title: Recombinant human endothelin-converting enzyme ECE-1b is located in an intracellular compartment when expressed in polarized Madin-Darby canine kidney cells.
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.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Montréal, Montréal, Québec, H3C 3J7, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Azarani
authors.forename: G
authors.initials: G
authors.lastname: Boileau
authors.forename: P
authors.initials: P
authors.lastname: Crine
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002451:Cell Compartmentation; D002460:Cell Line; D016764:Cell Polarity; D006224:Cricetinae; D004285:Dogs; D000072396:Endothelin-Converting Enzymes; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D006020:Glycopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon