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Article Dans Une Revue FEBS Letters Année : 1997

Construction, expression and characterization of a soluble form of human endothelin-converting-enzyme-1

Résumé

Endothelin-converting-enzyme-1 (ECE-1) belongs to the family of zinc metallopeptidases and is responsible for generating endothelin (ET) peptides from their inactive precursors the big endothelins (bigET). The enzyme is a type II integral membrane protein consisting of a short amino-terminal cytosolic domain of 56 amino acids, a single transmembrane domain and a large putative extracellular domain containing the catalytic site. Recombinant and native ECE-1 are expressed as a dimer. We have constructed a soluble form of ECE, named sECE*, by fusing the cleavable signal peptide of pro-opiomelanocortin in frame to the complete extracellular domain of human ECE-1. Stable expression of this construct in CHO cells resulted in the secretion of a fully active enzyme. In contrast to membrane-bound ECE, sECE* was expressed as a monomer, highly glycosylated, as assessed by gel filtration and Western blot. However, recombinant sECE* converted bigET-1 with similar specific activity as ECE-1a. This activity was completely inhibited by phosphoramidon, but not by thiorphan and captopril. sECE* was active in a broad range of pH, showing an optimum of 6.6-6.8 for bigET-1. Thus, the extracellular domain alone is sufficient for conferring full ECE-1 activity, inhibitors recognition and substrate specificity.

Dates et versions

hal-02279012 , version 1 (04-09-2019)

Identifiants

Citer

Petra Korth, Giorgia Egidy, Charles Parnot, Jean-Michel Lemoullec, Pierre Corvol, et al.. Construction, expression and characterization of a soluble form of human endothelin-converting-enzyme-1. FEBS Letters, 1997, 417 (3), pp.365-370. ⟨10.1016/s0014-5793(97)01323-9⟩. ⟨hal-02279012⟩

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