The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation
Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M)↓ motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies and/or Ala substitutions of the P1–P5 sites suggested that hydrophobic/aliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.
Proproteins are the fundamental units from which bioactive proteins and peptides are derived by limited proteolysis. Eight of the mammalian proprotein convertases (PCs) responsible for the tissue-specific processing of secretory precursors have been identified since 1990. Of these, seven belong to the yeast kexin subfamily of subtilases and exhibit cleavage- specificity for basic sites respecting the consensus (K,R)-(X)n-(K,R)↓, where n = 0, 2, 4, or 6. They are known as furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/LPC, respectively (1–3). Although less generalized, limited proteolysis also occurs at amino acids such as L, V, M, A, T, and S (4, 5). Recently, a subtilisin-kexin-like isozyme called SKI-1/S1P, belonging to the pyrolysin subfamily of subtilases was identified (6, 7). It cleaves at nonbasic residues in the (R,K)-X-(hydrophobic)-(L,T,K,F)↓ motif (3, 6–11).
Evidence for the existence of processing sites not recognized by the known PCs (12) (M. J. Vincent, personal communication) prompted us to mine databases. By using short, conserved segments of the SKI-1 catalytic subunit as baits and the protein blast program (www.ncbi.nlm.nih.gov/BLAST), we identified in the patented database a putative convertase called neural apoptosis-regulated convertase 1 (NARC-1; Millenium Pharmaceuticals, Cambridge, MA, patent no. WO 01/57081 A2) and LP251 (Eli Lilly, patent no. WO 02/14358 A2) recently cloned by two pharmaceutical companies. NARC-1 was identified via the cloning of cDNAs up-regulated after apoptosis induced by serum deprivation in primary cerebellar neurons, whereas LP251 was discovered via global cloning of secretory proteins. Aside from the fact that NARC-1 mRNA is expressed in liver ≫ testis > kidney and that the gene localizes to human chromosome 1p33-p34.3, no information is available on NARC-1 activity, cleavage specificity, cellular and tissue expression, and biological function.
In this article, we show that NARC-1 belongs to the proteinase K subfamily of subtilases. It is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum (ER) at the (Y,I)VV(V,L)(L,M)↓ motif, resulting in the cleavage of its prosegment that remains associated with the secreted enzyme. Tissue distribution analysis in adulthood and during ontogeny in mouse and rat by Northern blots and in situ hybridization histochemistry (ISH) revealed that NARC-1 mRNA is expressed in a limited number of tissues including the liver, kidney, cerebellum, and small intestine. Its expression is sometimes transient, as in kidney epithelial and brain telencephalon cells. On induction of liver regeneration after partial hepatectomy (PHx) in the rat, NARC-1 mRNA levels peaked on days 2–3, whereas those of PC5 were maximal on day 1. NARC-1 overexpression in primary embryonic telencephalon cells obtained from embryonic day 13.5 (E13.5) mice results in a higher percentage of differentiated neurons.
We acknowledge the technical help of A. Chen for microsequencing and PHx, M.-C. Asselin for cell cultures, A. Chamberland for Western blots and quantitative RT-PCR, and S. Basak for peptide synthesis and in vitro assays. The preparation of stable CHO transfectants by G. Siegfried, the bioinformatics assistance of J. Duhaime, and the advice of J. Cromlish and T. Reudelhuber are greatly appreciated. The secretarial work of B. Mary is greatly appreciated. This work was supported in part by Canadian Institutes of Health Research Grants GR11474 (to N.G.S. and M.C.) and MT-14766 (to N.G.S.) as well as Protein Engineering Network Centres of Excellence and Canadian Institutes of Health Research Grant MOP-42479 (to S.S.). S.S. is a Scholar of the Fonds de la Recherche en Sante du Quebec.
|ISH||in situ hybridization histochemistry|
|NARC-1||neural apoptosis-regulated convertase 1|
|CHO||Chinese hamster ovary|
|RE-OP||rostral extension of the olfactory peduncle|
This paper was submitted directly (Track II) to the PNAS office.
- 1. Steiner D F. Curr Opin Chem Biol. 1998;2:31–39.
- 2. Zhou A, Webb G, Zhu X, Steiner D F. J Biol Chem. 1999;274:20745–20748.
- 3. Seidah N G, Chretien M. Brain Res. 1999;848:45–62.
- 4. Docherty K, Steiner D F. Annu Rev Physiol. 1982;44:625–638.
- 5. Seidah N G, Mbikay M, Marcinkiewicz M, Chretien M In: Proteolytic and Cellular Mechanisms in Prohormone and Neuropeptide Precursor Processing. Hook V Y, editor. Georgetown, TX: Landes; 1998. pp. 49–76.
- 6. Seidah N G, Mowla S J, Hamelin J, Mamarbachi A M, Benjannet S, Toure B B, Basak A, Munzer J S, Marcinkiewicz J, Zhong M, et al Proc Natl Acad Sci USA. 1999;96:1321–1326.
- 7. Sakai J, Rawson R B, Espenshade P J, Cheng D, Seegmiller A C, Goldstein J L, Brown M S. Mol Cell. 1998;2:505–514.
- 8. Cheng D, Espenshade P J, Slaughter C A, Jaen J C, Brown M S, Goldstein J L. J Biol Chem. 1999;274:22805–22812.
- 9. Toure B B, Munzer J S, Basak A, Benjannet S, Rochemont J, Lazure C, Chretien M, Seidah N G. J Biol Chem. 2000;275:2349–2358.
- 10. Ye J, Rawson R B, Komuro R, Chen X, Dave U P, Prywes R, Brown M S, Goldstein J L. Mol Cell. 2000;6:1355–1364.
- 11. Elagoz A, Benjannet S, Mammarbassi A, Wickham L, Seidah N G. J Biol Chem. 2002;277:11265–11275.
- 12. Sanchez A J, Vincent M J, Nichol S T. J Virol. 2002;76:7263–7275.
- 13. Benjannet S, Elagoz A, Wickham L, Mamarbachi M, Munzer J S, Basak A, Lazure C, Cromlish J A, Sisodia S, Checler F, et al J Biol Chem. 2001;276:10879–10887.
- 14. Basak A, Chretien M, Seidah N G. FEBS Lett. 2002;514:333–339.
- 15. Seidah N G, Hamelin J, Mamarbachi M, Dong W, Tardos H, Mbikay M, Chretien M, Day R. Proc Natl Acad Sci USA. 1996;93:3388–3393.
- 16. Seidah N G, Chretien M, Day R. Biochimie. 1994;76:197–209.
- 17. Marcinkiewicz M, Seidah N G. J Neurochem. 2000;75:2133–2143.
- 18. Kountouras J, Boura P, Lygidakis N J. Hepatogastroenterology. 2001;48:556–562.
- 19. Ghosh A, Greenberg M E. Neuron. 1995;15:89–103.
- 20. Slack R S, El Bizri H, Wong J, Belliveau D J, Miller F D. J Cell Biol. 1998;140:1497–1509.
- 21. Nuthall H N, Husain J, McLarren K W, Stifani S. Mol Cell Biol. 2002;22:389–399.
- 22. Toma J G, El Bizri H, Barnabe-Heider F, Aloyz R, Miller F D. J Neurosci. 2000;20:7648–7656.
- 23. Siezen R J, Leunissen J A. Protein Sci. 1997;6:501–523.
- 24. Hospital V, Nishi E, Klagsbrun M, Cohen P, Seidah N G, Prat A. Biochem J. 2002;367:229–238.
- 25. Rovere C, Luis J, Lissitzky J C, Basak A, Marvaldi J, Chretien M, Seidah N G. J Biol Chem. 1999;274:12461–12467.
- 26. Lipkind G M, Zhou A, Steiner D F. Proc Natl Acad Sci USA. 1998;95:7310–7315.
- 27. Gluschankof P, Fuller R S. EMBO J. 1994;13:2280–2288.
- 28. Dragon N, Seidah N G, Lis M, Routhier R, Chretien M. Can J Biochem. 1977;55:666–670.
- 29. Benjannet S, Rondeau N, Paquet L, Boudreault A, Lazure C, Chretien M, Seidah N G. Biochem J. 1993;294:735–743.
- 30. Creemers J W, Vey M, Schafer W, Ayoubi T A, Roebroek A J, Klenk H D, Garten W, Van de Ven W J. J Biol Chem. 1995;270:2695–2702.
- 31. Creemers J W, Van de Loo J W, Plets E, Hendershot L M, Van de Ven W J. J Biol Chem. 2000;275:38842–38847.
- 32. Nagahama M, Taniguchi T, Hashimoto E, Imamaki A, Mori K, Tsuji A, Matsuda Y. FEBS Lett. 1998;434:155–159.
- 33. Yan W, Wu F, Morser J, Wu Q. Proc Natl Acad Sci USA. 2000;97:8525–8529.
- 34. Gritti A, Bonfanti L, Doetsch F, Cameron A, Caille I, Alvarez-Buylla A, Lim D A, Galli R, Verdugo J M, Herrera D G, et al J Neurosci. 2002;22:437–445.
- 35. Manitt C, Colicos M A, Thompson K M, Rousselle E, Peterson A C, Kennedy T E. J Neurosci. 2001;21:3911–3922.
- 36. Kubbutat M H, Key G, Duchrow M, Schluter C, Flad H D, Gerdes J. J Clin Pathol. 1994;47:524–528.
- 37. Munzer J S, Basak A, Zhong M, Mamarbachi A, Hamelin J, Savaria D, Lazure C, Hendy G N, Benjannet S, Chretien M, et al J Biol Chem. 1997;272:19672–19681.
- 38. Zhong M, Munzer J S, Basak A, Benjannet S, Mowla S J, Decroly E, Chretien M, Seidah N G. J Biol Chem. 1999;274:33913–33920.
- 39. Yabuta Y, Takagi H, Inouye M, Shinde U. J Biol Chem. 2001;276:44427–44434.
- 40. Dressler G. Trends Cell Biol. 2002;12:390–395.
- 41. Schuurmans C, Guillemot F. Curr Opin Neurobiol. 2002;12:26–34.
- 42. Fausto N. J Hepatol. 2000;32:19–31.
- 43. Sparks J D, Corsetti J P, Sparks C E. Metabolism. 1994;43:681–690.
- 44. Varret M, Rabes J P, Saint-Jore B, Cenarro A, Marinoni J C, Civeira F, Devillers M, Krempf M, Coulon M, Thiart R, et al Am J Hum Genet. 1999;64:1378–1387.
- 45. Hunt S C, Hopkins P N, Bulka K, McDermott M T, Thorne T L, Wardell B B, Bowen B R, Ballinger D G, Skolnick M H, Samuels M E. Arterioscler Thromb Vasc Biol. 2000;20:1089–1093.