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

Abstract

Big endothelin-1 (big ET-1) is converted to 21-amino acid residue endothelin-1 (ET-1) via a specific cleavage at Trp 21 -Val 22 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, Val , and Gly 34 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. 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, (Ala 21 )big ET-1, inhibits ECE-1a activity in a dose-dependent (K i =1 µM) and competitive manner.

Mutations in Endothelin 1 Cause Recessive Auriculocondylar Syndrome and Dominant Isolated Question-Mark Ears

“…In a mutagenesis study of selected residues in the C-terminal portion of bigEDN1, substitution of Pro77 (Pro25 numbering from the start of bigEDN1) to alanine resulted in a significant reduction in the conversion of bigEDN1 to EDN1, as assayed in the media of cells transfected with plasmids encoding wild-type or altered preproEDN1 and ECE1. 38 In addition, p.Pro25Alatransfected cells had less total recombinant protein (bigEDN1 plus EDN1) measured in the media than did cells transfected with wild-type preproEDN1, suggesting that this proline might play a role in bigEDN1 stability. These data argue that the p.Pro77His substitution in individual S1 is pathogenic.…”

Positron emission tomography of [18F]‐big endothelin‐1 reveals renal excretion but tissue‐specific conversion to [18F]‐endothelin‐1 in lung and liver

“…It has been shown that the His 27 ‐Gly 34 sequence in big ET‐1 is important for enzyme recognition and conversion to ET‐1 (Okada et al. , 1991; 1993; Brooks and Ergul, 1998). Our strategy avoids labelling within this His 27 ‐Gly 34 region, permitting both enzymic cleavage and formation of [ 18 F]‐ET‐1.…”

Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression

“…All three endothelins follow a similar pattern of biosynthesis with initial synthesis as precursor proteins (]200 amino acid residues) which undergo selective proteolysis to yield the biologically inactive intermediates called big endothelins (Yanagisawa et al 1988, Bloch et al 1989, Ohkubo et al 1990). The active endothelins are generated by hydrolysis of the Trp 21 -Val 22 bond in big ET-1 and big ET-2, or Trp 21 -Ile 22 in big ET-3 (Brooks & Ergul 1998). This cleavage is unique to the endothelins and hence the role of a specific endothelin-converting enzyme (ECE) was proposed (Yanagisawa et al 1988).…”