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 microM) and competitive manner.
Brooks
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Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Ergul
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D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D019332:Endothelin-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016232:Endothelins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D011994:Recombinant Proteins