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Originally published In Press as doi:10.1074/jbc.M505679200 on September 26, 2005

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 Meidan{ddagger}1, Eyal Klipper{ddagger}, Tamar Gilboa{ddagger}, Laurent Muller§, and Nitzan Levy{ddagger}

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelin-converting enzyme-1 (ECE-1)2 is a type II membrane protease that belongs to the neprilysin (NEP) family of zinc metallopeptidases (1, 2). ECE-1 is abundantly expressed in the vascular endothelial cells (EC) of all tissues but is also found in nonvascular cells (3, 4-6). 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 (7). ECE-1 is a glycosylated protein with 10 putative N-linked glycosylation sites (8). 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 (9, 10). 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 (11-13). The three ETs mediate their various effects via two G protein-coupled receptors: ETA and ETB (14, 15). 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 (1, 4)). ECE-1 then specifically hydrolyzes the Trp21-Val/Ile22 bonds of big-ETs to produce biologically active ETs (1, 16). 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 (17).

Four isoforms of human ECE-1 (1a, 1b, 1c, and 1d) have been identified to date (8, 18-20). 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 (8, 18-20). 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 (19, 21-23). 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 (24). An exciting novel substrate for ECE-1 is the {beta}-amyloid peptide that is implicated in the pathogenesis of Alzheimer disease (25, 26).

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 (27, 28). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (29-31), 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 (5, 32). 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.


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  		TABLE ONE
PCR primer list

 
Cell Fractionation and Western Blot Analysis—The procedure for total cell extracts was carried out as we have previously described (32, 33). 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 x 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 x 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 (34). 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 (23, 35). ECE-1 activity of HEK-293 cells stably expressing ECE-1d, SV, and SV cut was assayed as described by Luciani et al. (36). 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 x 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 x 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 ({lambda}ex = 330 nm; {lambda}em = 420 nm) in a multiwell plate reader fluorimeter (Varian/Cary EclipseTM 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.



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  		FIGURE 1.
Abundance of mRNA expression of each ECE-1 isoform (ECE-1a, -1b, -1c, and -1d) in different endothelial cell types. Total RNA was extracted from BAEC and freshly isolated and cultured luteal EC (LEC) and reverse-transcribed. Following real-time PCR, relative mean mRNA levels for each cell type were calculated using the {Delta}Ct method (see "Experimental Procedures"). Data are the means ± S.E. from four different experiments. Different letters indicate significant differences among isoforms within each cell type (p < 0.05).

 
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 (37). The sequence of the primers used in PCR reactions is shown in TABLE ONE.



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  		FIGURE 2.
Amplification of full-length ECE-1 isoforms and alternatively spliced forms in BAEC and freshly isolated and cultured luteal EC (LEC). Total RNA was extracted from the various endothelial cell types and reverse transcribed. The PCR reaction was conducted with a 5' primer specific for each ECE-1 isoform and a common reverse primer (see TABLE ONE). Inverse images of ethidium bromide-stained agarose gel show amplification of the various products.

 
Real-time PCR—The PCR reaction was performed as we described previously (33), 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 - {Delta}Ct, where {Delta}Ct = [(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 x 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.



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  		FIGURE 3.
A 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. (20). 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.

 
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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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).



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  		FIGURE 4.
The mRNA expression of total ECE-1 levels and the ratio (%) of SV to full-length ECE-1 isoforms in BAEC, luteal EC (LEC; freshly isolated and cultured), and in luteal steroidogenic cells (LSC). Data are the means ± S.E. from six different separate experiments. Following real-time PCR, relative mean mRNA levels for each cell type were calculated using the {Delta}Ct method (see "Experimental Procedures"). Different letters indicate significant differences among cell types (p < 0.05).

 
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 (38), and the lower bands were SVs lacking the same 142-bp sequence, corresponding to exon 3' found in all known ECE-1 isoforms (Ref. 18; 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).



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  		FIGURE 5.
Detection of ECE-1 proteins in HEK-293 cells stably expressing bECE-1d, SV, and SVcut plasmids. Total proteins were extracted in lysis buffer and processed as detailed under "Experimental Procedures." Twenty-five mg of each cell extract were separated by SDS-PAGE under reducing conditions. Proteins were detected by Western blots using COOH-terminal-specific antibody (A) and two specific NH2-terminal antibodies (B and C). NT, non-transfected cells.

 



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  		FIGURE 6.
Mutation of the putative bECE-1sv start codon. The following plasmids, bECE-1d, bECE-1d mut, SV, Svmut, and Svcut, were examined, either directly in a cell-free transcription/translation system (TNT) reaction (A) or after transfection into HEK-293 cells (B). Protein extracts of HEK-293 cells were detected by Western blot with an anti-COOH-terminal ECE-1 antibody. NT, non-transfected cells.

 



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  		FIGURE 7.
Biological activity of proteins extracted from HEK-293 cells overexpressing ECE-1d, SV, and SVcut plasmids and BAEC. Total protein extracts (20 µg) were incubated in Tris maleate buffer containing the substrate (BK-2, 30 µM) with or without phosphoramidon (100 µM). Fluorescence was measured ({lambda}ex = 330 nm; {lambda}em = 420 nm) in a multiwell plate reader fluorimeter. Data (means ± S.E.; n = 3) are the percentages of specific BK-2 breakdown. For each cell type (HEK-293 cells overexpressing ECE-1d, SV, and SVcut plasmids and BAEC) fluorescence was measured at several time points, and the data presented are from the time point that gave maximal specific fluorescence. NT, non-transfected cells. Different letters indicate significant differences among samples (p < 0.05).

 
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 forms were amplified with a primer that resides in the sequence of exon 3', which is absent from SV forms (TABLE ONE). Fig. 4 depicts the ratio of SV to full-length ECE-1 in endothelial (BAEC and luteal EC) and non-endothelial (luteal steroidogenic) cell types. In all EC types the expression of SV forms was high and ranged between 21 and 37% of that of the full-length forms of the enzyme. Interestingly, regardless of total ECE-1 levels, in luteal EC, whether cultured or freshly isolated, the proportion of SV was higher than in BAEC or luteal steroidogenic cells. For instance, in luteal steroidogenic cells expressing similar total ECE-1 levels as freshly isolated luteal EC, the SV forms were only 6% of full-length mRNA levels as compared with 22.5% in luteal EC (Fig. 4).

Characterization of Translated Forms of ECE-1—As ECE-1d was found to be the most abundant form (Fig. 1) we cloned and stably expressed its full-length and SV forms. An additional cDNA, in which part of the 5'-end of SV was deleted (SVcut) was stably expressed in HEK-293 cells as well. An antibody that recognizes the common COOH-terminal end of the enzyme identified a protein product in all cells expressing ECE-1d, SV, and SVcut (Fig. 5A). Cells transfected with full-length ECE-1d expressed as expected a protein of ~120 kDa, whereas SV and SVcut transfected cells both expressed a protein of ~75 kDa (Fig. 5A). Two different antisera directed against NH2-terminal parts of the molecule (one specific for ECE-1d; Fig. 5A and the second for forms b, c, and d; Fig. 5, B and C) were then examined in Western blot analysis. These two antibodies were readily bound to full-length ECE-1d but did not recognize the protein products of either SV or SVcut. These findings therefore suggest that SV forms share only the COOH-terminal end with ECE-1d. As NH2-terminal antibodies did not recognize the translated products of SV and SVcut (Fig. 5) it appeared that these forms have a different start codon from that used in ECE-1d. This was in fact expected, since splicing out of the 142-bp segment would modify the reading frame. Because cDNAs of both SV and the shorter form SVcut cDNA were translated into proteins with the same apparent molecular mass, it suggested that another start codon was located further downstream. ATG located between 207 and 209 bp in the cDNA sequence of the SV form of ECE-1d could drive translation in-frame with that of conventional ECE-1. Additionally, this ATG is a good candidate to be the translation initiator, since it lies within a Kozak sequence. To test this assumption, an SV construct mutated at ATG207 was produced. As shown in Fig. 6 mutating the ATG to TTT eliminated the SV protein, which suggests that ATG207 is indeed the putative start codon for the SV form. This was confirmed by Western blot analysis of cells expressing SV plasmid as well as in a cell-free translation system (TNT; Fig. 6). Mutating the same ATG in the ECE-1d sequence did not affect its translation (Fig. 6A).



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  		FIGURE 8.
Subcellular localization of ECE-1d and SV. A, immmunofluorescence. The subcellular distribution of ECE-1d (panels a and c) and of SV (panels b and d) was investigated in transiently transfected CHO (panels a and b) and HEK-293 (panels c and d) cells using the antibodies directed the COOH terminus of ECE-1. Immunoreactivity was detected with an antibody coupled to AlexaFluor-555 (red). Nuclei were stained with To-Pro-3 (blue). ECE-1d is present in intracellular vesicles concentrated in the perinuclear region. SV is detected as a diffuse staining of the cytosol. Bar = 10 mm. B, Western blot analysis. Upper panel, cytosolic fraction (supernatant of 44,000 x g); lower panel, particulate fraction (15,000 x g pellet). For details refer to "Experimental Procedures." Protein was detected by Western blot analysis using an antibody recognizing the common COOH-terminal end of ECE-1.

 



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  		FIGURE 9.
Native expression of ECE-1 sv protein in corpus luteum (CL) tissue, cultured LEC, and BAEC. Total protein was extracted in lysis buffer and detected by Western blot analysis with an anti COOH-terminal antibody. The double-headed arrow marks full-length ECE-1 forms; the shaded arrow indicates putative ECE-1sv.

 
Since the catalytic domain of the enzyme is found in its far COOH-terminal end, the translation product of SV and SVcut, designated from here on ECE-1sv, is expected to retain its bioactivity. Therefore we next examined the catalytic activity of extracts of HEK-293 that stably expressed ECE-1d, SV, and SVcut by using the quenched BK2 peptide (aminomethylcoumarin-RPPGFSAFR-dinitrophenyl) as a substrate (Fig. 7). While non-transfected cells had only marginal activity, SV- and SVcut- overexpressing cells exhibited enzymatic activity that was similar to that exhibited by full-length ECE-1d (Fig. 7). This enzymatic activity was inhibited by phosphoramidon, the common NEP/ECE-1 inhibitor. The catalytic activity of ECE-1sv was comparable on a total protein basis with that exhibited by cells expressing ECE-1d. These data further support those shown in Fig. 5b, suggesting that full-length ECE-1 and ECE-1sv share their COOH-terminal parts.

Localization of ECE-1sv and ECE-1d in Overexpressing Cells—The coding sequences of ECE-1d and its splice variant were transiently transfected in HEK 293 and CHO cells, and their intracellular localization was probed by indirect immunofluorescence using anti COOH-terminal antibody and Western blots pf extracts after diffeential centrifugation (Fig. 8, A and B). As expected, using immunofluorescence, ECE-1d was present in plasma membrane and vesicles (Ref. 23; Fig. 8A) and in the particulate cell fraction (15,000 x g pellet; Fig. 8B). ECE-1sv on the other hand exhibited a diffused cytosolic labeling by immunofluorescence (Fig. 8A), in agreement with Western blot data (Fig. 8B), demonstrated that most of ECE-1sv forms were detected in the 44,000 x g supernatant fraction, cytosol. The sequence of these two proteins predicts their different subcellular localization; while ECE-1d, as other full-length forms of ECE-1, contains the TM domain and is therefore a membrane-anchored protein, ECE-1sv lacks a TM sequence (or signal peptide) and therefore is expected to remain cytosolic.

Native Expression of ECE-1sv Protein in Normal Cells—Having demonstrated that EC abundantly expressed SV mRNA we next examined whether ECE-1sv, as characterized in cells overexpressing SV cDNA, was endogeneously present in normal cells. Data presented in Fig. 9 show a Western blot obtained by using an anti ECE-1 antibody. A 75-kDa protein was identified in BAEC, luteal EC, and also in corpus luteum, a highly vascular tissue. As expected these samples expressed full-length ECE-1 proteins, which appeared as two bands (Fig. 9), that most probably corresponded to different glycosylation forms of the enzyme.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study documented the prevalence of the various ECE-1 isoforms and found that ECE-1d was the most abundant and ECE-1a the least expressed. The mRNA of isoforms ECE-1b and -c were also rather scarce, with levels 6-10-fold lower than that of ECE-1d. However, these were not the only ECE-1 forms found in EC, we have also identified novel splice variants of ECE-1b, -c, and -d mRNA, which lacked exon 3' that codes for the TM domain of the enzyme. These mRNA species are abundantly expressed in EC, in microvascular EC they comprise up to 40% of full length ECE-1 mRNA. We used plasmid containing the coding sequence of the SV form of ECE-1d overexpressed in HEK cells and in a cell-free translation system to demonstrate that it is translated into a protein of apparent molecular mass of 75 kDa. This protein was recognized by anti-ECE-1 antibody raised against the COOH-terminal end of the enzyme. The start site of this protein was further downstream in a region common to all ECE-1 forms, which suggests that the three mRNA species were translated into one protein ECE-1sv. Since the catalytic domain of the enzyme is found in its far COOH-terminal end, the translation product is expected to retain its bioactivity. Indeed, the ECE-1sv protein was biologically active and cleaved a synthetic ECE-1 substrate as efficiently as the full-length enzyme. Last, we have identified a protein with an apparent molecular mass as ECE-1sv in normal EC and within a highly vascular endocrine gland such as the corpus luteum.

The hitherto known isoforms of ECE-1 arise from the use of alternative promoters upstream of exon 3' (18, 19). ECE-1sv is the first identified ECE-1 isoform that arises from an internal exon splicing. Alternative splicing is widespread in mammalian gene expression and is a major contributor to the functional complexity of mammalian genomes (39, 40). Variant splice patterns are often characteristic of specific stages of development, particular tissues, or a disease state (41). Whether the ratio of ECE-1 to its splice variant form also differs between different physiological or pathological conditions is as yet unknown. However, it is noteworthy that ECE-1sv was highly expressed in EC derived from macro and microvascular beds but much less so in steroidogenic cells that also express ECE-1, and this could imply that there is a cell-specific splicing mechanism. Since ECE-1sv is not routed to the secretory pathway it is expected to remain non-glycosylated as indeed occurred (TNT compared with Western blots), but nonetheless the protein retained its catalytic activity. Therefore these findings suggest that glycosylation may not be necessary for the enzyme to be bioactive. In agreement with this conclusion, deglycosylation of purified ECE-1 did not significantly alter its enzyme activity (42). Mutating two of ECE-1 glycosylation sites is, however, enough to render the enzyme inactive (43), which is expected considering the importance of glycosylation for proper protein folding in the secretory pathway (for review see (44). Our data thus suggest that molecular chaperones can efficiently compensate for the lack of glycosylation and promote proper folding of ECE-1sv in the cytosol.

Catalytic activity was demonstrated here by using BK-2 as a substrate, but its physiological substrates still remain to be determined. The catalytic site of full-length ECE-1 faces the extracelular milieu or the luminal side of vesicles, whereas the active site of ECE-1sv would resides within the cells cytoplasm. What are the putative substrates of this unique isoform? It is tempting to speculate that cytosolic ECE-1sv may degrade small peptides such as angiotensin I, bradykinin, neurotensin, and substance P, internalized via their receptors, or it could also cleave {beta}-amyloid peptide. All of these peptides (24, 25) were formerly shown to be very efficiently degraded by soluble ECE-1 engineered by truncating its TM domain. Breaking down these small peptides by ECE-1sv could act to terminate their signaling in a manner analogous to the action of insulin-degrading enzyme. Insulin-degrading enzyme is a neutral thiol metalloprotease, ubiquitously expressed, which is present mainly in cytosol and peroxisomes. The active site of insulin-degrading enzyme, HEXXH is very similar to that of ECE-1-HELTH, which could explain the overlapping specificity of these enzymes, i.e. cleavage of insulin and amyloid peptides.

Interestingly, the presence of a soluble intracellular form of ECE-1 in endothelial and vascular smooth muscle cells was postulated in the past (38, 45); however, no molecular identification was provided before.

The mechanism responsible for ECE-1sv translation most likely involves re-initiation of translation. This was shown to occur with mRNAs that have small open reading frames near the 5'-end (46). These short open reading frames are initiated at the respective start codons of isoforms ECE-1b, -c, and -d and terminated by a stop codon produced by splicing out exon 3' (~50 codons). It is also noteworthy that the start codon that initiates the translation of ECE-1sv, ccATGg, has a strong Kozak's motif (46).

The present findings concerning the prevalence of ECE-1 isoforms in EC may shed new light on these isoforms within their physiological context. Using quantitative real-time PCR we demonstrated here that ECE-1d is the dominant isoform. This was true for all EC types examined regardless of their origin. Currently, there is ambiguity regarding the abundance of ECE-1 isoforms in EC. Both ECE-1a and -1c were claimed to be the major isoforms (8, 47-50), and although these were based on either observations made prior to the discovery of ECE-1d sequence (19) or on the use methods that are quantitatively less reliable, it persisted in the literature.

The physiological significance of the different ECE-1 isoforms emanates from their distinct cellular and subcellular distribution. For example ECE-1a is only expressed in vascular EC and not in other cell types expressing the enzyme such as smooth muscle cells or steroiodgenic luteal cells (32, 50). Examination of the subcellular distribution of ECE-1 isoforms overexpressed in AtT-20 neuroendocrine cells (23) showed that ECE-1a and -1c were present at the plasma membrane, whereas ECE-1b and ECE-1d were retained inside the cells. However, in stably transfected CHO-K1 cells, ECE-1d was present at the plasma membrane yet at a lower level than ECE1a (19). Whether ECE-1d subcellular localization is cell dependent or not, being the dominant ECE-1 isoform in ECs, it seems imperative to gain a better understanding of its transcriptional regulation, sorting, and routing in naturally expressing EC.

The findings presented here, which demonstrate the plethora of different ECE-1 forms, full-length and spliced variants, that co-exist within EC offer a novel perspective on the physiological activation of ET-1 and of other substrates of ECE-1 family of proteins.


   FOOTNOTES
 
* 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. Back

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

2 The abbreviations used are: ECE-1, endothelin-converting enzyme-1; bECE-1, bovine ECE-1; EC, endothelial cells; DMEM, Dulbecco's minimum essential medium; BAEC, bovine aortic EC; CHO, Chinese hamster ovary; SV, splice variant; mut, mutated; TM, transmembrane; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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