title: Endothelin-converting enzyme-1, abundance of isoforms a-d and identification of a novel alternatively spliced variant lacking a transmembrane domain.
abstract: Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) 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 NH(2)-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.
authors.affiliation: Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. rina.meidan@huji.ac.il
authors.forename: Rina
authors.initials: R
authors.lastname: Meidan
authors.forename: Eyal
authors.initials: E
authors.lastname: Klipper
authors.forename: Tamar
authors.initials: T
authors.lastname: Gilboa
authors.forename: Laurent
authors.initials: L
authors.lastname: Muller
authors.forename: Nitzan
authors.initials: N
authors.lastname: Levy
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001011:Aorta; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000201:enzymology; D000072396:Endothelin-Converting Enzymes; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes