Disulfide Bonds in Big ET-1 Are Essential for the Specific Cleavage at the Trp21–Val22 Bond by Soluble Endothelin Converting Enzyme-1 from Baculovirus/Insect Cells

Endothelin converting enzyme-1 (ECE-1) is a type II integral membrane protein and a zinc metalloendopeptidase. ECE-1 generates endothelin-1 (ET-1), the most potent vasoconstrictor yet discovered, by specific proteolytic processing of a precursor peptide, big ET-1. An insect cell expression system, which generates up to 4.3 mg of a secreted, soluble form of ECE-1 (solECE-1) per liter culture medium, has been established and solECE-1 was purified to homogeneity using five chromatographic steps. SolECE-1 expressed in insect cells could be suitable for X-ray structure determination as it is much less glycosylated than solECE-1 from mammalian cells. SolECE-1 from both sources, nonetheless, has comparable enzymatic properties. Despite apparent structural similarities, ECE-1 cleaves big ET-1 exclusively between Trp21 and Val22, in contrast to neprilysin, which cleaves big ET-1 at various sites. However, when linear big ET-1, in which the formation of disulfide bonds has been prevented by alkylation of the four cysteines, was used as substrate, it was cleaved by solECE-1 at multiple sites. This result indicates that secondary/tertiary structure of big ET-1 induced by disulfide bonds is essential for the specific cleavage of the Trp21–Val22 bond by ECE-1. A continuous, fluorescent ECE-1 assay has been developed using a novel substrate, 2-aminobenzoyl–Arg–Pro–Pro–Gly–Phe–Ser–Pro–(p-nitro-Phe8)–Arg. This simple and rapid assay can greatly facilitate discovery of novel ECE inhibitors useful as pharmaceutical agents.