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. 1997 Jan 15;321(Pt 2):367–373. doi: 10.1042/bj3210367

Mutations within the propeptide, the primary cleavage site or the catalytic site, or deletion of C-terminal sequences, prevents secretion of proPC2 from transfected COS-7 cells.

N A Taylor 1, K I Shennan 1, D F Cutler 1, K Docherty 1
PMCID: PMC1218078  PMID: 9020868

Abstract

PC2 is a neuroendocrine endoprotease involved in the processing of prohormones and proneuropeptides. PC2 is synthesized as a proenzyme which undergoes proteolytic maturation within the cellular secretory apparatus. Cleavage occurs at specific sites to remove the N-terminal propeptide. The aim of the present study was to investigate structural requirements for the transfer of proPC2 through the secretory pathway. A series of mutant proPC2 constructs were transfected into COS-7 cells and the fate of the expressed proteins followed by pulse-chase analysis and immunocytochemistry. Human PC2 was secreted relatively slowly, and appeared in the medium primarily as proPC2 (75 kDa), together with much lower amounts of a processed intermediate (71 kDa) and mature PC2 (68 kDa). Mutations within the primary processing site or the catalytic triad caused the protein to accumulate intracellularly, whereas deletion of part of the propeptide, the P-domain or the C-terminal regions also prevented secretion. Immunocytochemistry showed that wild-type hPC2 was localized mainly in the Golgi, whereas two representative mutants showed a distribution typical of proteins resident in the endoplasmic reticulum. The results suggest that proenzyme processing is not essential for secretion of PC2, but peptides containing mutations that affect the ability of the propeptide (and cleavage sites) to fold within the catalytic pocket are not transferred beyond the early stages of the secretory pathway. C-terminal sequences may be involved in stabilizing such conformations.

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Selected References

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  1. Bruzzaniti A., Goodge K., Jay P., Taviaux S. A., Lam M. H., Berta P., Martin T. J., Moseley J. M., Gillespie M. T. PC8 [corrected], a new member of the convertase family. Biochem J. 1996 Mar 15;314(Pt 3):727–731. doi: 10.1042/bj3140727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Campbell M. J. Lipofection reagents prepared by a simple ethanol injection technique. Biotechniques. 1995 Jun;18(6):1027–1032. [PubMed] [Google Scholar]
  3. Creemers J. W., Siezen R. J., Roebroek A. J., Ayoubi T. A., Huylebroeck D., Van de Ven W. J. Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. J Biol Chem. 1993 Oct 15;268(29):21826–21834. [PubMed] [Google Scholar]
  4. Creemers J. W., Vey M., Schäfer W., Ayoubi T. A., Roebroek A. J., Klenk H. D., Garten W., Van de Ven W. J. Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J Biol Chem. 1995 Feb 10;270(6):2695–2702. doi: 10.1074/jbc.270.6.2695. [DOI] [PubMed] [Google Scholar]
  5. Davey J., Davis K., Imai Y., Yamamoto M., Matthews G. Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe. EMBO J. 1994 Dec 15;13(24):5910–5921. doi: 10.1002/j.1460-2075.1994.tb06936.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eder J., Rheinnecker M., Fersht A. R. Folding of subtilisin BPN': role of the pro-sequence. J Mol Biol. 1993 Sep 20;233(2):293–304. doi: 10.1006/jmbi.1993.1507. [DOI] [PubMed] [Google Scholar]
  7. Fuller R. S., Sterne R. E., Thorner J. Enzymes required for yeast prohormone processing. Annu Rev Physiol. 1988;50:345–362. doi: 10.1146/annurev.ph.50.030188.002021. [DOI] [PubMed] [Google Scholar]
  8. Gluschankof P., Fuller R. S. A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease. EMBO J. 1994 May 15;13(10):2280–2288. doi: 10.1002/j.1460-2075.1994.tb06510.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guest P. C., Arden S. D., Bennett D. L., Clark A., Rutherford N. G., Hutton J. C. The post-translational processing and intracellular sorting of PC2 in the islets of Langerhans. J Biol Chem. 1992 Nov 5;267(31):22401–22406. [PubMed] [Google Scholar]
  10. 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]
  11. Matthews G., Shennan K. I., Seal A. J., Taylor N. A., Colman A., Docherty K. Autocatalytic maturation of the prohormone convertase PC2. J Biol Chem. 1994 Jan 7;269(1):588–592. [PubMed] [Google Scholar]
  12. Meerabux J., Yaspo M. L., Roebroek A. J., Van de Ven W. J., Lister T. A., Young B. D. A new member of the proprotein convertase gene family (LPC) is located at a chromosome translocation breakpoint in lymphomas. Cancer Res. 1996 Feb 1;56(3):448–451. [PubMed] [Google Scholar]
  13. Mizuno K., Nakamura T., Ohshima T., Tanaka S., Matsuo H. Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun. 1988 Oct 14;156(1):246–254. doi: 10.1016/s0006-291x(88)80832-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Nakagawa T., Hosaka M., Torii S., Watanabe T., Murakami K., Nakayama K. Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4. J Biochem. 1993 Feb;113(2):132–135. doi: 10.1093/oxfordjournals.jbchem.a124015. [DOI] [PubMed] [Google Scholar]
  16. Nakayama K., Hosaka M., Hatsuzawa K., Murakami K. Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites. J Biochem. 1991 Jun;109(6):803–806. doi: 10.1093/oxfordjournals.jbchem.a123461. [DOI] [PubMed] [Google Scholar]
  17. Seidah N. G., Gaspar L., Mion P., Marcinkiewicz M., Mbikay M., Chrétien M. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Biol. 1990 Jul-Aug;9(6):415–424. doi: 10.1089/dna.1990.9.415. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Shen F. S., Seidah N. G., Lindberg I. Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells. J Biol Chem. 1993 Nov 25;268(33):24910–24915. [PubMed] [Google Scholar]
  21. Shennan K. I., Seal A. J., Smeekens S. P., Steiner D. F., Docherty K. Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes. J Biol Chem. 1991 Dec 15;266(35):24011–24017. [PubMed] [Google Scholar]
  22. Shennan K. I., Taylor N. A., Docherty K. Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2. J Biol Chem. 1994 Jul 15;269(28):18646–18650. [PubMed] [Google Scholar]
  23. Shennan K. I., Taylor N. A., Jermany J. L., Matthews G., Docherty K. Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events. J Biol Chem. 1995 Jan 20;270(3):1402–1407. doi: 10.1074/jbc.270.3.1402. [DOI] [PubMed] [Google Scholar]
  24. Siezen R. J., Creemers J. W., Van de Ven W. J. Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like proprotein convertases. Eur J Biochem. 1994 Jun 1;222(2):255–266. doi: 10.1111/j.1432-1033.1994.tb18864.x. [DOI] [PubMed] [Google Scholar]
  25. Smeekens S. P., Avruch A. S., LaMendola J., Chan S. J., Steiner D. F. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):340–344. doi: 10.1073/pnas.88.2.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Vey M., Schäfer W., Berghöfer S., Klenk H. D., Garten W. Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J Cell Biol. 1994 Dec;127(6 Pt 2):1829–1842. doi: 10.1083/jcb.127.6.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhu X. L., Ohta Y., Jordan F., Inouye M. Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature. 1989 Jun 8;339(6224):483–484. doi: 10.1038/339483a0. [DOI] [PubMed] [Google Scholar]
  29. van den Ouweland A. M., van Duijnhoven H. L., Keizer G. D., Dorssers L. C., Van de Ven W. J. Structural homology between the human fur gene product and the subtilisin-like protease encoded by yeast KEX2. Nucleic Acids Res. 1990 Feb 11;18(3):664–664. doi: 10.1093/nar/18.3.664. [DOI] [PMC free article] [PubMed] [Google Scholar]

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