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 endotheli n-1 (ET-1) via a specific cleavage at Trp(21)-Val(22) by endothelin convert ing enzyme (ECE). This conversion is an essential step to produce bioactive ET-1 and represents a regulatory site in the biosynthesis of this potent v asoconstrictor. ECE-1a, a unique membrane-bound enzyme, processes big ET-1 more efficiently than other big ET isoforms, which mainly differ in the C-t erminal tail (residues 22-38). In this study, each of the highly conserved residues, Val(22), Pro(25), Pro(30), Gly(32), Leu(33), and Gly(34) were rep laced with Ala in the preproendothelin-1 (PPET-1) cDNA using site-directed mutagenesis. The mutant and wild-type cDNAs were transiently transfected in to Chinese hamster ovary cells along with ECE-1a cDNAs, and concentrations of the resulting recombinant peptides, ET-1 and big ET-1, in the transfecti on media were then measured. The concentration of immunoreactive ET-1 in th e media from Val(22), pro(25), Pro(30), Gly(32), and Leu(33) mutant PPET-1- transfected cells was 4- to 6-fold lower than that of wild type and (Gly(34 )-->Ala)PPET-1. Moreover, with the exception of Gly(34), there was a corres ponding increase in the concentrations of immunoreactive big ET-1 in the me dia from mutants. Similar results were obtained when His(27),Val(28), and S er(35) 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 i mportant role in the intracellular processing of big ET-1 by ECE-la. Herein we also report that a recombinant big ET-1 analog we previously generated and characterized, (Ala(21))big ET-1, inhibits ECE-1a activity in a dose-de pendent (K-i=1 mu M) and competitive manner.