Identification of amino acid residues in the C-terminal tail of big endothelin-1 involved in processing to endothelin-1

Big endothelin-1 (big ET-1) is converted to 21-amino acid residue endothelin-1 (ET-1) via a specific cleavage at Trp²¹-Val²² by endothelin converting enzyme (ECE). This conversion is an essential step to produce bioactive ET-1 and represents a regulatory site in the biosynthesis of this potent vasoconstrictor. ECE-1a, a unique membrane-bound enzyme, processes big ET-1 more efficiently than other big ET isoforms, which mainly differ in the C-terminal tail (residues 22-38). In this study, each of the highly conserved residues, Val²², Pro²⁵, Pro³⁰, Gly³², Leu³³, and Gly³⁴ were replaced with Ala in the preproendothelin-1 (PPET-1) cDNA using site-directed mutagenesis. The mutant and wild-type cDNAs were transiently transfected into Chinese hamster ovary cells along with ECE-1a cDNAs, and concentrations of the resulting recombinant peptides, ET-1 and big ET-1, in the transfection media were then measured. The concentration of immunoreactive ET-1 in the media from Val²², Pro²⁵, Pro³⁰, Gly³², and Leu³³ mutant PPET-1- transfected cells was 4- to 6-fold lower than that of wild type and (Gly³⁴→Ala)PPET-1. Moreover, with the exception of Gly³⁴, there was a corresponding increase in the concentrations of immunoreactive big ET-1 in the media from mutants. Similar results were obtained when His²⁷, Val²⁸, and Ser³⁵ of big ET-1 were substituted with the corresponding residues in big ET-2 and big ET-3. These findings suggest that the C-terminal tail has an important role in the intracellular processing of big ET-1 by ECE-1a. Herein we alsO report that a recombinant big ET-1 analog we previously generated and characterized, (Ala²¹)big ET-1, inhibits ECE-1a activity in a dose- dependent (K(i)=1 μM) and competitive manner.