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-S, 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-l, 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-la, but has a slightly more acidic pH optimum of 6.1-6.4 than that of 6.7-6.9 for ECE-la. At its optimal pH of 6.4, solECE-1 cleaved big ET-l:big ET-a: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 mu M had a K-m value of 4.65 +/- 0.78 mu M for big ET-1, and had a k(cat) value of 5.82 +/- 0.21 min(-1), all values comparable to those for ECE-la at its optimal pH of 6.8. Phosphoramidon inhibition of both ECE-la and solECE-1 is highly pH-dependent. At pH 5.8, phosphoramidon inhibited ECE-la and solECE-1 with IC,, 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-la. 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. (C) 1998 Academic Press.