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. 1998 Jul 15;333(Pt 2):439–448. doi: 10.1042/bj3330439

Recombinant human endothelin-converting enzyme ECE-1b is located in an intracellular compartment when expressed in polarized Madin-Darby canine kidney cells.

A Azarani 1, G Boileau 1, P Crine 1
PMCID: PMC1219603  PMID: 9657986

Abstract

Endothelin-converting enzyme (ECE) is a phosphoramidon-sensitive membrane-bound metalloprotease responsible for the conversion of big-endothelins into endothelins [Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Yazaki, Goto and Masaki (1988) Nature (London) 332, 411-415]. Several distinct isoforms of ECE have been cloned and identified. ECE-1a, b and c have the same ectodomain and differ only by their cytosolic tails [Schweizer, Valdenaire, Nelböck, Deuschle, Edwards, Stumpf and Löffler (1997) Biochem. J. 328, 871-877]. The ectodomain common to ECE-1 a, b and c shares extensive sequence similarities with neprilysin, a major kidney brush border metallopeptidase. To study the sorting of ECE in polarized cells, ECE-1bcDNA was expressed by transfection in polarized Madin-Darby canine kidney (MDCK) cells. Cell-surface biotinylation and immunofluorescence studies showed that ECE-1b is not expressed on the cell-surface but was rather located in intracellular compartments that could also be labelled with anti-Rab-5 and Rab-7 antibodies and was thus tentatively identified as early and late endosomes. Similar results were also obtained when ECE-1b was expressed in non-polarized Chinese hamster ovary cells for comparison purposes. When MDCK or Chinese hamster ovary transfected cells were pre-treated with the ECE inhibitor phosphoramidon, a 3-fold increase in the level of ECE-1b was observed both by Western blotting and by enzymic activity. However, no change in the level of neprilysin or the beta-chain of meprin, two apical membrane metallopeptidases, was observed in MDCK cells transfected under similar conditions. Northern blotting showed that the increase in the level of ECE-1b was not owing to changes in the ECEmRNA transcription rate or stability. Rather, pulse-chase experiments followed by immunoprecipitation showed a decrease in the rate of degradation of ECE-1b in phosphoramidon-treated cells. Half-lives were determined to be 2.8 and 7.5 h for non-treated and phosphoramidon-treated cells, respectively. Confocal microscopy showed accumulation of ECE-1b immunoreactive material in the lysosomes of phosphoramidon-treated cells. Taken together, these results suggest that ECE-1b turns over very rapidly between endosomal and lysosomal compartments and that lysosomal degradation of the enzyme is slowed down by phosphoramidon.

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Selected References

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  1. Azarani A., Orlowski J., Goltzman D. Parathyroid hormone and parathyroid hormone-related peptide activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic cells (UMR-106) via a cAMP-dependent pathway. J Biol Chem. 1995 Sep 29;270(39):23166–23172. doi: 10.1074/jbc.270.39.23166. [DOI] [PubMed] [Google Scholar]
  2. Barnes K., Murphy L. J., Takahashi M., Tanzawa K., Turner A. J. Localization and biochemical characterization of endothelin-converting enzyme. J Cardiovasc Pharmacol. 1995;26 (Suppl 3):S37–S39. [PubMed] [Google Scholar]
  3. Barnes K., Shimada K., Takahashi M., Tanzawa K., Turner A. J. Metallopeptidase inhibitors induce an up-regulation of endothelin-converting enzyme levels and its redistribution from the plasma membrane to an intracellular compartment. J Cell Sci. 1996 May;109(Pt 5):919–928. doi: 10.1242/jcs.109.5.919. [DOI] [PubMed] [Google Scholar]
  4. Baynash A. G., Hosoda K., Giaid A., Richardson J. A., Emoto N., Hammer R. E., Yanagisawa M. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell. 1994 Dec 30;79(7):1277–1285. doi: 10.1016/0092-8674(94)90018-3. [DOI] [PubMed] [Google Scholar]
  5. Benlimame N., Simard D., Nabi I. R. Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle. J Cell Biol. 1995 Apr;129(2):459–471. doi: 10.1083/jcb.129.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caldwell P. R., Seegal B. C., Hsu K. C., Das M., Soffer R. L. Angiotensin-converting enzyme: vascular endothelial localization. Science. 1976 Mar 12;191(4231):1050–1051. doi: 10.1126/science.175444. [DOI] [PubMed] [Google Scholar]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen Y. G., Danoff A., Shields D. The propeptide of anglerfish preprosomatostatin-I rescues prosomatostatin-II from intracellular degradation. J Biol Chem. 1995 Aug 4;270(31):18598–18605. doi: 10.1074/jbc.270.31.18598. [DOI] [PubMed] [Google Scholar]
  9. Corbeil D., Boileau G., Lemay G., Crine P. Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains. J Biol Chem. 1992 Feb 5;267(4):2798–2801. [PubMed] [Google Scholar]
  10. Corder R., Harrison V. J., Khan N., Anggård E. E., Vane J. R. Effects of phosphoramidon in endothelial cell cultures on the endogenous synthesis of endothelin-1 and on conversion of exogenous big endothelin-1 to endothelin-1. J Cardiovasc Pharmacol. 1993;22 (Suppl 8):S73–S76. doi: 10.1097/00005344-199322008-00021. [DOI] [PubMed] [Google Scholar]
  11. Corder R., Khan N., Harrison V. J. A simple method for isolating human endothelin converting enzyme free from contamination by neutral endopeptidase 24.11. Biochem Biophys Res Commun. 1995 Feb 6;207(1):355–362. doi: 10.1006/bbrc.1995.1195. [DOI] [PubMed] [Google Scholar]
  12. Denault J. B., Claing A., D'Orléans-Juste P., Sawamura T., Kido T., Masaki T., Leduc R. Processing of proendothelin-1 by human furin convertase. FEBS Lett. 1995 Apr 10;362(3):276–280. doi: 10.1016/0014-5793(95)00249-9. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Goto K., Warner T. D. Molecular pharmacology. Endothelin versatility. Nature. 1995 Jun 15;375(6532):539–540. doi: 10.1038/375539a0. [DOI] [PubMed] [Google Scholar]
  15. Gottardi C. J., Caplan M. J. An ion-transporting ATPase encodes multiple apical localization signals. J Cell Biol. 1993 Apr;121(2):283–293. doi: 10.1083/jcb.121.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gottardi C. J., Dunbar L. A., Caplan M. J. Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol. 1995 Feb;268(2 Pt 2):F285–F295. doi: 10.1152/ajprenal.1995.268.2.F285. [DOI] [PubMed] [Google Scholar]
  17. Hosoda K., Hammer R. E., Richardson J. A., Baynash A. G., Cheung J. C., Giaid A., Yanagisawa M. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell. 1994 Dec 30;79(7):1267–1276. doi: 10.1016/0092-8674(94)90017-5. [DOI] [PubMed] [Google Scholar]
  18. Ingbar D. H., Hepler K., Dowin R., Jacobsen E., Dunitz J. M., Nici L., Jamieson J. D. gamma-Glutamyl transpeptidase is a polarized alveolar epithelial membrane protein. Am J Physiol. 1995 Aug;269(2 Pt 1):L261–L271. doi: 10.1152/ajplung.1995.269.2.L261. [DOI] [PubMed] [Google Scholar]
  19. Inoue A., Yanagisawa M., Kimura S., Kasuya Y., Miyauchi T., Goto K., Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2863–2867. doi: 10.1073/pnas.86.8.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jalal F., Dehbi M., Berteloot A., Crine P. Biosynthesis and polarized distribution of neutral endopeptidase in primary cultures of kidney proximal tubule cells. Biochem J. 1994 Sep 15;302(Pt 3):669–674. doi: 10.1042/bj3020669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jalal F., Lemay G., Zollinger M., Berteloot A., Boileau G., Crine P. Neutral endopeptidase, a major brush border protein of the kidney proximal nephron, is directly targeted to the apical domain when expressed in Madin-Darby canine kidney cells. J Biol Chem. 1991 Oct 15;266(29):19826–19832. [PubMed] [Google Scholar]
  22. Kennedy R. L., Haynes W. G., Webb D. J. Endothelins as regulators of growth and function in endocrine tissues. Clin Endocrinol (Oxf) 1993 Sep;39(3):259–265. doi: 10.1111/j.1365-2265.1993.tb02363.x. [DOI] [PubMed] [Google Scholar]
  23. Kurihara Y., Kurihara H., Suzuki H., Kodama T., Maemura K., Nagai R., Oda H., Kuwaki T., Cao W. H., Kamada N. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature. 1994 Apr 21;368(6473):703–710. doi: 10.1038/368703a0. [DOI] [PubMed] [Google Scholar]
  24. Masaki T., Yanagisawa M. Endothelins. Essays Biochem. 1992;27:79–89. [PubMed] [Google Scholar]
  25. Matsumura Y., Hisaki K., Takaoka M., Morimoto S. Phosphoramidon, a metalloproteinase inhibitor, suppresses the hypertensive effect of big endothelin-1. Eur J Pharmacol. 1990 Aug 21;185(1):103–106. doi: 10.1016/0014-2999(90)90216-s. [DOI] [PubMed] [Google Scholar]
  26. Matsumura Y., Tsukahara Y., Kojima T., Murata S., Murakami A., Takada K., Takaoka M., Morimoto S. Effects of phosphoramidon on endothelin-1 and big endothelin-1 production in human aortic endothelial cells. Biol Pharm Bull. 1995 Mar;18(3):401–406. doi: 10.1248/bpb.18.401. [DOI] [PubMed] [Google Scholar]
  27. Mostov K., Apodaca G., Aroeti B., Okamoto C. Plasma membrane protein sorting in polarized epithelial cells. J Cell Biol. 1992 Feb;116(3):577–583. doi: 10.1083/jcb.116.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Puffenberger E. G., Hosoda K., Washington S. S., Nakao K., deWit D., Yanagisawa M., Chakravart A. A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease. Cell. 1994 Dec 30;79(7):1257–1266. doi: 10.1016/0092-8674(94)90016-7. [DOI] [PubMed] [Google Scholar]
  29. Rawlings N. D., Barrett A. J. Evolutionary families of peptidases. Biochem J. 1993 Feb 15;290(Pt 1):205–218. doi: 10.1042/bj2900205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Romero G., Luttrell L., Rogol A., Zeller K., Hewlett E., Larner J. Phosphatidylinositol-glycan anchors of membrane proteins: potential precursors of insulin mediators. Science. 1988 Apr 22;240(4851):509–511. doi: 10.1126/science.3282305. [DOI] [PubMed] [Google Scholar]
  31. Rubanyi G. M., Polokoff M. A. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev. 1994 Sep;46(3):325–415. [PubMed] [Google Scholar]
  32. Schmidt M., Kröger B., Jacob E., Seulberger H., Subkowski T., Otter R., Meyer T., Schmalzing G., Hillen H. Molecular characterization of human and bovine endothelin converting enzyme (ECE-1). FEBS Lett. 1994 Dec 19;356(2-3):238–243. doi: 10.1016/0014-5793(94)01277-6. [DOI] [PubMed] [Google Scholar]
  33. Schweizer A., Valdenaire O., Nelböck P., Deuschle U., Dumas Milne Edwards J. B., Stumpf J. G., Löffler B. M. Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations. Biochem J. 1997 Dec 15;328(Pt 3):871–877. doi: 10.1042/bj3280871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shimada K., Matsushita Y., Wakabayashi K., Takahashi M., Matsubara A., Iijima Y., Tanzawa K. Cloning and functional expression of human endothelin-converting enzyme cDNA. Biochem Biophys Res Commun. 1995 Feb 15;207(2):807–812. doi: 10.1006/bbrc.1995.1258. [DOI] [PubMed] [Google Scholar]
  35. Shimada K., Takahashi M., Ikeda M., Tanzawa K. Identification and characterization of two isoforms of an endothelin-converting enzyme-1. FEBS Lett. 1995 Sep 4;371(2):140–144. doi: 10.1016/0014-5793(95)00886-e. [DOI] [PubMed] [Google Scholar]
  36. Shimada K., Takahashi M., Tanzawa K. Cloning and functional expression of endothelin-converting enzyme from rat endothelial cells. J Biol Chem. 1994 Jul 15;269(28):18275–18278. [PubMed] [Google Scholar]
  37. Takada Y., Hiwada K., Kokubu T. Isolation and characterization of angiotensin converting enzyme from human kidney. J Biochem. 1981 Nov;90(5):1309–1319. doi: 10.1093/oxfordjournals.jbchem.a133596. [DOI] [PubMed] [Google Scholar]
  38. Takahashi M., Fukuda K., Shimada K., Barnes K., Turner A. J., Ikeda M., Koike H., Yamamoto Y., Tanzawa K. Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells. Biochem J. 1995 Oct 15;311(Pt 2):657–665. doi: 10.1042/bj3110657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Valdenaire O., Rohrbacher E., Mattei M. G. Organization of the gene encoding the human endothelin-converting enzyme (ECE-1). J Biol Chem. 1995 Dec 15;270(50):29794–29798. doi: 10.1074/jbc.270.50.29794. [DOI] [PubMed] [Google Scholar]
  40. Waxman L., Doshi K. P., Gaul S. L., Wang S., Bednar R. A., Stern A. M. Identification and characterization of endothelin converting activity from EAHY 926 cells: evidence for the physiologically relevant human enzyme. Arch Biochem Biophys. 1994 Jan;308(1):240–253. doi: 10.1006/abbi.1994.1034. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Yazaki Y., Goto K., Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988 Mar 31;332(6163):411–415. doi: 10.1038/332411a0. [DOI] [PubMed] [Google Scholar]
  43. de Hoop M. J., Huber L. A., Stenmark H., Williamson E., Zerial M., Parton R. G., Dotti C. G. The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis. Neuron. 1994 Jul;13(1):11–22. doi: 10.1016/0896-6273(94)90456-1. [DOI] [PubMed] [Google Scholar]

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