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

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.