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 consi sting of an initial proteolytic cleavage of the proendothelin-1 precur sor to big endothelin-1-Lys-Arg, C-terminal trimming by a carboxypepti dase and further processing of the big endothelin-1 peptide to endothe lin-1 by endothelin-converting enzyme (ECE). To further clarify the me chanism of processing in the biosynthesis of endothelin-1, we introduc ed 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 ex pressed in Chinese hamster ovary (CHO)-K1 cells, they failed to be pro cessed at the mutated processing signal, suggesting that the Arg-Ser-L ys-Arg motifs of preproendothelin-1 are recognized by CHO-K1 furin-lik e convertase. Co-transfection with ECE-1 cDNA revealed that cleavage a t Arg52 is not essential for cleavage by ECE-1, but that cleavage at A rg92 is critical. Although a high-molecular-mass form of endothelin-1 is produced by processing by ECE-1 without cleavage at Arg52, it did n ot evoke Ca2+ transient in endothelin(A)-receptor-expressing cells. In conclusion, prior cleavage at Arg92 by furin-like convertase is absol utely necessary for cleavage by ECE-1 at Trp73 to produce mature endot helin-1.