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

C. Brooks, Adviye Ergul

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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 μM) and competitive manner.

Original languageEnglish (US)
Pages (from-to)307-315
Number of pages9
JournalJournal of Molecular Endocrinology
Volume21
Issue number3
DOIs
StatePublished - Dec 1 1998

Fingerprint

Endothelin-1
Tail
Amino Acids
Complementary DNA
Vasoconstrictor Agents
Site-Directed Mutagenesis
Cricetulus
Transfection
Ovary
Protein Isoforms
Endothelin-Converting Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

Cite this

Identification of amino acid residues in the C-terminal tail of big endothelin-1 involved in processing to endothelin-1. / Brooks, C.; Ergul, Adviye.

In: Journal of Molecular Endocrinology, Vol. 21, No. 3, 01.12.1998, p. 307-315.

Research output: Contribution to journalArticle

@article{ee53f7a8510346e7888bfb8770c6fea0,
title = "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 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 μM) and competitive manner.",
author = "C. Brooks and Adviye Ergul",
year = "1998",
month = "12",
day = "1",
doi = "10.1677/jme.0.0210307",
language = "English (US)",
volume = "21",
pages = "307--315",
journal = "Journal of Molecular Endocrinology",
issn = "0952-5041",
publisher = "Society for Endocrinology",
number = "3",

}

TY - JOUR

T1 - Identification of amino acid residues in the C-terminal tail of big endothelin-1 involved in processing to endothelin-1

AU - Brooks, C.

AU - Ergul, Adviye

PY - 1998/12/1

Y1 - 1998/12/1

N2 - 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 μM) and competitive manner.

AB - 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 μM) and competitive manner.

UR - http://www.scopus.com/inward/record.url?scp=0032416322&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032416322&partnerID=8YFLogxK

U2 - 10.1677/jme.0.0210307

DO - 10.1677/jme.0.0210307

M3 - Article

VL - 21

SP - 307

EP - 315

JO - Journal of Molecular Endocrinology

JF - Journal of Molecular Endocrinology

SN - 0952-5041

IS - 3

ER -