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• W2024895902 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, 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. 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, 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. Endothelin-converting enzyme-1 (ECE-1) 2The abbreviations used are: ECE-1endothelin-converting enzyme-1bECE-1bovine ECE-1ECendothelial cellsDMEMDulbecco's minimum essential mediumBAECbovine aortic ECCHOChinese hamster ovarySVsplice variantmutmutatedTMtransmembraneG3PDHglyceraldehyde-3-phosphate dehydrogenase.2The abbreviations used are: ECE-1endothelin-converting enzyme-1bECE-1bovine ECE-1ECendothelial cellsDMEMDulbecco's minimum essential mediumBAECbovine aortic ECCHOChinese hamster ovarySVsplice variantmutmutatedTMtransmembraneG3PDHglyceraldehyde-3-phosphate dehydrogenase. is a type II membrane protease that belongs to the neprilysin (NEP) family of zinc metallopeptidases (1Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (855) Google Scholar, 2Valdenaire O. Schweizer A. Biochem. Soc. Trans. 2000; 28: 426-430Crossref PubMed Scopus (17) Google Scholar). ECE-1 is abundantly expressed in the vascular endothelial cells (EC) of all tissues but is also found in nonvascular cells (3Maguire J.J. Johnson C.M. Mockridge J.W. Davenport A.P. Br. J. Pharmacol. 1997; 122: 1647-1654Crossref PubMed Scopus (41) Google Scholar, 4Sawamura T. Shinmi O. Kishi N. Sugita Y. Yanagisawa M. Goto K. Masaki T. Kimura S. Biochim. Biophys. Acta. 1993; 1161: 295-302Crossref PubMed Scopus (31) Google Scholar, 5Levy N. Gordin M. Smith M.F. Bolden-Tiller O.U. Meidan R. Biol. Reprod. 2003; 68: 1361-1368Crossref PubMed Scopus (22) Google Scholar, 6Korth P. Bohle R.M. Corvol P. Pinet F. J. Histochem. Cytochem. 1999; 47: 447-462Crossref PubMed Scopus (76) Google Scholar). This enzyme is characterized by a single transmembrane region, a short NH2-terminal cytosolic tail and a large COOH-terminal extracellular domain that contains the enzymatic active site (7Schmidt M. Kroger B. Jacob E. Seulberger H. Subkowski T. Otter R. Meyer T. Schmalzing G. Hillen H. FEBS Lett. 1994; 356: 238-243Crossref PubMed Scopus (183) Google Scholar). ECE-1 is a glycosylated protein with 10 putative N-linked glycosylation sites (8Turner A.J. Barnes K. Schweizer A. Valdenaire O. Trends Pharmacol. Sci. 1998; 19: 483-486Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The best characterized substrates are the ET family consisting of three isopeptides, termed ET-1, ET-2, and ET-3, which are derived from distinct genes (9Yanagisawa M. Kurihara H. Kimura S. Mitsui Y. Kobayashi M. Watanabe T. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10163) Google Scholar, 10Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2554) Google Scholar). ET-1, the most abundant of the three, is a pleiotropic peptide; although best known for its vasoconstricting activity it has diverse biological functions. These include roles in processes such as embryonic development, cardiovascular homeostasis, vascular permeability, and angiogenesis (11Kedzierski R.M. Yanagisawa M. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 851-876Crossref PubMed Scopus (608) Google Scholar, 12Kelly J.J. Whitworth J.A. Clin. Exp. Pharmacol. Physiol. 1999; 26: 158-161Crossref PubMed Scopus (37) Google Scholar, 13Grant K. Loizidou M. Taylor I. Br. J. Cancer. 2003; 88: 163-166Crossref PubMed Scopus (145) Google Scholar). The three ETs mediate their various effects via two G protein-coupled receptors: ETA and ETB (14Sakurai T. Yanagisawa M. Takuwa Y. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2353) Google Scholar, 15Arai H. Hori S. Aramori I. Ohkubo H. Nakanishi S. Nature. 1990; 348: 730-732Crossref PubMed Scopus (2501) Google Scholar). ETs are synthesized from ∼200-amino acid precursor-prepro ET (ppET). After removal of their signal peptide ETs are processed by dibasic pair-specific enzymatic activity to form the respective inactive big-ETs (38-41 residues long (1Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (855) Google Scholar, 4Sawamura T. Shinmi O. Kishi N. Sugita Y. Yanagisawa M. Goto K. Masaki T. Kimura S. Biochim. Biophys. Acta. 1993; 1161: 295-302Crossref PubMed Scopus (31) Google Scholar)). ECE-1 then specifically hydrolyzes the Trp21-Val/Ile22 bonds of big-ETs to produce biologically active ETs (1Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (855) Google Scholar, 16Shimada K. Takahashi M. Tanzawa K. J. Biol. Chem. 1994; 269: 18275-18278Abstract Full Text PDF PubMed Google Scholar). ECE-1 null mice exhibit a phenotype similar to that of ET-1- or ETA-deficient mice thus demonstrating the physiological relevance of ECE-1 in generating bioavailable ET-1 (17Yanagisawa H. Yanagisawa M. Kapur R.P. Richardson J.A. Williams S.C. Clouthier D.E. de Wit D. Emoto N. Hammer R.E. Development (Camb.). 1998; 125: 825-836Crossref PubMed Google Scholar). endothelin-converting enzyme-1 bovine ECE-1 endothelial cells Dulbecco's minimum essential medium bovine aortic EC Chinese hamster ovary splice variant mutated transmembrane glyceraldehyde-3-phosphate dehydrogenase. endothelin-converting enzyme-1 bovine ECE-1 endothelial cells Dulbecco's minimum essential medium bovine aortic EC Chinese hamster ovary splice variant mutated transmembrane glyceraldehyde-3-phosphate dehydrogenase. Four isoforms of human ECE-1 (1a, 1b, 1c, and 1d) have been identified to date (8Turner A.J. Barnes K. Schweizer A. Valdenaire O. Trends Pharmacol. Sci. 1998; 19: 483-486Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 19Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur. J. Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar, 20Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). The four proteins are encoded by one gene, but each is expressed from a distinct promoter that regulates the expression of the four unique amino termini (8Turner A.J. Barnes K. Schweizer A. Valdenaire O. Trends Pharmacol. Sci. 1998; 19: 483-486Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 19Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur. J. Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar, 20Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). Although the ectodomain containing the active site is identical in each of the isoforms, the amino-terminal sequences appear to be responsible for differences in subcellular localization (19Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur. J. Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar, 21Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike H. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 22Russell F.D. Skepper J.N. Davenport A.P. J. Cardiovasc. Pharmacol. 1998; 31: 424-430Crossref PubMed Scopus (65) Google Scholar, 23Muller L. Barret A. Etienne E. Meidan R. Valdenaire O. Corvol P. Tougard C. J. Biol. Chem. 2003; 278: 545-555Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). ECE-1 isoforms were mainly studied in cell lines overexpressing each isoform separately. This may explain why it is still unclear how abundant each of the ECE-1 isoforms is in naturally expressing cells. Several studies have shown that ECE-1 efficiently hydrolyzes a number of peptide hormones other than Ets, these include bradykinin, substance P, and neurotensin (24Johnson G.D. Stevenson T. Ahn K. J. Biol. Chem. 1999; 274: 4053-4058Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). An exciting novel substrate for ECE-1 is the β-amyloid peptide that is implicated in the pathogenesis of Alzheimer disease (25Eckman E.A. Reed D.K. Eckman C.B. J. Biol. Chem. 2001; 276: 24540-24548Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 26Funalot B. Ouimet T. Claperon A. Fallet C. Delacourte A. Epelbaum J. Subkowski T. Leonard N. Codron V. David J.P. Amouyel P. Schwartz J.C. Helbecque N. Mol. Psychiatry. 2004; 9: 1059Crossref Scopus (14) Google Scholar). Inhibitors of ECE-1 are considered to be valuable therapeutic agents and were developed for the treatment of various disorders linked to elevated ET-1 levels (27Jeng A.Y. Curr. Opin. Investig. Drugs. 2003; 4: 1076-1081PubMed Google Scholar, 28Jeng A.Y. Mulder P. Kwan A.L. Battistini B. Can. J. Physiol. Pharmacol. 2002; 80: 440-449Crossref PubMed Scopus (49) Google Scholar). Numerous peptides or non-peptidyl ECE-1 inhibitors have already been produced, but contrary to initial expectations, none is currently used for therapeutic purposes, perhaps because of insufficient knowledge of the ECE-1 family of proteins in naturally expressing cells. In this paper we report the prevalence of ECE-1 isoforms a-d in EC and the initial characterization of a novel splice variant of ECE-1 that lacks the transmembrane domain. Materials—Dulbecco's minimum essential medium (DMEM) low glucose, DMEM with Ham's F-12 1:1 (v/v) nutrient mixture, Super-ScriptII RNase H-reverse transcriptase, calf serum, and ultra pure electrophoresis agarose gel were obtained from Invitrogen. Vitrogen, type I collagen from Cohesion Technologies (Palo Alto, CA). Penicillin, streptomycin, and fetal calf serum were from Biological Industries (Beit Haemek, Israel). TRI Reagent from was from Molecular Research Center (Cincinnati, OH). Deoxynucleotide triphosphates, random hexamer oligodeoxynucleotides, and TaqDNA polymerase were from Fermentas (Vilnius, Lithuania). Oligo(dT) and oligonucleotide primers were synthesized by MWG Biotech AG (Ebersberg, Germany). The real-time PCR SYBR-Green master-mix kit was from Eurogentec (Seraing, Belgium). Protease inhibitor mixture for mammalian cell extracts and horseradish peroxidase-conjugated goat anti-rabbit IgG were from Sigma. The protein quantification kit was from Bio-Rad. Hifidelity Taq polymerase was from Takara (Otsu, Shiga, Japan). Restriction enzymes were from Fermentas (Hanover, MD). DpnI was from New England Biolabs (Beverly, MA). FuGENE 6 transfection reagent was from Roche Applied Science. pGEM-T vector, pcDNA6/V5-HiS version C, and blasticidin were from Invitrogen. N-Octyl glucoside and phosphoramidon were from Sigma. BK-2 was synthesized by Sigma-Genosys (The Woodlands, TX). Cell Cultures—Bovine aortic EC (BAEC) were kindly provided by I. Vlodavsky of the Hadassah-Hebrew University Hospital, (Jerusalem, Israel), and the cells were grown in complete DMEM containing 10% calf serum and 2 mm glutamine. Microvascular EC derived from the bovine corpus luteum (29Spanel-Borowski K. Fenyves A. Arzneimittelforschung. 1994; 44: 385-391PubMed Google Scholar, 30Spanel-Borowski K. Cell Tissue Res. 1991; 266: 37-49Crossref PubMed Scopus (34) Google Scholar, 31Lehmann I. Brylla E. Sittig D. Spanel-Borowski K. Aust G. J. Vasc. Res. 2000; 37: 408-416Crossref PubMed Scopus (34) Google Scholar), termed luteal EC, was grown in complete DMEM Ham's F-12 containing 10% fetal calf serum and 2 mm glutamine on plates precoated with 2% Vitrogen. Experiments were carried out on cells from passages 5-12, with 70-80% confluence. Human embryonic kidney cell cultures (HEK-293) and Chinese hamster ovary (CHO) cell cultures were cultured in complete DMEM Ham's F-12 containing 10% fetal calf serum and 2 mm glutamine. Enrichment of Luteal Steroidogenic and Endothelial Cells—For enrichment of luteal cell subpopulations, mid-cycle corpora lutea were dispersed by using collagenase IV as described previously (5Levy N. Gordin M. Smith M.F. Bolden-Tiller O.U. Meidan R. Biol. Reprod. 2003; 68: 1361-1368Crossref PubMed Scopus (22) Google Scholar, 32Levy N. Gordin M. Mamluk R. Yanagisawa M. Smith M.F. Hampton J.H. Meidan R. Endocrinology. 2001; 142: 5254-5260Crossref PubMed Scopus (43) Google Scholar). Briefly, corpora lutea were sliced and dispersed in M-199 containing 0.5% bovine serum albumin and collagenase (420 units/ml). Dispersed luteal cells were mixed with epoxy magnetic beads precoated with Bandeiraea simplicifolia lectin-1 (BS-1), a lectin specific for bovine EC. Both BS-1-positive cells (EC) and non-adherent cells (enriched steroidogenic cells) were collected and further processed for RNA extraction. Production of bECE-1 Constructs—The cDNA sequences of full-length bovine ECE-1d and ECE-1d splice variant (SV) were amplified with 1d and ECE-1-end as primers (TABLE ONE). The amplification products were separated on agarose gels and the corresponding single bands were extracted and cloned onto pGEM-TEasy vector. Inserts were subsequently subcloned into pcDNA vector (pcDNA6/V5) and sequenced. ECE-1d and SV plasmids were mutated (mut) at the putative start site of the latter (ATG located between bases 207-209) as follows: 26-bp complementary sense and antisense oligonucleotides, containing the desired mutation (ATG to TTT), were used in a PCR reaction with the original SV plasmid as a template. Template plasmid was then digested with DpnI. A shorter SVcut construct lacking the first 169 bp of SV was generated by digesting SV with Eco91I (BstEII). HEK-293 cells were transfected by FuGENE 6 transfection reagent. Stably transfected cells lines (containing bECE-1d, SV, and SVcut) were established using blasticidin (1 μg/ml) as a selective antibiotic.TABLE ONEPCR primer listGenePrimerSequenceProduct lengthG3PDHForward5′-GGCGTGAACCACGAGAAGTAT-3′141Reverse5′-CGTGGACAGTGGTCATAAGT-3′ECE-1, totalForward5′-TGTGGCGGCTGGATCAAAGC-3′103Reverse5′-AGGTGCTTGATGATGGCTTG-3′ECE-1aForward5′-GTTCCTCTCCTGGATTAG-3′178Reverse5′-CTTGTCTGGTATTGGATGC-3′ECE-1bForward5′-TGTCGGCGCTGGGGATGTC-3′113ECE-1cForward5′-CGGAGCGCGCGAGCGAT-3′109ECE-1dForward5′-CCATGGAGGCGCTAAGAGAGT-3′142ECE-1b, -c, and -dReverse5′-GAAGTTCACCTGCAGGTGGT-3′ECE-svForward5′-TACCCCAACCACCTGCAGGAACG-3′226Reverse5′-AGGTGCTTGATGATGGCTTG-3′ECE-1, full- lengthForward5′-GCAGGTGAACTTCCGAGG-3′150Reverse5′-CTTGTCTGGTATTGGATGC-3′ECE-1, commonReverse5′-GAAGGGGGAGGTGTGGTAGT-3′1a: 5711b: 5031c: 5071d: 533ECE-1, endReverse5′-CCCTTCACCAGACTTCACACT-3′1d: 2312SV: 2170 Open table in a new tab Cell Fractionation and Western Blot Analysis—The procedure for total cell extracts was carried out as we have previously described (32Levy N. Gordin M. Mamluk R. Yanagisawa M. Smith M.F. Hampton J.H. Meidan R. Endocrinology. 2001; 142: 5254-5260Crossref PubMed Scopus (43) Google Scholar, 33Klipper E. Gilboa T. Levy N. Kisliouk T. Spanel-Borowski K. Meidan R. Reproduction. 2004; 128: 463-473Crossref PubMed Scopus (53) Google Scholar). Briefly, cells were homogenized in lysis buffer (25 mm Tris-HCl, 100 mm NaCl, 0.5% deoxycholate, 0.5% Nonidet P-40, 5 mm EDTA, at pH 7.5 and 10% protease inhibitor mixture). Cell extracts were sonicated on ice for 10 s at low speed. For subcellular localization of ECE-1 forms, HEK-293 cells were homogenized in lysis buffer without detergents and then centrifuged for 15 min at 15,000 × g. The resulting pellet (containing particulate fraction) was dissolved in lysis buffer containing 0.5% deoxycholate and 0.5% Nonidet P-40. The supernatant was centrifuged at 44,000 × g for 1 h. The supernatant obtained after ultracentrifugation was defined as the cytosolic fraction. The cytosol and cell particulate fractions were separated by 7.5% SDS-PAGE, under reducing conditions. Protein concentrations were determined by using Bio-Rad DC reagents. All steps were performed on ice, and samples were kept at -80 °C until use. Proteins were electrically transferred to nitrocellulose membranes. After 2 h blocking in TBST (20 mm Tris, 150 mm NaCl, and 0.05% Tween 20) + 5% low fat milk, membranes were incubated with the appropriate ECE-1 antibodies. Anti-total ECE-1 antiserum (anti COOH-terminal antibody: 4788) was raised against a synthetic peptide comprising the last 16 amino acids of ECE-1 and was affinity purified by means of the immunizing peptide immobilized on a Sepharose 4B column. Two antibodies raised against NH2-terminal sequences of ECE-1 were also used: ds-90, which recognizes the cytosolic sequence of ECE-1d, and 1207, which recognizes ECE-1bcd (34Muller L. Valdenaire O. Barret A. Korth P. Pinet F. Corvol P. Tougard C. J. Cardiovasc. Pharmacol. 2000; 36: S15-S18Crossref PubMed Google Scholar). The membranes were washed three times and then incubated with horseradish peroxidase-conjugated goat anti rabbit IgG for 1 h at room temperature. A chemiluminescent signal was generated with SuperSignal and the membranes were exposed to x-ray film. Cell-free Transcription/Translation System (TNT)—The T7 transcription/translation system with [35S]methionine was used to probe translated products of the various plasmids (bECE-1d, bECE-1d-mut SV, Svmut, and SVcut). Briefly, 1 mg of plasmid DNA was incubated with TNT master mix (Promega) and [35S]methionine, 90 min at 30 °C. The resulting proteins were then run on an SDS-PAGE gel under reducing conditions, and the gel was dried (Bio-Rad gel dryer) and exposed to x-rays film. Immunofluorescence—CHO or HEK-293 cells were seeded on 14-mm coverslips and transfected with plasmids coding for either ECE-1d or SV. They were cultivated for 48 h before fixation with cold methanol for 5 min. Nonspecific binding was saturated with 10% normal goat serum in phosphate-buffered saline. Cells were then incubated with the primary antibodies directed against the COOH terminus of ECE-1 in 1% normal goat serum in phosphate-buffered saline. Secondary goat antibodies directed against rabbit IgG were coupled to AlexaFluor-555 (Molecular Probes). Nuclei were labeled using To-Pro-3 (Molecular Probes). Coverslips were mounted with Mowiol and observed with a TCS SP2 confocal microscope (Leica Microsystems). Biological Activity—ECE-1 activity was measured using BK2 peptide (aminomethylcoumarin-RPPGFSAFR-dinitrophenyl) as a substrate. The proteolysis of this quenched peptide at the Ala7-Phe8 bond by ECE-1 has already been characterized (23Muller L. Barret A. Etienne E. Meidan R. Valdenaire O. Corvol P. Tougard C. J. Biol. Chem. 2003; 278: 545-555Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 35Johnson G.D. Ahn K. Anal. Biochem. 2000; 286: 112-118Crossref PubMed Scopus (47) Google Scholar). ECE-1 activity of HEK-293 cells stably expressing ECE-1d, SV, and SV cut was assayed as described by Luciani et al. (36Luciani N. de Rocquigny H. Turcaud S. Romieu A. Roques B.P. Biochem. J. 2001; 356: 813-819Crossref PubMed Google Scholar). Non-transfected cells served as a negative control and BAEC as a positive control (endogenous activity). Cells were grown to 80% confluence, washed twice with phosphate-buffered saline, and harvested. The cells were pelleted at 300 × g, re-suspended in 200 μl of ice-cold 50 mm Tris/maleate, pH 6.8, containing 1% (w/v) N-octyl glucoside (as permeabilization agent), and protease inhibitor mixture and sonicated. Following 1 h of incubation on ice, the extracts were centrifuged (15,000 × g, 15 min, 4 °C), and the protein content of the supernatants was measured with Bio-Rad DC reagents. ECE-1 activity was assayed in white 96-well microplates in a final volume of 100 μl. Substrate (BK-2, final concentration 30 μm), and 20 μg of cellular protein extracts were incubated in Tris maleate with or without phosphoramidon (100 μm). Fluorescence was measured (λex = 330 nm; λem = 420 nm) in a multiwell plate reader fluorimeter (Varian/Cary Eclipse™ Fluorimeter, Melbourne, Australia). Blanks consisting of all reagents except either cell extract or substrate gave only negligible conversion. Fluorescence was measured at several time points for each cell type until complete hydrolysis was achieved. An incubation time of 90 min was chosen as it gave the maximal activity. Relative BK-2 breakdown was calculated by subtracting the values obtained in the presence of phosphoramidon from the total fluorescence at the corresponding time point. RNA Extraction and Reverse Transcription-PCR—Total RNA was extracted from the cells using TRI Reagent. One microgram of total RNA was reverse-transcribed, and semiquantitative PCR was performed as described previously (37Mamluk R. Chen D. Greber Y. Davis J.S. Meidan R. Biol. Reprod. 1998; 58: 849-856Crossref PubMed Scopus (82) Google Scholar). The sequence of the primers used in PCR reactions is shown in TABLE ONE. Real-time PCR—The PCR reaction was performed as we described previously (33Klipper E. Gilboa T. Levy N. Kisliouk T. Spanel-Borowski K. Meidan R. Reproduction. 2004; 128: 463-473Crossref PubMed Scopus (53) Google Scholar), using the SYBR-Green I PCR kit as recommended by the manufacturer with ROX passive reference. The fold change of the target gene normalized to an endogenous reference, G3PDH, and was calculated by the following equation = 2 - ΔCt, where ΔCt = [(Ct target - Ct G3PDH)]. To compare the amplification efficiency of the primers for different ECE-1 isoforms, plasmids containing inserts of each ECE isoform, ECE-1a, ECE-1b, ECE-1c, and ECE-1d, were generated by PCR with the corresponding 5′ primers and ECE-1 common reverse primer (TABLE ONE). These constructs were electroporated into CHO cells as follows: 10 × 106 cells were electroporated by using a Gene-Pulser (370V, capacitance 960 microfarads) with 10 μg of plasmid DNA. Cells were then transferred into complete DMEM/F-12 medium and cultured for 16 and 48 h. RNA was extracted from the cells and cDNA was analyzed by real-time PCR with two sets of primers. The first primer set included isoform specific primers (TABLE ONE), the second set comprised primers amplifying the common sequence of ECE-1 and was used to normalize for the total amount of ECE-1 expressed in the transfected cells. The ratios of the expression of ECE-1a, -b, -c, and -d to that of total ECE-1 was 2.95, 0.72, 1.24, and 0.47, respectively. The results obtained were corrected for primer efficiency. Statistical Analysis—The differences between groups were analyzed by one-way analysis of variance, employing the post hoc multiple comparisons Dunnett's test. Analysis was performed using SPSS (SPSS Inc. (Chicago, IL) Version 10.05 for Windows). Differences were considered significant if p < 0.05. Abundance of the Various ECE-1 Isoforms in Bovine EC—To determine the abundance of the ECE-1 isoforms in EC we used quantitative real-time PCR with specific primers (TABLE ONE). In all the cell types examined, including macro- and microvascular EC (BAEC and luteal EC, respectively), ECE-1d was by far the most abundant type. Its expression levels were 14-25 times higher than those of the least expressed form 1a (Fig. 1). The mRNA levels of forms b and c were similarly expressed in all three cell types, but the relative expression level of 1c was higher in BAEC than in the two luteal EC types. Nevertheless in BAEC as in other cell types, the levels of isoform 1d expression were four to eight times as high as those in isoforms c and b, respectively (Fig. 1). Identification and Occurrence of the Spliced ECE-1 Forms of mRNA—PCR of cDNA derived from several EC types; BAEC and luteal EC with a 5′ primer specific for each ECE-1 isoform and a common reverse primer located ∼500 bp downstream (TABLE ONE) generated two products for isoforms b, c, and d (Fig. 2). The upper bands were of the expected size and the lower ones appeared ∼140 bp shorter. The existence of these two PCR products was also observed when other 3′ primers of ECE-1 were used (data not shown). The upper and lower bands were excised, cloned, and sequenced. The upper bands were the expected PCR products based on the known sequences of bECE-1 b-d (38Barker S. Khan N.Q. Wood E.G. Corder R. Mol. Pharmacol. 2001; 59: 163-169Crossref PubMed Scopus (15) Google Scholar), and the lower bands were SVs lacking the same 142-bp sequence, corresponding to exon 3′ found in all known ECE-1 isoforms (Ref. 18Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar; Fig. 3A). This region in isoform ECE-1d is highlighted (in Fig. 3B). No such splice variant (lacking exon 3′) was observed for ECE-1a isoform whose transcription begins further downstream, in exon 3. Similar shorter spliced variants were also detected in human umbilical vein EC (data not shown).FIGURE 3A schematic representation of ECE-1 gene structure and its mRNA. A, ECE-1 gene structure showing the first alternative (1c, 1b, 2, 3) exons and their promoters (p). Exons 4-19, common to all isoforms and encoding the major part of ECE-1 cDNA, are not represented at the same scale. Exons are numbered according to Valdenaire et al. (20Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). Exons of the four different full-length ECE-1 mRNAs together with those of the spliced variants mRNAs are depicted below. B, the sequence of the first ∼300 nucleotides of ECE-1d showing the spliced region (gray) corresponding to exon 3′. The putative ATG start codon of the spliced variant is underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next sought to determine the total ECE-1 expression and the ratio between the expression of SV mRNAs (of ECE-1b, -c, and -d) and the full-length forms of ECE-1. For that we designed primers that spanned a unique sequence of SV forms (produced by the end of exon 2′ merged with exon 4; Fig. 3A). This primer (with a reverse primer spanning the region between 333 and 352 in the SV sequence of ECE-1d; TABLE ONE) should amplify all three SV forms. Full-length ECE-1" @default.
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• W2024895902 date "2005-12-01" @default.
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• W2024895902 title "Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain" @default.
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