title: Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum.
abstract: Endothelins (ET) are a family of potent vasoactive peptides that are produced from biologically inactive intermediates, termed big endothelins, via a proteolytic processing at Trp21-Val/Ile22. We recently cloned and characterized a membrane-bound metalloprotease that catalyzes this proteolytic activation, endothelin-converting enzyme-1 (ECE-1) (Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M. (1994) Cell 78, 473-485). This enzyme was shown to function in the secretory pathway as well as on the cell surface. Here we report molecular cloning of another novel enzyme, ECE-2, that produces mature ET-1 from big ET-1 both in vitro and in transfected cells. The cDNA sequence predicts that bovine ECE-2 is a metalloprotease structurally related to ECE-1, neutral endopeptidase 24.11, and human Kell blood group protein. The deduced amino acid sequence of ECE-2 is most similar to ECE-1, with an overall identity of 59%. ECE-2 resembles ECE-1 in that it is inhibited in vitro by phosphoramidon and FR901533 but not by thiorphan or captopril, and it converts big ET-1 more efficiently than big ET-2 or big ET-3. However, ECE-2 also exhibits the following striking differences from ECE-1. (i) The sensitivity of ECE-2 to phosphoramidon is 250-fold higher as compared with ECE-1, while FR901533 inhibits both enzymes at similar concentrations. (ii) ECE-2 has an acidic pH optimum at pH 5.5, which is in sharp contrast to the neutral pH optimum of ECE-1. ECE-2 has a narrow pH profile and is virtually inactive at neutral pH. Chinese hamster ovary (CHO) cells, which lack detectable levels of endogenous ECE activity, secrete mature ET-1 into the medium when doubly transfected with ECE-2 and prepro-ET-1 cDNAs. However, ECE-2-transfected CHO cells do not efficiently produce mature ET-1 when present with an exogenous source of big ET-1 through coculture with prepro-ET-1-transfected CHO cells. These findings suggest that ECE-2 acts as an intracellular enzyme responsible for the conversion of endogenously synthesized big ET-1 at the trans-Golgi network, where the vesicular fluid is acidified.
authors.affiliation: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235-9050, USA.
authors.forename: N
authors.initials: N
authors.lastname: Emoto
authors.forename: M
authors.initials: M
authors.lastname: Yanagisawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D017931:DNA Primers; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D000906:Antibodies; D017931:DNA Primers; D016232:Endothelins; D006020:Glycopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon