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. 2002 May 1;363(Pt 3):697–705. doi: 10.1042/0264-6021:3630697

Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin.

Christiane Rose 1, Stéphanie Voisin 1, Claude Gros 1, Jean-Charles Schwartz 1, Tanja Ouimet 1
PMCID: PMC1222522  PMID: 11964170

Abstract

Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.

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Selected References

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  1. Baumer P., Danquechin Dorval E., Bertrand J., Vetel J. M., Schwartz J. C., Lecomte J. M. Effects of acetorphan, an enkephalinase inhibitor, on experimental and acute diarrhoea. Gut. 1992 Jun;33(6):753–758. doi: 10.1136/gut.33.6.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bralet J., Schwartz J. C. Vasopeptidase inhibitors: an emerging class of cardiovascular drugs. Trends Pharmacol Sci. 2001 Mar;22(3):106–109. doi: 10.1016/s0165-6147(00)01644-8. [DOI] [PubMed] [Google Scholar]
  3. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  4. Devault A., Lazure C., Nault C., Le Moual H., Seidah N. G., Chrétien M., Kahn P., Powell J., Mallet J., Beaumont A. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA. EMBO J. 1987 May;6(5):1317–1322. doi: 10.1002/j.1460-2075.1987.tb02370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Du L., Desbarats M., Viel J., Glorieux F. H., Cawthorn C., Ecarot B. cDNA cloning of the murine Pex gene implicated in X-linked hypophosphatemia and evidence for expression in bone. Genomics. 1996 Aug 15;36(1):22–28. doi: 10.1006/geno.1996.0421. [DOI] [PubMed] [Google Scholar]
  6. Emoto N., Yanagisawa M. Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. J Biol Chem. 1995 Jun 23;270(25):15262–15268. doi: 10.1074/jbc.270.25.15262. [DOI] [PubMed] [Google Scholar]
  7. Fink C. A., Carlson J. E., McTaggart P. A., Qiao Y., Webb R., Chatelain R., Jeng A. Y., Trapani A. J. Mercaptoacyl dipeptides as orally active dual inhibitors of angiotensin-converting enzyme and neutral endopeptidase. J Med Chem. 1996 Aug 2;39(16):3158–3168. doi: 10.1021/jm960323z. [DOI] [PubMed] [Google Scholar]
  8. Gafford J. T., Skidgel R. A., Erdös E. G., Hersh L. B. Human kidney "enkephalinase", a neutral metalloendopeptidase that cleaves active peptides. Biochemistry. 1983 Jun 21;22(13):3265–3271. doi: 10.1021/bi00282a035. [DOI] [PubMed] [Google Scholar]
  9. Ghaddar G., Ruchon A. F., Carpentier M., Marcinkiewicz M., Seidah N. G., Crine P., Desgroseillers L., Boileau G. Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family. Biochem J. 2000 Apr 15;347(Pt 2):419–429. doi: 10.1042/0264-6021:3470419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorman C. M., Gies D., Schofield P. R., Kado-Fong H., Malfroy B. Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells. J Cell Biochem. 1989 Mar;39(3):277–284. doi: 10.1002/jcb.240390307. [DOI] [PubMed] [Google Scholar]
  11. Gros C., Noël N., Souque A., Schwartz J. C., Danvy D., Plaquevent J. C., Duhamel L., Duhamel P., Lecomte J. M., Bralet J. Mixed inhibitors of angiotensin-converting enzyme (EC 3.4.15.1) and enkephalinase (EC 3.4.24.11): rational design, properties, and potential cardiovascular applications of glycopril and alatriopril. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4210–4214. doi: 10.1073/pnas.88.10.4210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gros C., Souque A., Schwartz J. C. Degradation of atrial natriuretic factor in mouse blood in vitro and in vivo: role of enkephalinase (EC 3.4.24.11). Neuropeptides. 1990 Sep;17(1):1–5. doi: 10.1016/0143-4179(90)90133-j. [DOI] [PubMed] [Google Scholar]
  13. Gros C., Souque A., Schwartz J. C., Duchier J., Cournot A., Baumer P., Lecomte J. M. Protection of atrial natriuretic factor against degradation: diuretic and natriuretic responses after in vivo inhibition of enkephalinase (EC 3.4.24.11) by acetorphan. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7580–7584. doi: 10.1073/pnas.86.19.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hersh L. B., Morihara K. Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin. J Biol Chem. 1986 May 15;261(14):6433–6437. [PubMed] [Google Scholar]
  15. Ikeda K., Emoto N., Raharjo S. B., Nurhantari Y., Saiki K., Yokoyama M., Matsuo M. Molecular identification and characterization of novel membrane-bound metalloprotease, the soluble secreted form of which hydrolyzes a variety of vasoactive peptides. J Biol Chem. 1999 Nov 5;274(45):32469–32477. doi: 10.1074/jbc.274.45.32469. [DOI] [PubMed] [Google Scholar]
  16. Kenny A. J., Stephenson S. L. Role of endopeptidase-24.11 in the inactivation of atrial natriuretic peptide. FEBS Lett. 1988 May 9;232(1):1–8. doi: 10.1016/0014-5793(88)80375-2. [DOI] [PubMed] [Google Scholar]
  17. Kerr M. A., Kenny A. J. The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem J. 1974 Mar;137(3):477–488. doi: 10.1042/bj1370477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lafrance M. H., Vézina C., Wang Q., Boileau G., Crine P., Lemay G. Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11. Biochem J. 1994 Sep 1;302(Pt 2):451–454. doi: 10.1042/bj3020451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee S., Zambas E. D., Marsh W. L., Redman C. M. Molecular cloning and primary structure of Kell blood group protein. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6353–6357. doi: 10.1073/pnas.88.14.6353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Malfroy B., Kado-Fong H., Gros C., Giros B., Schwartz J. C., Hellmiss R. Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases. Biochem Biophys Res Commun. 1989 May 30;161(1):236–241. doi: 10.1016/0006-291x(89)91586-6. [DOI] [PubMed] [Google Scholar]
  21. Malfroy B., Kuang W. J., Seeburg P. H., Mason A. J., Schofield P. R. Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase). FEBS Lett. 1988 Feb 29;229(1):206–210. doi: 10.1016/0014-5793(88)80828-7. [DOI] [PubMed] [Google Scholar]
  22. Malfroy B., Schofield P. R., Kuang W. J., Seeburg P. H., Mason A. J., Henzel W. J. Molecular cloning and amino acid sequence of rat enkephalinase. Biochem Biophys Res Commun. 1987 Apr 14;144(1):59–66. doi: 10.1016/s0006-291x(87)80475-8. [DOI] [PubMed] [Google Scholar]
  23. Malfroy B., Schwartz J. C. Enkephalinase from rat kidney. Purification, characterization, and study of substrate specificity. J Biol Chem. 1984 Dec 10;259(23):14365–14370. [PubMed] [Google Scholar]
  24. Malfroy B., Swerts J. P., Guyon A., Roques B. P., Schwartz J. C. High-affinity enkephalin-degrading peptidase in brain is increased after morphine. Nature. 1978 Nov 30;276(5687):523–526. doi: 10.1038/276523a0. [DOI] [PubMed] [Google Scholar]
  25. Matsas R., Fulcher I. S., Kenny A. J., Turner A. J. Substance P and [Leu]enkephalin are hydrolyzed by an enzyme in pig caudate synaptic membranes that is identical with the endopeptidase of kidney microvilli. Proc Natl Acad Sci U S A. 1983 May;80(10):3111–3115. doi: 10.1073/pnas.80.10.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsas R., Kenny A. J., Turner A. J. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. Biochem J. 1984 Oct 15;223(2):433–440. doi: 10.1042/bj2230433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mauborgne A., Bourgoin S., Benoliel J. J., Hamon M., Cesselin F. Is substance P released from slices of the rat spinal cord inactivated by peptidase(s) distinct from both 'enkephalinase' and 'angiotensin-converting enzyme'? Neurosci Lett. 1991 Feb 25;123(2):221–225. doi: 10.1016/0304-3940(91)90935-m. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Orlowski M., Wilk S. Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. Biochemistry. 1981 Aug 18;20(17):4942–4950. doi: 10.1021/bi00520a021. [DOI] [PubMed] [Google Scholar]
  30. Ouimet T., Facchinetti P., Rose C., Bonhomme M. C., Gros C., Schwartz J. C., Tanja O. Neprilysin II: A putative novel metalloprotease and its isoforms in CNS and testis. Biochem Biophys Res Commun. 2000 May 19;271(3):565–570. doi: 10.1006/bbrc.2000.2664. [DOI] [PubMed] [Google Scholar]
  31. Patey G., De La Baume S., Schwartz J. C., Gros C., Roques B., Fournie-Zaluski M. C., Soroca-Lucas E. Selective protection of methionine enkephalin released from brain slices by enkephalinase inhibition. Science. 1981 Jun 5;212(4499):1153–1155. doi: 10.1126/science.7015510. [DOI] [PubMed] [Google Scholar]
  32. Raharjo S. B., Emoto N., Ikeda K., Sato R., Yokoyama M., Matsuo M. Alternative splicing regulates the endoplasmic reticulum localization or secretion of soluble secreted endopeptidase. J Biol Chem. 2001 May 7;276(27):25612–25620. doi: 10.1074/jbc.M101703200. [DOI] [PubMed] [Google Scholar]
  33. Roques B. P., Fournié-Zaluski M. C., Soroca E., Lecomte J. M., Malfroy B., Llorens C., Schwartz J. C. The enkephalinase inhibitor thiorphan shows antinociceptive activity in mice. Nature. 1980 Nov 20;288(5788):286–288. doi: 10.1038/288286a0. [DOI] [PubMed] [Google Scholar]
  34. Schwartz J. C., Gros C., Lecomte J. M., Bralet J. Enkephalinase (EC 3.4.24.11) inhibitors: protection of endogenous ANF against inactivation and potential therapeutic applications. Life Sci. 1990;47(15):1279–1297. doi: 10.1016/0024-3205(90)90192-t. [DOI] [PubMed] [Google Scholar]
  35. Spillantini M. G., Sicuteri F., Salmon S., Malfroy B. Characterization of endopeptidase 3.4.24.11 ("enkephalinase") activity in human plasma and cerebrospinal fluid. Biochem Pharmacol. 1990 Apr 15;39(8):1353–1356. doi: 10.1016/0006-2952(90)90012-a. [DOI] [PubMed] [Google Scholar]
  36. Stephenson S. L., Kenny A. J. The metabolism of neuropeptides. Hydrolysis of peptides by the phosphoramidon-insensitive rat kidney enzyme 'endopeptidase-2' and by rat microvillar membranes. Biochem J. 1988 Oct 1;255(1):45–51. doi: 10.1042/bj2550045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Valdenaire O., Richards J. G., Faull R. L., Schweizer A. XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in the CNS. Brain Res Mol Brain Res. 1999 Feb 5;64(2):211–221. doi: 10.1016/s0169-328x(98)00321-0. [DOI] [PubMed] [Google Scholar]
  38. Xu D., Emoto N., Giaid A., Slaughter C., Kaw S., deWit D., Yanagisawa M. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell. 1994 Aug 12;78(3):473–485. doi: 10.1016/0092-8674(94)90425-1. [DOI] [PubMed] [Google Scholar]
  39. Zuzel K. A., Rose C., Schwartz J. C. Assessment of the role of "enkephalinase" in cholecystokinin inactivation. Neuroscience. 1985 May;15(1):149–158. doi: 10.1016/0306-4522(85)90129-0. [DOI] [PubMed] [Google Scholar]

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