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, A.
PY - 1998
Y1 - 1998
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.
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U2 - 10.1677/jme.0.0210307
DO - 10.1677/jme.0.0210307
M3 - Article
C2 - 9845671
AN - SCOPUS:0032416322
SN - 0952-5041
VL - 21
SP - 307
EP - 315
JO - Journal of Molecular Endocrinology
JF - Journal of Molecular Endocrinology
IS - 3
ER -