International Journal of Biological Macromolecules 72 (2015) 673–679
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Serine proteases as candidates for proteolytic processing of angiotensin-I converting enzyme Danielle S. Aragão a , Maria Claudina C. de Andrade c , Fabiana Ebihara a , Ingrid K.M. Watanabe a , Dayane C.B.P. Magalhães a , Maria Aparecida Juliano b , Izaura Yoshico Hirata b , Dulce Elena Casarini a,∗ a
Department of Medicine, Nephrology Division, Federal University of São Paulo, Brazil Department of Biophysics, Federal University of São Paulo, Brazil c Albert Einstein Research Institute, Brazil b
a r t i c l e
i n f o
Article history: Received 23 April 2014 Received in revised form 4 September 2014 Accepted 5 September 2014 Available online 28 September 2014 Keywords: Angiotensin-I converting enzyme Shedding, Mesangial cells
a b s t r a c t Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. Ndomain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Angiotensin-I converting enzyme (ACE) (EC 3.4.15.1) is a broadly distributed zinc metalloendopeptidase which plays a role in blood pressure and electrolyte homeostasis [1]. Primarily responsible for the conversion of angiotensin I (Ang I) into angiotensin II (Ang II) and the inactivation of bradykinin, ACE is also involved in immunity, reproduction and neuropeptide metabolism [2,3]. Two ACE membrane-bound isoenzymes have been described in the literature. A somatic form, known as somatic ACE (sACE), is widely distributed and comprises two homologous domains (N and C domains) presenting 60% of sequence identity and each bearing a functional catalytic site [2,4–6]. A second form is only expressed in testis (tACE) and presents a unique domain identical
∗ Corresponding author at: Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Medicina, Disciplina de Nefrologia, Rua Botucatu 740, CEP 04023-900, São Paulo-SP, Brasil. Tel.: +55 11 59041684; fax: +55 11 59041683. E-mail addresses: [email protected], [email protected] (D.E. Casarini). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.017 0141-8130/© 2014 Elsevier B.V. All rights reserved.
to the C domain of sACE, except for 36 amino acid residues at its N-terminus [7,8]. The transmembrane protein ACE can be proteolytically shed from the cell surface by the action of a unidentified protease, resulting in soluble forms found in blood plasma, amniotic fluid, seminal plasma [3,9], ileal fluid [10], urine [11–13] and mesangial cells (MC) [14,15]. In the shedding process, the entire extracellular domain is released to the external environment, converting the membraneanchored protein into a soluble form [2,16]. Previous studies indicate that ACE sheddase a metalloprotease that is stimulated by phorbol esters (PMA) and inhibited by hydroxamates (TAPI-1) [17–19]. N-domain soluble ACE isoforms of 65 and 90 kDa present a different pattern of distribution between normotensive and hypertensive subjects. The 90-kDa isoform described as a genetic marker of hypertension was detected only in cells and urine from hypertensive humans and animals [11–15]. The soluble 65 kDa and 90 kDa ACE isoforms are found in cell lysates and culture media of MC, which exert multiple functions in the glomerular physiology and pathophysiology [20]. MC
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also express other components of renin-angiotensin system (RAS), including sACE [14,15], ACE2 [21], NEP [22] and renin [23–25]. Andrade et al. (1998) purified and characterized an active 65 kDa ACE from Wistar rats primary MC. These isoforms were able to cleave Ang I into Ang II and hydrolyze angiotensin 1–7 (Ang 1–7) [14]. In 2006, the 90 kDa isoform was isolated from cell lysate and culture media from primary MC from SHR. Besides the presence of these nACE in culture media, their location in the MC nuclei indicates a proteolytic processing of the sACE at the intracellular environment or a post-transcriptional modification of the enzyme [15]. Since cultured MC have been considered a valid model for the study of synthesis and release of bioactive products in the kidney, which could act as autocrine, intracrine and/or paracrine mediators [26–28], and based on the fact that MC presents somatic and soluble forms of ACE, this cell line can be introduced as a suitable in vitro model in order to improve knowledge on the components and functions related to the ACE shedding. The aim of this study was to evaluate the existence of ACE sheddases in mice immortalized MC (IMC) and then, to isolate and identify these enzymes.
2.4.2. ACE activity ACE catalytic activity was measured as described by Friedland and Silverstein [31] and Piquilloud [32]. Aliquots of cell lysate (50 L) and culture media (5 L) were incubated with 200 L of 100 mM Borate, 300 nM NaCl, 0.1 nM ZnSO4 buffer, pH 8.3, containing 1 mM of Z-Phe-His-Leu (ZPhe-HL) or 5 mM of Hippuryl-His-Leu (HHL) (Bachem, USA) for 2 h at 37 ◦ C. The reaction was stopped by the addition of 1.5 mL of 280 nM NaOH and incubated with 100 mL of 20 mg/mL o-phtaldialdehyde (Sigma, USA) diluted in methanol for 10 min followed by 200 L of 3N HCl. The liberated dipeptide -HL was fluorometrically measured using F-2000 Fluorescent Spectrophotometer (TECAN, USA) with an excitation of 360 nm and emission of 500 nm. The standard curve was obtained using different concentrations of -HL (His-Leu, Sigma-Aldrich, USA) in the blank reaction mixture and it showed a linear relation between relative fluorescence and -HL concentration.
2. Materials and methods
Besides the design of specific substrates for ACE sheddases, cells were treated with PMA and TAPI-1, a shedding activator and inhibitor, respectively, to confirm the presence of ACE sheddases in IMC. Prior to reach confluence, IMC were incubated with DMEN/F12 (3:1) with 2% FBS for 12 h. Subsequently, the cells were maintained for 3 h with DMEN/F12 (3:1) with FBS (control group), DMEN/F12 (3:1) with 2% FBS containing 10 M of Phorbol 12-myristate 13acetate (Sigma-Aldrich, USA) (PMA group) and DMEN/F12 (3:1) with 2% FBS containing 100 M of TAPI-1 (Calbiochem, USA) (TAPI1 group) (N = 3). After the treatment, the culture media and the cell lysates were collected as previously described. The sheddases and ACE enzymatic activities were determined using fluorogenic substrates S1 and S2 and ACE substrates ZPhe-HL and HHL. The catalytic activity of each ACE domain was determined using the same quantity of enzyme for each assay and the results expressed by ZPhe-HL/HHL ratio that suggest the presence of nACE.
This study was approved by the Ethics Committee of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil. 2.1. Immortalized mesangial cells culture (IMC) IMC from mice were purchased from The American Type of Culture Collection (ATCC: CRL-1927). Cells were grown in DMEM/F12 (3:1) (Gibco, USA) supplemented with 5% fetal bovine serum (FBS), 5000 U/L penicillin and 50 mg/L streptomycin. Before the experiments, IMC were incubated with DEMEN/F12 without FBS for 24 h. 2.2. Culture media and cell lysate from IMC preparation After the removal of the culture media, the culture flasks were rinsed twice with 5 mL of 0.025 M Tris-HCl buffer pH 8.0 and scraped with 1 mL of 0.05 M HEPES, 0.5 M NaCl buffer, pH 7.5 containing 1% of Triton X-114 at 4 ◦ C. The lysate was collected, sonicated at 4 ◦ C for 20 min and centrifuged at 12,000 rpm for 5 min. The supernatant was fractioned and stored at −20 ◦ C. 2.3. Synthesis of fluorogenic substrates for ACE sheddases based on the C-terminal sequence of 65 and 90 kDa N-terminal ACE from MC The C-terminal amino acid sequence of the 65 and 90 kDa Nterminal soluble ACE were determined as previously described [29]. Based on these C-terminal sequences, two fluorogenic substrates (S1: Abz-RWGVFSGRTPQ-EDDnp and S2: Abz-EYQWHPPLPDNYQEDDnp) for the sheddases responsible for the generation of 65 and 90 kDa isoforms, respectively, were constructed using Fmoc synthesis protocol and H-Pro-2-chlorotrityl resin [30] (Fig. 1). 2.4. Enzymatic activity assay 2.4.1. ACE sheddase activity Sheddases catalytic activities were measured fluorometrically using the substrates described above. Aliquots (50 L) of chromatography fractions, cell lysate or culture media were incubated in 1 mL of 0.05 M Tris-HCl, 0.15 M NaCl buffer, pH 8.0, containing 2 M of fluorogenic substrates at 37 ◦ C for 2 h. The fluorescence was measured using F-2000 Fluorescent Spectrophotometer (TECAN, USA) with an excitation of 320 nm and emission of 420 nm.
2.5. Effects of the treatment with PMA or TAPI-1 on ACE sheddases activity and the ratio ZPhe-HL/HHL ACE activities from the cell lysate and culture media of IMC
2.6. Partial purification of ACE sheddases from IMC IMC cell lysates were submitted to gel filtration chromatography on 120 mL AcA-34 resin column (Sigma-Aldrich, USA) coupled to a peristaltic pump (Amersham Pharmacia Biotech-GE, USA) equilibrated with 0.05 M Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl. The enzymes were eluted with the same buffer and 1 mL fractions were collected at a flow rate of 20 mL/h. The optical density of each fraction was quantified at 280 nm and the chromatographic fractions were pooled according to detection of enzymatic activity for S1 and S2 fluorogenic substrates. 2.7. Analysis of the somatic ACE cleavage by the proteins presents in the chromatographic pools 1 and 2 The chromatographic pools 1 and 2 presenting catalytic activity for the S1 and S2 fluorogenic substrates were dialyzed and concentrated to 0.1 mL against 0.05 M Tris-HCl buffer, pH 8.0 using Amicon Ultra with a 10-kDa exclusion membrane (Millipore, USA). Aliquots of pool 1 and pool 2 were incubated in the same buffer with 0.25 mL of somatic ACE (ACE elevated control-Sigma Diagnostics, USA) diluted as recommended by the manufacturers, for 24 h at 37 ◦ C. Aliquots were taken at times 0 h and 24 h and the reaction was stopped with the addition of 0.1% orthophosphoric acid (Merck, USA). The products of the somatic ACE hydrolysis by the enzymes in pools 1 and 2 were analyzed by Western blotting.
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Fig. 1. Sequence alignment of rat (UniProtKB/Swiss-Prot: P47820.1) and mouse (UniProtKB/Swiss-Prot: P09470.3) sACE. The C-terminal alignment of 65 kDa rat nACE ended at Ser482 . The same analysis for 90 kDa rat nACE showed that the enzyme sequence ended at Pro629 [29].
2.8. SDS-PAGE and Western blotting analysis Proteins were analyzed by sodium dodecil sulfatepolyacrylamide gel (7.5%) electrophoresis (SDS-PAGE) under reduced condition using -mercaptoethanol. Electrophoretic transfer was performed for 60 min under constant voltage (60 V), into a nitrocellulose membrane (Millipore, USA) using transfer buffer (0.015 M Tris, 0.190 M glycine, and 0.1% SDS). The membrane was incubated in 0.1 M TBS with 0.1% of tween containing 0.5% of bovine albumin (Sigma, USA) for 1 h before 2-h incubation with anti-ACE monoclonal antibodies 9B9 (1:2000), 5F1 (1:500) (Chemicon International, USA) or BAF 929 (1:500) (RD System, USA) specific to the N-domain portion of the molecule. Subsequent steps were performed using the Amershan ECL Select Western blotting detection reagent kit (GE Healthcare, EUA) or the streptavidin/phosphatase alkaline system (Amershan Pharmacia Biosciences, USA) using BCIP/NBT as substrate (Bio-Rad Laboratories, USA) as recommended by the manufacturers.
on a PPSQ-23 Automated Protein Sequencer (Shimadzu Corporation, Japan). The results were submitted to the BLAST platform (www.nbci.com/blast) in order to identify the proteins.
2.10. Analysis of serine proteases SH1 and STH2 previously purified from human urine as ACE sheddases Aliquots of the endopeptidases, serine protease SH1 and serine thiol protease STH2 previously purified and characterized from human urine by Casarini et al. in 1992 and 1993 and Quinto et al. in 1999 and 2004 [33–36] were incubated in 0.05 M Tris-HCl buffer, pH 8.0 at 37 ◦ C for 24 h with 100 g of somatic ACE from rabbit lung (Sigma-Aldrich, USA). Aliquots were taken at times 0 h and 24 h and the reaction was stopped with the addition of 0.1% orthophosphoric acid (Merck, USA). The products of the hydrolysis of the somatic ACE by SH1 and STH2 enzymes were analyzed by Western blotting.
2.9. Amino-terminal sequence of the proteins from pool 2 3. Results The chromatographic pool presenting higher catalytic activity for the S1 and S2 fluorogenic substrates was dialyzed against water using Amicon Ultra with a 10-kDa pore membrane (Millipore, USA) and subsequently dried. The sample (50 g) was ressuspended in Laemmli sample buffer (Bio-Rad, USA) and submitted to SDS-PAGE. Protein gel staining was performed using Silver Staining Protein kit (Amershan Biosciences, USA) according to the manufactures instructions. The same procedure was repeated and proteins were electroblotted to a PVDF protein sequencing membrane (Bio-Rad Laboratories, USA) using a 0.1 M CAPS buffer, pH 11.0. Membrane was stained with 2% Ponceau-S in 1% acetic acid targeting the protein localization. Protein bands were excised from the membrane and submitted to N-terminal amino acid sequencing
3.1. Enzymatic activity The ACE sheddase enzymatic activity was determined in culture media and cell lysate from IMC. The cell lysate showed specific activity of 205.7 (±24.2) and 62.4 (±3.2) nM/min/mg for the S1 and S2 fluorogenic substrates, respectively, whereas the culture media showed specific activity of 46.9 (±33.7) and 43.8 (±6.8) nM/min/mg for the S1 and S2, respectively. The cell lysate showed specific ACE activity of 0.30 (±0.07) and 0.21 (±0.10) nmol/min/mg for the ZPhe-HL and HHL, respectively, and the ratio ZPhe-HL/HHL was 1.2 (±0.14). The culture media showed specific activity of 2.67 (±0.31) and 0.43
Fig. 2. Specific activity nM/min/mg of A: 65 kDA nACE sheddase and B: 90 kDA nACE sheddase from IMC lysate and culture media. Results are mean ± SE. * p < 0.05 vs CT; # p < 0.05 vs TAPI-1.
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Pool 1
70
Pool 2
0.6
50
0.4
40 30 0.2
20
OD 280 nm
nM/min/mL
60
10 0
0.0 20
30
40
50
60
70
80
Fractions
90
100 110 120
Fig. 5. Gel filtration chromatography using AcA-34 column of IMC lysate. ACE ) Abzsheddases were eluted in two peaks with enzymatic activity using ( ) Abz-EYQWHPPLPDNYQ-EDDnp (S2) as RWGVFSGRTPQ-EDDnp (S1) and ( ) OD 280 nm. substrates (
Fig. 3. Ratio of Zphe-HL/HHL ACE substrates from IMC lysates and culture media. Results are mean ± SE. * p < 0.05 vs CT, # p < 0.05 vs PMA.
Fig. 6. Western blotting analysis of incubates of sACE with pool 1 and 2 from gel filtration using specific ACE antibody (5F1). Lane 1: pool 1; lane 2: pool 2; lane 3: pool 2 + ACE 0 h; lane 4: pool 2 + ACE 24 h; lane 5: pool 1 + ACE 0 h; lane 6: pool 1 + ACE 24 h.
The cell lysate with enzymatic activity was applied to an AcA-34 gel filtration column equilibrated with 0.05 M Tris-HCl, 0.15 M NaCl buffer, pH 8.0. ACE sheddases were eluted in two peaks denominated as pool 1 and 2. The pool 1 (fractions 41–59) presented specific activity of 241.9 and 122.8 nM/mg using S1 and S2 as substrates, respectively. The pool 2 (fractions 72–99) specific activity was 1618.2 and 522.7 nM/mg for S1 and S2 substrates, respectively (Fig. 5, Table 1).
Fig. 4. Western blotting of IMC lysate using 9B9 antibody. Lane 1: Molecular weight marker; Lanes 2 and 3: IMC lysate.
(±0.09) nmol/min/mg for the ZPhe-HL and HHL, respectively, and the ratio ZPhe-HL/HHL was 5.82 (±0.60). The treatment with PMA activated the sheddase responsible for the generation of the 65 and 90 kDa isoforms in IMC cell lysate when compared to the control group, whereas TAPI-1 did not affect the enzymatic activity. In the culture media, the enzymatic activity able to hydrolyze S1 substrate was higher in PMA treatment than in TAPI-1 (Fig. 2). The ACE activity ratio ZPhe-HL/HHL was higher in the culture media of group treated with PMA when compared to control and TAPI-1 groups suggesting the presence of nACE (Fig. 3).
3.3. Cleavage of somatic ACE by enzymes from pools 1 and 2 After 24 h of incubation of sACE with the enzyme from pools 1 and 2, we immunodetected ACE isoforms with molecular weight of 65 and 90 kDa (Fig. 6). 3.4. N-terminal sequence of protein from purified pool 2
3.2. Partial purification of ACE sheddases from IMC Based on the results obtained after incubation of pool 1 and pool 2 with sACE, we decided to analyze the N-terminal sequence of the enzyme purified from pool 2. The protein was identified as serine protease as described in Table 2.
Prior to the purification performance, ACE protein expression was assessed by Western blotting. The IMC lysate expresses sACE, presenting 130 kDa and nACE form with 65 kDa (Fig. 4). Table 1 Partial purification of ACE sheddases from IMC using S1 and S2 fluorogenic substrates. Step
Volume (mL)
Protein (mg/mL)
Total protein (mg)
Enzymatic activity (nM/min/mL)
Specific activity (nM/mg)
Purification (X)
Recovery (%)
S1 Pool 1 Pool 2
1.2 7.5 15.0
2.3 0.036 0.033
2.8 0.27 0.50
357.45 8.7 53.4
154.1 241.9 1618.2
1 1.6 10.5
100 9.7 17.8
S2 Pool 1 Pool 2
1.2 7.5 15.0
2.3 0.036 0.033
2.8 0.27 0.50
80.04 4.4 17.3
34.5 122.8 522.7
1 3.6 15.2
100 9.7 17.8
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Fig. 7. Western blotting analysis of incubates of the serine proteases SH1 and STH2 with sACE using specific ACE antibodies (5F1 or BAF 929). Lane 1: molecular weight marker; lane 2: SH1 + ACE 0 h; lane 3: SH1 + ACE 24 h; lane 4: STH2 + ACE 0 h; lane 5: STH2 + ACE 24 h; lane 6: ACE 0 h; lane 7: ACE 24 h. The arrows indicate proteins with 65, 75 and 90 kDa on lane 2 and with 65 and 90 kDa on lane 4.
Table 2 Amino-terminal sequence of the protein bands found in partial purification of ACE sheddases. Protein
Homologues sequences
Description and species
Band 1 DRXEDTWPW
(601–609) DSQEGTWPW
Transmenbrane protease serine 7 (Matriptase-3)-Rattus novergicus Transmenbrane protease serine 7 (Matriptase-3)-Mus musculus
(597–605) DSQEGTWPW
Band 2 TDYRED
(231–236) TNYRED
Serine protease 45-Bos taurus
Band 3 HPXTPXNE
(326–333) HPGSPENE
Serine protease 40-Mus musculus
3.5. Hydrolysis of S1 and S2 substrates by SH1 e STH2 from human urine Based on results obtained from N-terminal sequence that identified the protein from pool 2 as serine protease, we decided to study the action of SH1 and STH2 enzymes previously purified from human urine upon S1 and S2 fluorogenic substrates. The specific activity of SH1 was 7.34 and 4.22 nM/min/mg for the S1 and S2, respectively, and for STH2 was 34.35 and 11.02 nM/min/mg for S1 and S2, respectively. 3.6. Analysis of the sACE cleavage by the serine proteases SH1 and STH2 by Western Blotting Purified SH1 and STH2 were able to hydrolyze sACE in vitro. After 24 h of incubation of SH1 and STH2 with sACE, separately, we detected the presence of protein bands with 65, 75 and 90 kDa in the incubation of SH1+ sACE and 65 and 90 kDa in the incubation of STH2+ sACE, confirming the cleavage of sACE (Fig. 7). 4. Discussion Active soluble nACE isoforms with 65 and 90 kDa were purified from cell lysates and culture media from Wistar or spontaneously hypertensive rat (SHR) primary MC, being the 90 kDa isoform only expressed in SHR cells [14,15]. Andrade and Affonso et al. (2010) [29] analyzed the secondary structure and structural organization of 130 kDa sACE and 65
and 90 kDa nACE. They determined the C-terminal amino acids sequence of nACE with 65 kDa as GYLVDQXRXGVFS and of nACE with 90 kDa as EVLGXPEYQXHPP. In this study they demonstrated that sACE is cleaved at Pro628 -Pro629 ® Lys630 and Phe481 -Ser482 ® Gly483 generating the isoforms with 90 and 65 kDa, respectively. The cause of this difference in the occurrence of these isoforms between non-hypertensive (with 130 and 65 kDa) and SHR (90 and 65 kDa) is not clear, although it might be linked to the processing of the enzymes upon shedding from the cell membrane. It has been suggested that either differences in glycosylation and/or dimerization may contribute to the complex nature of the activity and stability of these isoforms. Two different fluorogenic substrates were constructed for detection of ACE sheddases in IMC based on the cleavage site of sACE for the enzymes responsible for the generation of 65 and 90 kDa ACE isoforms. Despite we immunodetected only a soluble ACE form with 65 kDa, we detected enzymatic activity for both fluorogenic substrates (S1 and S2) in cell lysate and culture media, indicating, although these cells express only the nACE with 65 kDa, they also express the enzyme responsible for the formation of 90 kDa nACE form. The presence of 90 kDa nACE sheddase in these cells suggests that this enzyme can be regulated in a different manner in normotensive or hypertensive humans and animals, remaining inactive in subjects genetically not programmed to hypertension. Further studies are necessary to better understand the mechanism of this process. Besides the enzymatic activity detected in IMC lysates and culture media using the specific substrates for ACE sheddases, the cells in culture were treated with PMA and TAPI-1, a shedding activator and inhibitor, respectively. PMA treatment increased the enzymatic activity upon S1 and S2 fluorogenic substrates in the cell lysates and a higher nACE activity was detected in the culture media according to the ratio of ZPhe-HL/HHL, confirming the presence of ACE sheddases in these cells. The ratio ZPhe-HL/HHL can distinguish the activities of the N and C domains. ZPhe-HL is hydrolyzed at an equal rate by both domains [37], whereas HHL is hydrolyzed at a faster rate by the C domain active site [5]. If the ZPhe-HL/HHL ratio is higher than 1, this result indicates higher N-domain activity compared to the C-domain activity. Therefore, a higher ratio of ZPhe-HL/HHL indicates an augmented release of nACE isoforms to the culture media. Previous studies showed that TAPI treatment inhibits ACE shedding. An addition of 50 M of TAPI resulted in an inhibition of the soluble form of ACE in 10% of control levels [38]. Also, the ectodomain shedding processes of wild type somatic and germinal ACE were inhibited by 10–20% of control levels [18]. Surprisingly TAPI treatment did not affect either sheddase activity or the ratio
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of ZPhe-HL/HHL in both cells lysates and culture media when compared to control group, indicating that sheddases found in these cells are not metalloproteases. ACE sheddase was initially described as a membrane-associated metalloprotease [16,19,39–43], but recent studies also suggest that serine proteases may be involved in the shedding of ACE process regarding to the germinal form of the enzyme [44–46]. Takeuchi et al. (2009) [44] synthesized a fluorogenic substrate based on the cleavage site of the germinal ACE. This substrate was used to characterize the protease involved in the ACE shedding process being able to generate the soluble isoform present in the seminal fluid. Sheddase activity was strongly inhibited by AEBSF and antipain, and not by EDTA, leupeptin and E-64, suggesting that this enzyme is a serine protease. These data support our findings that serine proteases SH1 and STH2 in addition to the enzyme present in pool 2 similar to matriptase-3 are capable of cleaving somatic isoform and generate the N-domain ACE isoforms. In the partial purification of ACE sheddases, the chromatographic pool 2 with higher activity for the substrates studied was submitted to SDS-PAGE. The bands were subjected to Nterminal sequencing and subsequently analyzed by BLAST database of NCBI nucleotide sequence (http://www.ncbi.nml.gov/BLAST). The sequences showed homology with the enzyme matriptase-3 and serine proteases 40 and 45. The matriptase-3 is a TTPS family member anchored transmembrane serine protease capable of cleaving macromolecular substrates such as denatured collagen (gelatin), casein and bovine serum albumin through hydrolysis of peptide bounds containing Arg or Lys at the P1 position [47]. Work-related to the homologous protein band present in pool 2 showed that other serine proteases, representatives of the subfamily of TTPS, have higher catalytic efficiency for peptic substrates containing Arg or Lys (Pro-Pro-Arg or Lys) in position P1 [48]. Based on that, the purified enzyme is possibly a serine protease like matriptase-3 and the cleavage of S2 fluorogenic substrate possibly occurs at Lys-Pro bound. In order to evaluate if one of these identified proteins could be an ACE sheddase, pools 1 and 2 were incubated with sACE and the following generation of 65 and 90 kDa ACE forms indicates the presence of an ACE sheddase.Based on the identification and specificity of serine proteases that could be able to cleave sACE and generate 65 and 90 kDa soluble ACEs, we selected two enzymes produced in the kidney, named SH1 and STH2 purified from human urine by our group which have similar characteristics to matriptase-3. These enzymes were isolated from human urine and characterized as serine proteases as described by Casarini et al., 1992 and 1993 [34,49] and Quinto et al. in 1999 and 2004 [35,36]. SH1 preferentially hydrolyzes bradykinin between amino acids Phe5 -Ser6 , while the STH2 cleaves the same peptide bounds between amino acids Phe5 -Ser6 , Phe8 -Arg9 and Pro3 -Gly4 [34,36,49]. The enzyme is able to hydrolyze synthetic substrates containing proline in their sequence, for example, Abz-RPPGFQ-EDDnp and Abz-RPPAQEDDnp [35]. SH1 and STH2 were capable to hydrolyze S1 and S2 substrates constructed for sheddases identification, indicating that both enzymes could also hydrolyze sACE. In fact, the incubation of SH1 and STH2 with sACE showed the generation of isoforms with 65 and 90 kDa, suggesting that these enzymes are strong candidates for ACE shedding. In the present study, we described for the first time the presence of ACE sheddases in IMC. The identified sheddases are serine proteases able to hydrolyze sACE in vitro. The mechanisms and functions of the ACE shedding remains unclear, considering that 65 and 90 kDa ACEs were produced by cleavage in the middle of sACE anchored in the membrane. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation
of nACEs especially the 90 kDa form possibly related to genetically programmed hypertension. Acknowledgments We acknowledge the financial support from the Brazilian funding agency ‘Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo’ (FAPESP) (2008/55771-3 and 2010/51904-9). References [1] R.L. Soffer, Ann. Rev. Biochem. 45 (1976) 73–94. [2] M.R. Ehlers, Y.N. Chen, J.F. Riordan, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 1009–1013. [3] N.M. Hooper, Int. J. Biochem. 23 (1991) 641–647. [4] F. Soubrier, F. Alhenc-Gelas, C. Hubert, J. Allegrini, M. John, G. Tregear, P. Corvol, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 9386–9390. [5] L. Wei, F. Alhenc-Gelas, P. Corvol, E. Clauser, J. Biol. Chem. 266 (1991) 9002–9008. [6] T.A. Williams, S. Danilov, F. Alhenc-Gelas, F. Soubrier, Biochem. Pharmacol. 51 (1996) 11–14. [7] A.L. Lattion, F. Soubrier, J. Allegrini, C. Hubert, P. Corvol, F. Alhenc-Gelas, FEBS Lett. 252 (1989) 99–104. [8] M.R. Ehlers, E.A. Fox, D.J. Strydom, J.F. Riordan, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 7741–7745. [9] E.G. Erdos, R.A. Skidgel, Lab. Invest.; J. Techn. Methods Pathol. 56 (1987) 345–348. [10] P.A. Deddish, J. Wang, B. Michel, P.W. Morris, N.O. Davidson, R.A. Skidgel, E.G. Erdos, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 7807–7811. [11] D.E. Casarini, F.L. Plavinik, M.T. Zanella, O. Marson, J.E. Krieger, I.Y. Hirata, R.C. Stella, Int. J. Biochem. Cell Biol. 33 (2001) 75–85. [12] L.C. Maluf-Meiken, F.B. Fernandes, D.S. Aragao, F.A. Ronchi, M.C. Andrade, M.C. Franco, A.C. Febba, F.L. Plavnik, J.E. Krieger, J.G. Mill, R.C. Sesso, D.E. Casarini, Int. J. Hypertension 2012 (2012) 581780. [13] G.D. Marques, B.M. Quinto, F.L. Plavinik, J.E. Krieger, O. Marson, D.E. Casarini, Hypertension 42 (2003) 693–701. [14] M.C. Andrade, B.M. Quinto, A.K. Carmona, O.S. Ribas, M.A. Boim, N. Schor, D.E. Casarini, J. Hypertension 16 (1998) 2063–2074. [15] M.C. Camargo de Andrade, G.S. Di Marco, V. de Paulo Castro Teixeira, R.A. Mortara, R.A. Sabatini, J.B. Pesquero, M.A. Boim, A.K. Carmona, N. Schor, D.E. Casarini, Am. J. Physiol. Renal Physiol. 290 (2006) F364–F375. [16] N.M. Hooper, E.H. Karran, A.J. Turner, Biochem. J. 321 (2) (1997) 265–279. [17] S.L. Schwager, A.J. Chubb, R.R. Scholle, W.F. Brandt, C. Eckerskorn, E.D. Sturrock, M.R. Ehlers, Biochemistry 37 (1998) 15449–15456. [18] S.L. Schwager, A.J. Chubb, R.R. Scholle, W.F. Brandt, R. Mentele, J.F. Riordan, E.D. Sturrock, M.R. Ehlers, Biochemistry 38 (1999) 10388–10397. [19] Z.L. Woodman, S.L. Schwager, P. Redelinghuys, A.K. Carmona, M.R. Ehlers, E.D. Sturrock, Biochem. J. 389 (2005) 739–744. [20] H.E. Abboud, Exp. Cell Res. 318 (2012) 979–985. [21] D.S. Aragao, T.S. Cunha, D.Y. Arita, M.C. Andrade, A.B. Fernandes, I.K. Watanabe, R.A. Mortara, D.E. Casarini, Int. J. Biol. Macromol. 49 (2011) 79–84. [22] F. Ebihara, G.S. Di Marco, M.A. Juliano, D.E. Casarini, J. Renin-angiotensinaldosterone Syst. JRAAS 4 (2003) 228–233. [23] A.Q. Andrade, D.E. Casarini, N. Schor, M.A. Boim, Brazil. J. Med. Biol. Res.=Rev. Brasil. Pesquisas Med. Biol./Soc. Brasil. Biofis. 35 (2002) 17–24. [24] D.E. Casarini, M.A. Boim, R.C. Stella, M.H. Krieger-Azzolini, J.E. Krieger, N. Schor, Am. J. Physiol. 272 (1997) F405–F409. [25] D.B. Vidotti, D.E. Casarini, P.C. Cristovam, C.A. Leite, N. Schor, M.A. Boim, Am. J. Physiol. Renal Physiol. 286 (2004) F1039–F1045. [26] D.Y. Arita, G.S. Di Marco, N. Schor, D.E. Casarini, J. Cell. Biochem. 87 (2002) 58–64. [27] G.S. Di Marco, C.P. Vio, O.F. Dos Santos, N. Schor, D.E. Casarini, Cell. Physiol. Biochem.: Int. J. Exp. Cell. Physiol., Biochem., Pharmacol. 20 (2007) 919–924. [28] P. Mene, M.S. Simonson, M.J. Dunn, Physiol. Rev. 69 (1989) 1347–1424. [29] M.C. de Andrade, R. Affonso, F.B. Fernandes, A.C. Febba, I.D. da Silva, R.C. Stella, O. Marson, G.N. Jubilut, I.Y. Hirata, A.K. Carmona, H. Corradi, K.R. Acharya, E.D. Sturrock, D.E. Casarini, Int. J. Biol. Macromol. 47 (2010) 238–243. [30] G.D.K. Barlos, S. Rapolos, G. Papaphotiu, W. Schafer, W.Q. Yao, Tetrahedron Lett. 30 (1989) 3. [31] J. Friedland, E. Silverstein, Am. J. Clin. Pathol. 66 (1976) 416–424. [32] Y. Piquilloud, A. Reinharz, M. Roth, Biochim. Biophys. Acta 206 (1970) 136–142. [33] D.E. Casarini, K.B. Alves, M.S. Araujo, R.C. Stella, Brazil. J. Med. Biol. Res.=Rev. Brasil. Pesquisas Med. Biol./Soc. Brasil. Biofis. 25 (1992) 219–229. [34] D.E. Casarini, R.C. Stella, M.S. Araujo, C.A. Sampaio, Brazil. J. Med. Biol. Res.=Rev. Brasil. Pesquisas Med. Biol./Soc. Brasil. Biofis. 26 (1993) 15–29. [35] B.M. Quinto, L. Juliano, M. Juliano, A.K. Carmona, R.C. Stella, D.E. Casarini, Immunopharmacology 45 (1999) 223–228. [36] B.M. Quinto, M.A. Juliano, I. Hirata, A.K. Carmona, L. Juliano, D.E. Casarini, Int. J. Biochem. Cell Biol. 36 (2004) 1933–1944. [37] S. Danilov, E. Jaspard, T. Churakova, H. Towbin, F. Savoie, L. Wei, F. Alhenc-Gelas, J. Biol. Chem. 269 (1994) 26806–26814.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Serine proteases as candidates for proteolytic processing of angiotensin-I converting enzyme Danielle S. Aragão a , Maria Claudina C. de Andrade c , Fabiana Ebihara a , Ingrid K.M. Watanabe a , Dayane C.B.P. Magalhães a , Maria Aparecida Juliano b , Izaura Yoshico Hirata b , Dulce Elena Casarini a,∗ a
Department of Medicine, Nephrology Division, Federal University of São Paulo, Brazil Department of Biophysics, Federal University of São Paulo, Brazil c Albert Einstein Research Institute, Brazil b
a r t i c l e
i n f o
Article history: Received 23 April 2014 Received in revised form 4 September 2014 Accepted 5 September 2014 Available online 28 September 2014 Keywords: Angiotensin-I converting enzyme Shedding, Mesangial cells
a b s t r a c t Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. Ndomain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Angiotensin-I converting enzyme (ACE) (EC 3.4.15.1) is a broadly distributed zinc metalloendopeptidase which plays a role in blood pressure and electrolyte homeostasis [1]. Primarily responsible for the conversion of angiotensin I (Ang I) into angiotensin II (Ang II) and the inactivation of bradykinin, ACE is also involved in immunity, reproduction and neuropeptide metabolism [2,3]. Two ACE membrane-bound isoenzymes have been described in the literature. A somatic form, known as somatic ACE (sACE), is widely distributed and comprises two homologous domains (N and C domains) presenting 60% of sequence identity and each bearing a functional catalytic site [2,4–6]. A second form is only expressed in testis (tACE) and presents a unique domain identical
∗ Corresponding author at: Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Medicina, Disciplina de Nefrologia, Rua Botucatu 740, CEP 04023-900, São Paulo-SP, Brasil. Tel.: +55 11 59041684; fax: +55 11 59041683. E-mail addresses: [email protected], [email protected] (D.E. Casarini). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.017 0141-8130/© 2014 Elsevier B.V. All rights reserved.
to the C domain of sACE, except for 36 amino acid residues at its N-terminus [7,8]. The transmembrane protein ACE can be proteolytically shed from the cell surface by the action of a unidentified protease, resulting in soluble forms found in blood plasma, amniotic fluid, seminal plasma [3,9], ileal fluid [10], urine [11–13] and mesangial cells (MC) [14,15]. In the shedding process, the entire extracellular domain is released to the external environment, converting the membraneanchored protein into a soluble form [2,16]. Previous studies indicate that ACE sheddase a metalloprotease that is stimulated by phorbol esters (PMA) and inhibited by hydroxamates (TAPI-1) [17–19]. N-domain soluble ACE isoforms of 65 and 90 kDa present a different pattern of distribution between normotensive and hypertensive subjects. The 90-kDa isoform described as a genetic marker of hypertension was detected only in cells and urine from hypertensive humans and animals [11–15]. The soluble 65 kDa and 90 kDa ACE isoforms are found in cell lysates and culture media of MC, which exert multiple functions in the glomerular physiology and pathophysiology [20]. MC
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also express other components of renin-angiotensin system (RAS), including sACE [14,15], ACE2 [21], NEP [22] and renin [23–25]. Andrade et al. (1998) purified and characterized an active 65 kDa ACE from Wistar rats primary MC. These isoforms were able to cleave Ang I into Ang II and hydrolyze angiotensin 1–7 (Ang 1–7) [14]. In 2006, the 90 kDa isoform was isolated from cell lysate and culture media from primary MC from SHR. Besides the presence of these nACE in culture media, their location in the MC nuclei indicates a proteolytic processing of the sACE at the intracellular environment or a post-transcriptional modification of the enzyme [15]. Since cultured MC have been considered a valid model for the study of synthesis and release of bioactive products in the kidney, which could act as autocrine, intracrine and/or paracrine mediators [26–28], and based on the fact that MC presents somatic and soluble forms of ACE, this cell line can be introduced as a suitable in vitro model in order to improve knowledge on the components and functions related to the ACE shedding. The aim of this study was to evaluate the existence of ACE sheddases in mice immortalized MC (IMC) and then, to isolate and identify these enzymes.
2.4.2. ACE activity ACE catalytic activity was measured as described by Friedland and Silverstein [31] and Piquilloud [32]. Aliquots of cell lysate (50 L) and culture media (5 L) were incubated with 200 L of 100 mM Borate, 300 nM NaCl, 0.1 nM ZnSO4 buffer, pH 8.3, containing 1 mM of Z-Phe-His-Leu (ZPhe-HL) or 5 mM of Hippuryl-His-Leu (HHL) (Bachem, USA) for 2 h at 37 ◦ C. The reaction was stopped by the addition of 1.5 mL of 280 nM NaOH and incubated with 100 mL of 20 mg/mL o-phtaldialdehyde (Sigma, USA) diluted in methanol for 10 min followed by 200 L of 3N HCl. The liberated dipeptide -HL was fluorometrically measured using F-2000 Fluorescent Spectrophotometer (TECAN, USA) with an excitation of 360 nm and emission of 500 nm. The standard curve was obtained using different concentrations of -HL (His-Leu, Sigma-Aldrich, USA) in the blank reaction mixture and it showed a linear relation between relative fluorescence and -HL concentration.
2. Materials and methods
Besides the design of specific substrates for ACE sheddases, cells were treated with PMA and TAPI-1, a shedding activator and inhibitor, respectively, to confirm the presence of ACE sheddases in IMC. Prior to reach confluence, IMC were incubated with DMEN/F12 (3:1) with 2% FBS for 12 h. Subsequently, the cells were maintained for 3 h with DMEN/F12 (3:1) with FBS (control group), DMEN/F12 (3:1) with 2% FBS containing 10 M of Phorbol 12-myristate 13acetate (Sigma-Aldrich, USA) (PMA group) and DMEN/F12 (3:1) with 2% FBS containing 100 M of TAPI-1 (Calbiochem, USA) (TAPI1 group) (N = 3). After the treatment, the culture media and the cell lysates were collected as previously described. The sheddases and ACE enzymatic activities were determined using fluorogenic substrates S1 and S2 and ACE substrates ZPhe-HL and HHL. The catalytic activity of each ACE domain was determined using the same quantity of enzyme for each assay and the results expressed by ZPhe-HL/HHL ratio that suggest the presence of nACE.
This study was approved by the Ethics Committee of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil. 2.1. Immortalized mesangial cells culture (IMC) IMC from mice were purchased from The American Type of Culture Collection (ATCC: CRL-1927). Cells were grown in DMEM/F12 (3:1) (Gibco, USA) supplemented with 5% fetal bovine serum (FBS), 5000 U/L penicillin and 50 mg/L streptomycin. Before the experiments, IMC were incubated with DEMEN/F12 without FBS for 24 h. 2.2. Culture media and cell lysate from IMC preparation After the removal of the culture media, the culture flasks were rinsed twice with 5 mL of 0.025 M Tris-HCl buffer pH 8.0 and scraped with 1 mL of 0.05 M HEPES, 0.5 M NaCl buffer, pH 7.5 containing 1% of Triton X-114 at 4 ◦ C. The lysate was collected, sonicated at 4 ◦ C for 20 min and centrifuged at 12,000 rpm for 5 min. The supernatant was fractioned and stored at −20 ◦ C. 2.3. Synthesis of fluorogenic substrates for ACE sheddases based on the C-terminal sequence of 65 and 90 kDa N-terminal ACE from MC The C-terminal amino acid sequence of the 65 and 90 kDa Nterminal soluble ACE were determined as previously described [29]. Based on these C-terminal sequences, two fluorogenic substrates (S1: Abz-RWGVFSGRTPQ-EDDnp and S2: Abz-EYQWHPPLPDNYQEDDnp) for the sheddases responsible for the generation of 65 and 90 kDa isoforms, respectively, were constructed using Fmoc synthesis protocol and H-Pro-2-chlorotrityl resin [30] (Fig. 1). 2.4. Enzymatic activity assay 2.4.1. ACE sheddase activity Sheddases catalytic activities were measured fluorometrically using the substrates described above. Aliquots (50 L) of chromatography fractions, cell lysate or culture media were incubated in 1 mL of 0.05 M Tris-HCl, 0.15 M NaCl buffer, pH 8.0, containing 2 M of fluorogenic substrates at 37 ◦ C for 2 h. The fluorescence was measured using F-2000 Fluorescent Spectrophotometer (TECAN, USA) with an excitation of 320 nm and emission of 420 nm.
2.5. Effects of the treatment with PMA or TAPI-1 on ACE sheddases activity and the ratio ZPhe-HL/HHL ACE activities from the cell lysate and culture media of IMC
2.6. Partial purification of ACE sheddases from IMC IMC cell lysates were submitted to gel filtration chromatography on 120 mL AcA-34 resin column (Sigma-Aldrich, USA) coupled to a peristaltic pump (Amersham Pharmacia Biotech-GE, USA) equilibrated with 0.05 M Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl. The enzymes were eluted with the same buffer and 1 mL fractions were collected at a flow rate of 20 mL/h. The optical density of each fraction was quantified at 280 nm and the chromatographic fractions were pooled according to detection of enzymatic activity for S1 and S2 fluorogenic substrates. 2.7. Analysis of the somatic ACE cleavage by the proteins presents in the chromatographic pools 1 and 2 The chromatographic pools 1 and 2 presenting catalytic activity for the S1 and S2 fluorogenic substrates were dialyzed and concentrated to 0.1 mL against 0.05 M Tris-HCl buffer, pH 8.0 using Amicon Ultra with a 10-kDa exclusion membrane (Millipore, USA). Aliquots of pool 1 and pool 2 were incubated in the same buffer with 0.25 mL of somatic ACE (ACE elevated control-Sigma Diagnostics, USA) diluted as recommended by the manufacturers, for 24 h at 37 ◦ C. Aliquots were taken at times 0 h and 24 h and the reaction was stopped with the addition of 0.1% orthophosphoric acid (Merck, USA). The products of the somatic ACE hydrolysis by the enzymes in pools 1 and 2 were analyzed by Western blotting.
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Fig. 1. Sequence alignment of rat (UniProtKB/Swiss-Prot: P47820.1) and mouse (UniProtKB/Swiss-Prot: P09470.3) sACE. The C-terminal alignment of 65 kDa rat nACE ended at Ser482 . The same analysis for 90 kDa rat nACE showed that the enzyme sequence ended at Pro629 [29].
2.8. SDS-PAGE and Western blotting analysis Proteins were analyzed by sodium dodecil sulfatepolyacrylamide gel (7.5%) electrophoresis (SDS-PAGE) under reduced condition using -mercaptoethanol. Electrophoretic transfer was performed for 60 min under constant voltage (60 V), into a nitrocellulose membrane (Millipore, USA) using transfer buffer (0.015 M Tris, 0.190 M glycine, and 0.1% SDS). The membrane was incubated in 0.1 M TBS with 0.1% of tween containing 0.5% of bovine albumin (Sigma, USA) for 1 h before 2-h incubation with anti-ACE monoclonal antibodies 9B9 (1:2000), 5F1 (1:500) (Chemicon International, USA) or BAF 929 (1:500) (RD System, USA) specific to the N-domain portion of the molecule. Subsequent steps were performed using the Amershan ECL Select Western blotting detection reagent kit (GE Healthcare, EUA) or the streptavidin/phosphatase alkaline system (Amershan Pharmacia Biosciences, USA) using BCIP/NBT as substrate (Bio-Rad Laboratories, USA) as recommended by the manufacturers.
on a PPSQ-23 Automated Protein Sequencer (Shimadzu Corporation, Japan). The results were submitted to the BLAST platform (www.nbci.com/blast) in order to identify the proteins.
2.10. Analysis of serine proteases SH1 and STH2 previously purified from human urine as ACE sheddases Aliquots of the endopeptidases, serine protease SH1 and serine thiol protease STH2 previously purified and characterized from human urine by Casarini et al. in 1992 and 1993 and Quinto et al. in 1999 and 2004 [33–36] were incubated in 0.05 M Tris-HCl buffer, pH 8.0 at 37 ◦ C for 24 h with 100 g of somatic ACE from rabbit lung (Sigma-Aldrich, USA). Aliquots were taken at times 0 h and 24 h and the reaction was stopped with the addition of 0.1% orthophosphoric acid (Merck, USA). The products of the hydrolysis of the somatic ACE by SH1 and STH2 enzymes were analyzed by Western blotting.
2.9. Amino-terminal sequence of the proteins from pool 2 3. Results The chromatographic pool presenting higher catalytic activity for the S1 and S2 fluorogenic substrates was dialyzed against water using Amicon Ultra with a 10-kDa pore membrane (Millipore, USA) and subsequently dried. The sample (50 g) was ressuspended in Laemmli sample buffer (Bio-Rad, USA) and submitted to SDS-PAGE. Protein gel staining was performed using Silver Staining Protein kit (Amershan Biosciences, USA) according to the manufactures instructions. The same procedure was repeated and proteins were electroblotted to a PVDF protein sequencing membrane (Bio-Rad Laboratories, USA) using a 0.1 M CAPS buffer, pH 11.0. Membrane was stained with 2% Ponceau-S in 1% acetic acid targeting the protein localization. Protein bands were excised from the membrane and submitted to N-terminal amino acid sequencing
3.1. Enzymatic activity The ACE sheddase enzymatic activity was determined in culture media and cell lysate from IMC. The cell lysate showed specific activity of 205.7 (±24.2) and 62.4 (±3.2) nM/min/mg for the S1 and S2 fluorogenic substrates, respectively, whereas the culture media showed specific activity of 46.9 (±33.7) and 43.8 (±6.8) nM/min/mg for the S1 and S2, respectively. The cell lysate showed specific ACE activity of 0.30 (±0.07) and 0.21 (±0.10) nmol/min/mg for the ZPhe-HL and HHL, respectively, and the ratio ZPhe-HL/HHL was 1.2 (±0.14). The culture media showed specific activity of 2.67 (±0.31) and 0.43
Fig. 2. Specific activity nM/min/mg of A: 65 kDA nACE sheddase and B: 90 kDA nACE sheddase from IMC lysate and culture media. Results are mean ± SE. * p < 0.05 vs CT; # p < 0.05 vs TAPI-1.
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Pool 1
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50
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40 30 0.2
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Fig. 5. Gel filtration chromatography using AcA-34 column of IMC lysate. ACE ) Abzsheddases were eluted in two peaks with enzymatic activity using ( ) Abz-EYQWHPPLPDNYQ-EDDnp (S2) as RWGVFSGRTPQ-EDDnp (S1) and ( ) OD 280 nm. substrates (
Fig. 3. Ratio of Zphe-HL/HHL ACE substrates from IMC lysates and culture media. Results are mean ± SE. * p < 0.05 vs CT, # p < 0.05 vs PMA.
Fig. 6. Western blotting analysis of incubates of sACE with pool 1 and 2 from gel filtration using specific ACE antibody (5F1). Lane 1: pool 1; lane 2: pool 2; lane 3: pool 2 + ACE 0 h; lane 4: pool 2 + ACE 24 h; lane 5: pool 1 + ACE 0 h; lane 6: pool 1 + ACE 24 h.
The cell lysate with enzymatic activity was applied to an AcA-34 gel filtration column equilibrated with 0.05 M Tris-HCl, 0.15 M NaCl buffer, pH 8.0. ACE sheddases were eluted in two peaks denominated as pool 1 and 2. The pool 1 (fractions 41–59) presented specific activity of 241.9 and 122.8 nM/mg using S1 and S2 as substrates, respectively. The pool 2 (fractions 72–99) specific activity was 1618.2 and 522.7 nM/mg for S1 and S2 substrates, respectively (Fig. 5, Table 1).
Fig. 4. Western blotting of IMC lysate using 9B9 antibody. Lane 1: Molecular weight marker; Lanes 2 and 3: IMC lysate.
(±0.09) nmol/min/mg for the ZPhe-HL and HHL, respectively, and the ratio ZPhe-HL/HHL was 5.82 (±0.60). The treatment with PMA activated the sheddase responsible for the generation of the 65 and 90 kDa isoforms in IMC cell lysate when compared to the control group, whereas TAPI-1 did not affect the enzymatic activity. In the culture media, the enzymatic activity able to hydrolyze S1 substrate was higher in PMA treatment than in TAPI-1 (Fig. 2). The ACE activity ratio ZPhe-HL/HHL was higher in the culture media of group treated with PMA when compared to control and TAPI-1 groups suggesting the presence of nACE (Fig. 3).
3.3. Cleavage of somatic ACE by enzymes from pools 1 and 2 After 24 h of incubation of sACE with the enzyme from pools 1 and 2, we immunodetected ACE isoforms with molecular weight of 65 and 90 kDa (Fig. 6). 3.4. N-terminal sequence of protein from purified pool 2
3.2. Partial purification of ACE sheddases from IMC Based on the results obtained after incubation of pool 1 and pool 2 with sACE, we decided to analyze the N-terminal sequence of the enzyme purified from pool 2. The protein was identified as serine protease as described in Table 2.
Prior to the purification performance, ACE protein expression was assessed by Western blotting. The IMC lysate expresses sACE, presenting 130 kDa and nACE form with 65 kDa (Fig. 4). Table 1 Partial purification of ACE sheddases from IMC using S1 and S2 fluorogenic substrates. Step
Volume (mL)
Protein (mg/mL)
Total protein (mg)
Enzymatic activity (nM/min/mL)
Specific activity (nM/mg)
Purification (X)
Recovery (%)
S1 Pool 1 Pool 2
1.2 7.5 15.0
2.3 0.036 0.033
2.8 0.27 0.50
357.45 8.7 53.4
154.1 241.9 1618.2
1 1.6 10.5
100 9.7 17.8
S2 Pool 1 Pool 2
1.2 7.5 15.0
2.3 0.036 0.033
2.8 0.27 0.50
80.04 4.4 17.3
34.5 122.8 522.7
1 3.6 15.2
100 9.7 17.8
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Fig. 7. Western blotting analysis of incubates of the serine proteases SH1 and STH2 with sACE using specific ACE antibodies (5F1 or BAF 929). Lane 1: molecular weight marker; lane 2: SH1 + ACE 0 h; lane 3: SH1 + ACE 24 h; lane 4: STH2 + ACE 0 h; lane 5: STH2 + ACE 24 h; lane 6: ACE 0 h; lane 7: ACE 24 h. The arrows indicate proteins with 65, 75 and 90 kDa on lane 2 and with 65 and 90 kDa on lane 4.
Table 2 Amino-terminal sequence of the protein bands found in partial purification of ACE sheddases. Protein
Homologues sequences
Description and species
Band 1 DRXEDTWPW
(601–609) DSQEGTWPW
Transmenbrane protease serine 7 (Matriptase-3)-Rattus novergicus Transmenbrane protease serine 7 (Matriptase-3)-Mus musculus
(597–605) DSQEGTWPW
Band 2 TDYRED
(231–236) TNYRED
Serine protease 45-Bos taurus
Band 3 HPXTPXNE
(326–333) HPGSPENE
Serine protease 40-Mus musculus
3.5. Hydrolysis of S1 and S2 substrates by SH1 e STH2 from human urine Based on results obtained from N-terminal sequence that identified the protein from pool 2 as serine protease, we decided to study the action of SH1 and STH2 enzymes previously purified from human urine upon S1 and S2 fluorogenic substrates. The specific activity of SH1 was 7.34 and 4.22 nM/min/mg for the S1 and S2, respectively, and for STH2 was 34.35 and 11.02 nM/min/mg for S1 and S2, respectively. 3.6. Analysis of the sACE cleavage by the serine proteases SH1 and STH2 by Western Blotting Purified SH1 and STH2 were able to hydrolyze sACE in vitro. After 24 h of incubation of SH1 and STH2 with sACE, separately, we detected the presence of protein bands with 65, 75 and 90 kDa in the incubation of SH1+ sACE and 65 and 90 kDa in the incubation of STH2+ sACE, confirming the cleavage of sACE (Fig. 7). 4. Discussion Active soluble nACE isoforms with 65 and 90 kDa were purified from cell lysates and culture media from Wistar or spontaneously hypertensive rat (SHR) primary MC, being the 90 kDa isoform only expressed in SHR cells [14,15]. Andrade and Affonso et al. (2010) [29] analyzed the secondary structure and structural organization of 130 kDa sACE and 65
and 90 kDa nACE. They determined the C-terminal amino acids sequence of nACE with 65 kDa as GYLVDQXRXGVFS and of nACE with 90 kDa as EVLGXPEYQXHPP. In this study they demonstrated that sACE is cleaved at Pro628 -Pro629 ® Lys630 and Phe481 -Ser482 ® Gly483 generating the isoforms with 90 and 65 kDa, respectively. The cause of this difference in the occurrence of these isoforms between non-hypertensive (with 130 and 65 kDa) and SHR (90 and 65 kDa) is not clear, although it might be linked to the processing of the enzymes upon shedding from the cell membrane. It has been suggested that either differences in glycosylation and/or dimerization may contribute to the complex nature of the activity and stability of these isoforms. Two different fluorogenic substrates were constructed for detection of ACE sheddases in IMC based on the cleavage site of sACE for the enzymes responsible for the generation of 65 and 90 kDa ACE isoforms. Despite we immunodetected only a soluble ACE form with 65 kDa, we detected enzymatic activity for both fluorogenic substrates (S1 and S2) in cell lysate and culture media, indicating, although these cells express only the nACE with 65 kDa, they also express the enzyme responsible for the formation of 90 kDa nACE form. The presence of 90 kDa nACE sheddase in these cells suggests that this enzyme can be regulated in a different manner in normotensive or hypertensive humans and animals, remaining inactive in subjects genetically not programmed to hypertension. Further studies are necessary to better understand the mechanism of this process. Besides the enzymatic activity detected in IMC lysates and culture media using the specific substrates for ACE sheddases, the cells in culture were treated with PMA and TAPI-1, a shedding activator and inhibitor, respectively. PMA treatment increased the enzymatic activity upon S1 and S2 fluorogenic substrates in the cell lysates and a higher nACE activity was detected in the culture media according to the ratio of ZPhe-HL/HHL, confirming the presence of ACE sheddases in these cells. The ratio ZPhe-HL/HHL can distinguish the activities of the N and C domains. ZPhe-HL is hydrolyzed at an equal rate by both domains [37], whereas HHL is hydrolyzed at a faster rate by the C domain active site [5]. If the ZPhe-HL/HHL ratio is higher than 1, this result indicates higher N-domain activity compared to the C-domain activity. Therefore, a higher ratio of ZPhe-HL/HHL indicates an augmented release of nACE isoforms to the culture media. Previous studies showed that TAPI treatment inhibits ACE shedding. An addition of 50 M of TAPI resulted in an inhibition of the soluble form of ACE in 10% of control levels [38]. Also, the ectodomain shedding processes of wild type somatic and germinal ACE were inhibited by 10–20% of control levels [18]. Surprisingly TAPI treatment did not affect either sheddase activity or the ratio
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of ZPhe-HL/HHL in both cells lysates and culture media when compared to control group, indicating that sheddases found in these cells are not metalloproteases. ACE sheddase was initially described as a membrane-associated metalloprotease [16,19,39–43], but recent studies also suggest that serine proteases may be involved in the shedding of ACE process regarding to the germinal form of the enzyme [44–46]. Takeuchi et al. (2009) [44] synthesized a fluorogenic substrate based on the cleavage site of the germinal ACE. This substrate was used to characterize the protease involved in the ACE shedding process being able to generate the soluble isoform present in the seminal fluid. Sheddase activity was strongly inhibited by AEBSF and antipain, and not by EDTA, leupeptin and E-64, suggesting that this enzyme is a serine protease. These data support our findings that serine proteases SH1 and STH2 in addition to the enzyme present in pool 2 similar to matriptase-3 are capable of cleaving somatic isoform and generate the N-domain ACE isoforms. In the partial purification of ACE sheddases, the chromatographic pool 2 with higher activity for the substrates studied was submitted to SDS-PAGE. The bands were subjected to Nterminal sequencing and subsequently analyzed by BLAST database of NCBI nucleotide sequence (http://www.ncbi.nml.gov/BLAST). The sequences showed homology with the enzyme matriptase-3 and serine proteases 40 and 45. The matriptase-3 is a TTPS family member anchored transmembrane serine protease capable of cleaving macromolecular substrates such as denatured collagen (gelatin), casein and bovine serum albumin through hydrolysis of peptide bounds containing Arg or Lys at the P1 position [47]. Work-related to the homologous protein band present in pool 2 showed that other serine proteases, representatives of the subfamily of TTPS, have higher catalytic efficiency for peptic substrates containing Arg or Lys (Pro-Pro-Arg or Lys) in position P1 [48]. Based on that, the purified enzyme is possibly a serine protease like matriptase-3 and the cleavage of S2 fluorogenic substrate possibly occurs at Lys-Pro bound. In order to evaluate if one of these identified proteins could be an ACE sheddase, pools 1 and 2 were incubated with sACE and the following generation of 65 and 90 kDa ACE forms indicates the presence of an ACE sheddase.Based on the identification and specificity of serine proteases that could be able to cleave sACE and generate 65 and 90 kDa soluble ACEs, we selected two enzymes produced in the kidney, named SH1 and STH2 purified from human urine by our group which have similar characteristics to matriptase-3. These enzymes were isolated from human urine and characterized as serine proteases as described by Casarini et al., 1992 and 1993 [34,49] and Quinto et al. in 1999 and 2004 [35,36]. SH1 preferentially hydrolyzes bradykinin between amino acids Phe5 -Ser6 , while the STH2 cleaves the same peptide bounds between amino acids Phe5 -Ser6 , Phe8 -Arg9 and Pro3 -Gly4 [34,36,49]. The enzyme is able to hydrolyze synthetic substrates containing proline in their sequence, for example, Abz-RPPGFQ-EDDnp and Abz-RPPAQEDDnp [35]. SH1 and STH2 were capable to hydrolyze S1 and S2 substrates constructed for sheddases identification, indicating that both enzymes could also hydrolyze sACE. In fact, the incubation of SH1 and STH2 with sACE showed the generation of isoforms with 65 and 90 kDa, suggesting that these enzymes are strong candidates for ACE shedding. In the present study, we described for the first time the presence of ACE sheddases in IMC. The identified sheddases are serine proteases able to hydrolyze sACE in vitro. The mechanisms and functions of the ACE shedding remains unclear, considering that 65 and 90 kDa ACEs were produced by cleavage in the middle of sACE anchored in the membrane. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation
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