Processing of Proendothelin‐1 at the C‐Terminus of Big Endothelin‐1 is Essential for Proteolysis by Endothelin‐Converting Enzyme‐1 in vivo

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Abstract
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.

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