Processing of proendothelin-1 at the C-terminus of big endothelin-1 is essential for proteolysis by endothelin-converting enzyme-1 in vivo.
Production of endothelin-1 is thought to be a three-step process consisting of an initial proteolytic cleavage of the proendothelin-1 precursor to big endothelin-1-Lys-Arg, C-terminal trimming by a carboxypeptidase and further processing of the big endothelin-1 peptide to endothelin-1 by endothelin-converting enzyme (ECE). To further clarify the mechanism of processing in the biosynthesis of endothelin-1, we introduced a point mutation into endothelin-1 cDNA to replace the Arg in the -4 position of the recognition motifs of furin-like convertase in human preproendothelin-1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in Chinese hamster ovary (CHO)-K1 cells, they failed to be processed at the mutated processing signal, suggesting that the Arg-Ser-Lys-Arg motifs of preproendothelin-1 are recognized by CHO-K1 furin-like convertase. Co-transfection with ECE-1 cDNA revealed that cleavage at Arg52 is not essential for cleavage by ECE-1, but that cleavage at Arg92 is critical. Although a high-molecular-mass form of endothelin-1 is produced by processing by ECE-1 without cleavage at Arg52, it did not evoke Ca2+ transient in endothelinA-receptor-expressing cells. In conclusion, prior cleavage at Arg92 by furin-like convertase is absolutely necessary for cleavage by ECE-1 at Trp73 to produce mature endothelin-1.
Kido
T
T
Department of Pharmacology, Faculty of Medicine, Kyoto University, Japan.
Sawamura
T
T
Hoshikawa
H
H
D'Orléans-Juste
P
P
Denault
J B
JB
Leduc
R
R
Kimura
J
J
Masaki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D011994:Recombinant Proteins; C093240:proendothelin 1; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes