Endothelin-converting enzyme-1, abundance of isoforms a-d and identification of a novel alternatively spliced variant lacking a transmembrane domain

Rina Meidan*, Eyal Klipper, Tamar Gilboa, Laurent Muller, Nitzan Levy

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and β-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH2 terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternalively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3′, which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH2-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Original languageEnglish
Pages (from-to)40867-40874
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number49
DOIs
StatePublished - 9 Dec 2005

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