem. 2000 Aug 4;275(31):23596–23601. doi: 10.1074/jbc.M001583200. [DOI] [PubMed] [Google Scholar]
  26. Rovere C., Viale A., Nahon J., Kitabgi P. Impaired processing of brain proneurotensin and promelanin-concentrating hormone in obese fat/fat mice. Endocrinology. 1996 Jul;137(7):2954–2958. doi: 10.1210/endo.137.7.8770919. [DOI] [PubMed] [Google Scholar]
  27. Sayah M., Fortenberry Y., Cameron A., Lindberg I. Tissue distribution and processing of proSAAS by proprotein convertases. J Neurochem. 2001 Mar;76(6):1833–1841. doi: 10.1046/j.1471-4159.2001.00165.x. [DOI] [PubMed] [Google Scholar]
  28. Seidah N. G., Hamelin J., Mamarbachi M., Dong W., Tardos H., Mbikay M., Chretien M., Day R. cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3388–3393. doi: 10.1073/pnas.93.8.3388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seidah N. G., Marcinkiewicz M., Benjannet S., Gaspar L., Beaubien G., Mattei M. G., Lazure C., Mbikay M., Chrétien M. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol. 1991 Jan;5(1):111–122. doi: 10.1210/mend-5-1-111. [DOI] [PubMed] [Google Scholar]
  30. Sigafoos J., Chestnut W. G., Merrill B. M., Taylor L. C., Diliberto E. J., Jr, Viveros O. H. Novel peptides from adrenomedullary chromaffin vesicles. J Anat. 1993 Oct;183(Pt 2):253–264. [PMC free article] [PubMed] [Google Scholar]
  31. Smeekens S. P., Steiner D. F. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem. 1990 Feb 25;265(6):2997–3000. [PubMed] [Google Scholar]
  32. Strub J. M., Garcia-Sablone P., Lonning K., Taupenot L., Hubert P., Van Dorsselaer A., Aunis D., Metz-Boutigue M. H. Processing of chromogranin B in bovine adrenal medulla. Identification of secretolytin, the endogenous C-terminal fragment of residues 614-626 with antibacterial activity. Eur J Biochem. 1995 Apr 15;229(2):356–368. doi: 10.1111/j.1432-1033.1995.tb20476.x. [DOI] [PubMed] [Google Scholar]
  33. Udupi V., Gomez P., Song L., Varlamov O., Reed J. T., Leiter E. H., Fricker L. D., Greeley G. H., Jr Effect of carboxypeptidase E deficiency on progastrin processing and gastrin messenger ribonucleic acid expression in mice with the fat mutation. Endocrinology. 1997 May;138(5):1959–1963. doi: 10.1210/endo.138.5.5113. [DOI] [PubMed] [Google Scholar]
  34. Udupi V., Townsend C. M., Jr, Gomez G., Zhang T., Greeley G. H., Jr Downregulation of prohormone convertase-1 by a phorbol ester. Biochem Biophys Res Commun. 1995 Dec 14;217(2):495–500. doi: 10.1006/bbrc.1995.2803. [DOI] [PubMed] [Google Scholar]
  35. Varlamov O., Fricker L. D. Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface. J Cell Sci. 1998 Apr;111(Pt 7):877–885. doi: 10.1242/jcs.111.7.877. [DOI] [PubMed] [Google Scholar]
  36. Varlamov O., Leiter E. H., Fricker L. Induced and spontaneous mutations at Ser202 of carboxypeptidase E. Effect on enzyme expression, activity, and intracellular routing. J Biol Chem. 1996 Jun 14;271(24):13981–13986. doi: 10.1074/jbc.271.24.13981. [DOI] [PubMed] [Google Scholar]
  37. Vilim F. S., Aarnisalo A. A., Nieminen M. L., Lintunen M., Karlstedt K., Kontinen V. K., Kalso E., States B., Panula P., Ziff E. Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli. Mol Pharmacol. 1999 May;55(5):804–811. [PubMed] [Google Scholar]
  38. Zhou A., Webb G., Zhu X., Steiner D. F. Proteolytic processing in the secretory pathway. J Biol Chem. 1999 Jul 23;274(30):20745–20748. doi: 10.1074/jbc.274.30.20745. [DOI] [PubMed] [Google Scholar]
  39. van Horssen A. M., van den Hurk W. H., Bailyes E. M., Hutton J. C., Martens G. J., Lindberg I. Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2. J Biol Chem. 1995 Jun 16;270(24):14292–14296. doi: 10.1074/jbc.270.24.14292. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES