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J. Biol. Chem., Vol. 280, Issue 49, 40867-40874, December 9, 2005

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Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain^*

Rina Meidan1, Eyal Klipper, Tamar Gilboa, Laurent Muller, and Nitzan Levy

From the Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel and the INSERM U 36, College de France (Paris), 75005 Paris, France

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and -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, alternatively 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.

Received for publication, May 24, 2005 , and in revised form, September 7, 2005.

^* This work was supported by a grant from the Israel Science Foundation (ISF 0396189). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 972-89489394; Fax: 972-89465763; E-mail: rina.meidan{at}huji.ac.il.

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