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

FEBS Letters

LeMoullec, Jean-Michel¹  Pinet, Florence²  Korth, Petra²  Egidy, Giorgia²  Corvol, Pierre²  Parnot, Charles² 

[1] Roussel Uclaf, 102 route de Noisy, 93235 Romainville, France;INSERM U36, Collège de France, 3 rue d'Ulm, 75005 Paris, France

关键词: Human;    ECE-1;    Endothelin;    Soluble enzyme;    Immunoprecipitation;    Glycosylation;    ECE-1;    endothelin-converting-enzyme-1;    ET;    endothelin;    NEP;    neutral endopeptidase;    ACE;    angiotensin-converting enzyme;    POMC;    pro-opiomelanocortin;   

DOI  :  10.1016/S0014-5793(97)01323-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown