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

Maria Serena Fabbrini, Alessandro Vitale, Emanuela Pedrazzini, Gianpaolo Nitti, Moreno Zamai, Monica Tamburin, Valeria R. Caiolfa, Carlo Patrono, Luca Benatti

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)3923-3927
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number9
Publication statusPublished - May 1 1993

Keywords

  • Basic-pair site processing
  • Big-endothelin
  • Endothelin
  • Endothelin-converting enzyme
  • Xenopus oocytes

ASJC Scopus subject areas

  • General
  • Genetics

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