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 endothelin(A)-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.