In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.

Abstract

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg --Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.The 21-residue vasoconstrictor peptide human endothelin-1 (ET-1), like many growth factors and peptide hormones, is first synthesized as a larger inactive precursor (preproET-1, see Fig. 1) (1). While ET-1 seems to exert a wide range of diverse biological activities (2), the 38-amino acid intermediate big ET-1 is at least 2 orders of magnitude less potent than ET-1 in contractile activity, and it is still not clear whether it plays some pathophysiologic role. Many efforts have been made to search for a specific endothelinconverting enzyme (ECE) responsible for the cleavage of the unusual Trp-Val site of big ET-1 as a key element in the regulation of ET-1 biosynthesis (3, 4). However, expression of human preproET-1 in Xenopus oocytes (5) or insect cells (6) results in constitutive secretion of peptides with immunological and biological characteristics of ET-1 and big ET-1.For further study of the processing events and molecular requirements leading to big ET-1 and ET-1 formation, we report expression of a set of mutated preproendothelins in Xenopus oocytes. We demonstrate that big ET-1 is a necessary intermediate in the formation of ET-1, whereas the second part of preproET-1 (after the C terminus of big ET-1) is not necessary for the synthesis of these two peptides.

SOX10 mutations in chronic intestinal pseudo-obstruction suggest a complex physiopathological mechanism

“…The cysteine newly generated is located in a region, between the mature endothelin sequence and the endothelin-like (ET-like) peptide, which is normally devoid of cysteine, suggesting that the introduction of this new amino acid may alter disulphide bond formation in the endothelin and/or the ET-like peptide regions. Although this hypothesis has to be confirmed, it is consistent with the previously reported role of endothelin and ET-like peptide disulphide bonds in the secondary structure of the preproendothelin that allow enzymatic processing of an inactive protein into a mature active peptide, and with the characterisation of other EDN3 mutations involving cysteines (Fabbrini et al 1993;Hofstra et al 1996Hofstra et al , 1997Pingault et al 2001). The mutation segregates at the heterozygous state in the mother's and in the father's families.…”

Cytosolic immunization allows the expression of preATF‐saporin chimeric toxin in eukaryotic cells

“…In some experiments 10 M monensin (Sigma, St. Louis, Mo.) was included in the chase medium (24). The concentration of secretory ATF-SAP in the oocyte incubation medium was estimated by an enzyme-linked immunoassay (8), using a biotinylated monoclonal antibody to human uPA (Monozyme, Denmark) and human recombinant Pro-uPA as standard.…”

A heterozygous endothelin 3 mutation in Waardenburg-Hirschsprung disease: is there a dosage effect of EDN3/EDNRB gene mutations on neurocristopathy phenotypes?

“…The ET-like peptide might play a role in the first enzymatic cleavage step. The absence of this first cleavage step impairs the final clipping step by endothelin conversion enzyme (ECE-1) 34. As a result, the C169X mutation, by substituting the third cysteine of the ET-like peptide, prevents the disulphide bonds and probably generates an inappropriately cleaved, inactive proendothelin.…”