Soluble human endothelin-converting enzyme-1: expression, purification, and demonstration of pronounced pH sensitivity.
Endothelin-converting enzyme-1 (ECE-1) is a type II integral membrane protein that belongs to a family of metalloproteases which includes ECE-2, neprilysin (neutral endopeptidase 24.11, EC 3.4.24. 11), and Kell blood group protein. ECE-1 cleaves its biologically inactive native substrate, big endothelin-1, to generate a powerful vasoactive 21-amino acid peptide, endothelin-1. ECE-1 consists of a short N-terminal cytoplasmic tail, a transmembrane hydrophobic domain, and a large extracellular domain containing the catalytic site with a conserved Zn-binding motif. We have constructed a secreted, soluble form of ECE-1 (solECE-1) by fusing the cleavable N-terminal signal sequence of human alkaline phosphatase in frame with the entire extracellular domain of ECE-1. Stable transfectant CHO cell lines expressing up to 6.1 mg of solECE-1 per liter culture medium were established and solECE-1 was purified to homogeneity using three chromatographic steps with a 24% yield. SolECE-1 behaves as a dimer of 110-kDa subunits. SolECE-1 has a sharp pH optimum, similar to the native form, ECE-1a, but has a slightly more acidic pH optimum of 6.1-6.4 than that of 6.7-6.9 for ECE-1a. At its optimal pH of 6.4, solECE-1 cleaved big ET-1:big ET-2:big ET-3 in a ratio of 8.1:1:1.4, was inhibited by phosphoramidon with an IC50 value of 0.35 +/- 0.05 microM, had a Km value of 4.65 +/- 0.78 microM for big ET-1, and had a kcat value of 5.82 +/- 0.21 min-1, all values comparable to those for ECE-1a at its optimal pH of 6.8. Phosphoramidon inhibition of both ECE-1a and solECE-1 is highly pH-dependent. At pH 5.8, phosphoramidon inhibited ECE-1a and solECE-1 with IC50 values of 14 and 33 nM, respectively, which are 49- and 1224-fold more potent than at pH 7.2. SolECE-1 is highly glycosylated, similar to ECE-1a. Deglycosylation of solECE-1 by peptide N-glycosidase F shifted the apparent molecular weight of solECE-1 to approximately 80 kDa and the deglycosylated form(s) of solECE-1 preserved at least 72% of the activity of the glycosylated form.
Ahn
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Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan, 48105, USA. ahnk@aa.wl.com
Herman
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Fahnoe
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D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D004789:Enzyme Activation / Q000187:drug effects; D006020:Glycopeptides / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity; D014162:Transfection
D006020:Glycopeptides; D011480:Protease Inhibitors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon