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

Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):439-48. doi: 10.1042/bj3330439.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aspartic Acid Endopeptidases / antagonists & inhibitors
  • Aspartic Acid Endopeptidases / genetics
  • Aspartic Acid Endopeptidases / metabolism*
  • CHO Cells
  • Cell Compartmentation
  • Cell Line
  • Cell Polarity
  • Cricetinae
  • Dogs
  • Endothelin-Converting Enzymes
  • Glycopeptides / pharmacology
  • Humans
  • Kidney / cytology
  • Kidney / enzymology
  • Metalloendopeptidases / antagonists & inhibitors
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism*
  • Protease Inhibitors / pharmacology
  • Recombinant Proteins / metabolism
  • Transfection


  • Glycopeptides
  • Protease Inhibitors
  • Recombinant Proteins
  • Aspartic Acid Endopeptidases
  • Metalloendopeptidases
  • ECE1 protein, human
  • Endothelin-Converting Enzymes
  • phosphoramidon