Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations

Abstract

Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (K_m, k_cat and V_max) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.