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.
Korth
P
P
INSERM U36, Collège de France, Paris.
Egidy
G
G
Parnot
C
C
LeMoullec
J M
JM
Corvol
P
P
Pinet
F
F
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes