B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

Takafumi Minato; Satoru Nirasawa; Teruki Sato; Tomokazu Yamaguchi; Midori Hoshizaki; Tadakatsu Inagaki; Kazuhiko Nakahara; Tadashi Yoshihashi; Ryo Ozawa; Saki Yokota; Miyuki Natsui; Souichi Koyota; Taku Yoshiya; Kumiko Yoshizawa-Kumagaye; Satoru Motoyama; Takeshi Gotoh; Yoshikazu Nakaoka; Josef M. Penninger; Hiroyuki Watanabe; Yumiko Imai; Saori Takahashi; Keiji Kuba

doi:10.1038/s41467-020-14867-z

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

Minato, Takafumi; Nirasawa, Satoru; Sato, Teruki; Yamaguchi, Tomokazu; Hoshizaki, Midori; Inagaki, Tadakatsu; Nakahara, Kazuhiko; Yoshihashi, Tadashi; Ozawa, Ryo; Yokota, Saki; Natsui, Miyuki; Koyota, Souichi; Yoshiya, Taku; Yoshizawa-Kumagaye, Kumiko; Motoyama, Satoru; Gotoh, Takeshi; Nakaoka, Yoshikazu; Penninger, Josef M.; Watanabe, Hiroyuki; Imai, Yumiko; Takahashi, Saori; Kuba, Keiji 2020-02-26 00:00:00 ARTICLE https://doi.org/10.1038/s41467-020-14867-z OPEN B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction 1,13 2,13 1,3,13 1 4 Takafumi Minato , Satoru Nirasawa , Teruki Sato , Tomokazu Yamaguchi , Midori Hoshizaki , 5 2 2 1 6 1 Tadakatsu Inagaki , Kazuhiko Nakahara , Tadashi Yoshihashi , Ryo Ozawa , Saki Yokota , Miyuki Natsui , 7 8 8 9 6 Souichi Koyota , Taku Yoshiya , Kumiko Yoshizawa-Kumagaye , Satoru Motoyama , Takeshi Gotoh , 5 10,11 3 4 12 Yoshikazu Nakaoka , Josef M. Penninger , Hiroyuki Watanabe , Yumiko Imai , Saori Takahashi & 1,13 Keiji Kuba Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1–7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angio- tensin II-induced hypertension, cardiac hypertrophy, and ﬁbrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial ﬁbrosis, and car- diac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carbox- ypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure. 1 2 Department of Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. 3 4 Department of Cardiovascular Medicine, Akita University Graduate School of Medicine, Akita, Japan. Laboratory of Regulation of Intractable Infectious Diseases, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Department of Vascular Physiology, Research Institute National Cerebral and Cardiovascular Center, 6-1 Kishibe Shinmachi, Suita, Osaka 564-8565, Japan. Department of Materials Science, Applied Chemistry Course, Graduate School of Engineering Science, Akita University, 1-1 Tegatagakuen-machi, Akita 010-8502, Japan. Molecular Medicine Laboratory, Bioscience Education and Research Support Center, Akita University, 1-1-1 Hondo, Akita 010-8543, Japan. Peptide Institute, Inc., 7-2-9 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Department of Surgery, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. 10 11 IMBA -Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Campus Vienna BioCenter, Vienna 1030, Austria. Department of Medical Genetics, Life Science Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan. These authors contributed equally: Takafumi Minato, Satoru Nirasawa, Teruki Sato, Keiji Kuba. email: stnirasa@affrc.go.jp; kuba@med.akita-u.ac.jp NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 1 1234567890():,; ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z he renin–angiotensin system (RAS) has an essential role in searched published crystal structures of M32 carboxypeptidase in maintaining blood pressure (BP) homeostasis, as well as the database of MEROPS (http://merops.sanger.ac.uk/) and found 1–3 Tﬂuid and salt balance . When the RAS is activated, that three microbial M32 carboxypeptidases are reported for their angiotensin-converting enzyme (ACE) cleaves the C-terminus of three-dimensional (3D) structures, including BS-CAP (or BsuCP) deca-peptide angiotensin I (Ang I or Ang 1–10) to generate a (B. subtilis), Taq (Thermus aquaticus), and Pfu (P. furiosus). vasopressor octa-peptide angiotensin II (Ang II or Ang 1–8). Although BS-CAP, Taq, or Pfu showed low sequence identity to 4,5 ACE2 was discovered as a human homolog of ACE in 2000 . ACE2, we examined whether BS-CAP, Taq, or Pfu has structural ACE2 is a negative regulator of the RAS, which catalyzes the similarity to human ACE2 by using the program of Molecular conversion of Ang II to angiotensin 1–7 (Ang 1–7) and down- Operating Environment (Chemical Computing Group, Canada). 4–6 regulates Ang II levels, thereby counterbalancing ACE activity . The program detected only BS-CAP as a protein structurally Physiological function of ACE2 was initially identiﬁed as a related to ACE2 and the 3D structures of both proteins are mostly regulator of heart function and BP, and ACER, a ﬂy homolog of merged (Fig. 1a). Importantly, the position of key amino acids ACE2, was shown to be essential for heart morphogenesis and constituting the catalytic site (His-Glu-X-X-His motif) and 7,8 cardiac functions in ﬂies . Although activation of the RAS and substrate-binding region (Arg273/348, His345/234, His505/408, generation of Ang II worsen cardiovascular pathologies, such as and Tyr515/420 in ACE2/BS-CAP) were almost identical between cardiac ﬁbrosis and pathological hypertrophy in heart failure, the both proteins, implicating that BS-CAP may have similar sub- enzymatic activity of ACE2 exhibits a protective role in cardio- strate preference to ACE2 (Fig. 1a, Supplementary Fig. 1, and 9,10 vascular diseases . ACE2 also has protective roles to improve Supplementary Table 1). Indeed, a previous study on BS-CAP the pathologies in acute respiratory distress syndrome (ARDS)/ structure had predicted that there may be structural similarity acute lung injury and diabetic nephropathy, in which Ang II is between ACE2 and BS-CAP . We further searched for bacterial 11–13 overproduced or its signaling enhanced . Loss of ACE2 can be proteins that exhibit high sequence identity to BS-CAP in the detrimental, as it leads to progression of cardiac, renal, and pul- BLAST and found that BA-CAP, a carboxypeptidase derived from 11,14,15 monary pathologies . Treatment with recombinant human Bacillus amyloliquefaciens, is homologous to BS-CAP (Fig. 1b; ACE2 protein (rhACE2), which is devoid of its membrane- Supplementary Fig. 1; Supplementary Table 1). Furthermore, we anchored domain thus soluble, has been demonstrated to exhibit found that our recently identiﬁed bacterial strain, Paenibacillus beneﬁcial effects in various animal models including heart failure, sp. B38, also has a similar M32 carboxypeptidase to BS-CAP with 11,13,16 acute lung injury, and diabetic nephropathy, and so forth . high sequence identity (Fig. 1b, Supplementary Fig. 1, and Sup- rhACE2 is currently tested in the clinic to treat ARDS patients . plementary Table 1). We termed this Paenibacillus sp. B38- Despite its beneﬁcial effects, rhACE2 is a glycosylated protein derived M32 carboxypeptidase as B38-CAP hereafter. Despite and thus its preparation requires time- and cost-consuming pro- evolutionally distant relationship to ACE2 (Fig. 1b), these bac- tein expression system with mammalian or insect cells, which terial enzymes are likely homologs of ACE2 with divergent may not be advantageous in drug development and medical evolution. 6,18–20 economy . We prepared recombinant proteins of BS-CAP, BA-CAP, and Both ACE2 and ACE proteins belong to the M2 family of zinc- B38-CAP in the Escherichia coli protein expression system (Fig. 1c) binding metallopeptidases containing the HEXXH metal- and all of the proteins were highly expressed and soluble in E. coli coordinating motif, although the biological activities of these and easily puriﬁed with anion-exchange and gel ﬁltration two enzymes are different; ACE2 functions as a mono-carbox- chromatography (Supplementary Fig. 2a). Indeed, the production 2,4,5 ypeptidase, whereas ACE is a dipeptidyl-carboxypeptidase . of recombinant B38-CAP in E. coli (16.8 mg protein yield per Structural analyses had revealed signiﬁcant homology between culture volume (L)) was more efﬁcient in terms of the recovered ACE and a carboxypeptidase from the hyperthermophilic protein amount compared with the production of His-tagged archaeon Pfu (Pyrococcus furiosus), which is a member of the rhACE2 in baculovirus-Sf9 insect cells (5.42 mg protein yield per M32 family of carboxypeptidases belonging to the family of culture volume (L)). Moreover, the time for culture and metallopeptidases with the HEXXH active-site motif . The two puriﬁcation of B38-CAP (2 days) was shorter than that of rhACE2 enzymes share little amino acid sequence identity, yet they exhibit (6 days, not including baculovirus preparation) (Supplementary similarities in core structure and the active-site regions .In Fig. 2a–d). We ﬁrst tested whether these enzymes have ACE2-like addition, a structural similarity within the active-site region proteolytic activity to hydrolyze the ﬂuorogenic peptide Nma-His- between ACE2 and the M32 carboxypeptidase from the bacter- Pro-Lys(Dnp), which we had previously developed as a speciﬁc 21 19 ium Bacillus subtilis has been reported , suggesting that the ACE2 substrate . As a result, all the enzymes were revealed to functions might be conserved. We had previously cloned a D- catalyze the hydrolysis of the ACE2 substrate Nma-His-Pro-Lys aspartyl endopeptidase (paenidase I) from Paenibacillus sp. B38, a (Dnp) (Fig. 1d and Table 1). When we incubated Ang II peptide 22,23 new substrain of B. subtilis . Paenidase I cleaves D-α-Asp- with BS-CAP, BA-CAP, or B38-CAP in vitro, all of the enzymes containing amyloid-β peptide, which is detected in Alzheimer’s converted Ang II to Ang 1–7 (Fig. 1e and Supplementary Fig. 3a). disease, suggesting a potential application as a therapeutic . On the other hand, the dependency of ACE2-like enzymatic In this study, we show that B38-CAP, a Paenibacillus sp. B38- activity on anion (Cl ) concentration is much higher in B38-CAP derived carboxypeptidase, is an ACE2-like enzyme, which cleaves than in BS-CAP and BA-CAP (Fig. 1d), suggesting that B38-CAP both Ang I and Ang II to Ang 1–7. We show that recombinant is the most potent in ACE2-like activity under physiological B38-CAP protein downregulates Ang II levels in mice and conditions of mammals. Analysis for kinetic constants with the antagonizes Ang II-induced hypertension, pathological cardiac ﬂuorogenic ACE2 substrate revealed that B38-CAP has the same hypertrophy, and myocardial ﬁbrosis. We also show beneﬁcial potency as rhACE2 protein (Table 1 and Supplementary Fig. 3b). effects of B38-CAP on the pathology of pressure overload- Consistently, the IC values of ACE2 inhibitor (MLN-4760) for induced heart failure in mice without overt toxicities. B38-CAP and human ACE2 were almost equivalent (Table 1 and Supplementary Fig. 3c). In addition, the dependence of B38-CAP proteolytic activity on pH and temperature was also similar to that 6,19 Results of ACE2 (Supplementary Fig. 3d, e). Moreover, when various Identiﬁcation of B38-CAP as an ACE2-like enzyme. To address ACE2-substrate biological peptides were treated with B38-CAP, all whether there are any ACE2-like proteins in bacteria, we ﬁrst the peptides tested were C-terminally cleaved by B38-CAP (Table 2 2 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE a b 0.252 B38-CAP His Glu 0.195 0.090 BA-CAP 0.150 His 0.092 BS-CAP Zn Tyr 0.092 Human ACE2 0.221 His 0.047 Glu Rat ACE2 Arg 0.126 0.037 0.049 Mouse ACE2 0.05 0.328 His Fly ACER ACE2 f i 0 min BS-CAP Ang 1–9 40 min 90 min 600 100 c e Ang II + vehicle (kDa) 0246 4 68 10 Retention time (min) 0 Ang 1–7 Ang I Ang 1–9 Ang II Ang 1–7 Ang II + ACE2 h 0 102030405060708090 Ang I Ang II + B38-CAP B38-CAP Leu His Phe 600 600 BS-CAP 4 200 20 BA-CAP 0 0246 0 0.5 1 1.5 2 Retention time (min) Retention time (min) NaCl (M) 0 102030405060708090 Vehicle; B38-CAP Minutes after reaction initiation Fig. 1 B38-CAP, a bacteria-derived carboxypeptidase, is Angiotensin-converting enzyme 2 (ACE2)-like enzyme. a Crystal structures of BS-CAP and human ACE2 proteins. Inset: metal-coordinating residues (red) and substrate-binding residues (black) are shown. b Phylogenetic tree of ACE2 and bacterial ACE2-like carboxypeptidases. c SDS-PAGE analysis of recombinant proteins of BS-CAP, BA-CAP, and B38-CAP. d Dependence of ACE2-like proteolytic activity of BS-CAP, BA-CAP, and B38-CAP on anion concentration. ACE2 activity was measured with hydrolysis rate of the ﬂuorogenic ACE2 substrate Nma-His-Pro-Lys(Dnp). e–h HPLC analysis of B38-CAP-treated angiotensin peptides. Ang II (e), Ang 1–9(f), Ang 1–7(g), or Ang I (h) (5 nmol each) was incubated with vehicle, recombinant B38-CAP protein, or recombinant ACE2 protein (5 μg each) for 90 min, then subjected to HPLC analysis. i, j Kinetic analysis for hydrolysis of Ang I with B38-CAP. HPLC analysis of angiotensin peptides generated after incubating Ang I with B38-CAP (i, j, upper panel). Amino acids in the same samples were quantiﬁed with LC-MS system (j, lower panel). Experiments were repeated more than three times and representative chromatography charts are shown. j Values are means ± SEM. n = 3 independent experiments. increased and ﬁnally reached the same levels of the initial Ang I Table 1 Kinetic constants for hydrolysis of ACE2 substrate amount, whereas Ang I was undetectable at 90 min (Fig. 1i, j). On by B38-CAP and IC of MLN-4760, an ACE2 inhibitor. the other hand, Ang 1–9 and Ang II exhibited a minor peak at 20 min and 60 min, respectively, and both peptides became −1 −1 −1 K (μM) k (s ) k /K (s μM )IC (pM) m cat cat m 50 undetectable at the end of the reaction period (Fig. 1j). Consistent B38-CAP 23.3 ± 1.70 188 ± 6.87 8.07 ± 0.295 710 ± 173 with peptide kinetics, the amino acids leucine (Leu), histidine ACE2 23.8 ± 1.91 168 ± 6.18 7.08 ± 0.260 340 ± 50.1 (His), and phenylalanine (Phe) were generated in the same order as mono-carboxyl proteolysis of Ang I, Ang 1–9, and Ang II, respectively (Fig. 1j), indicating that the conversion of Ang I to Ang 1–7 by B38-CAP is mediated through three steps of mono- and Supplementary Fig. 4a). For non-ACE2-substrate peptides carboxyl proteolysis. Therefore, B38-CAP has an ACE2-like Ang 1–7 and angiotensin 1–9(Ang 1–9), however, B38-CAP did activity, which converts both Ang II and Ang I peptides to Ang cleave Ang 1–9, whereas it did not affect Ang 1–7 (Fig. 1f, g and 1–7invitro. Table 2). Consistently, B38-CAP converted Ang I to Ang 1–7 (Fig. 1h), which is distinct from ACE2 conversion of Ang I to Ang 1–9 . To address how B38-CAP converts Ang I to Ang 1–7, we B38-CAP suppresses Ang II-induced cardiovascular pathology. conducted kinetic analysis (Fig. 1i, j and Supplementary Fig. 4b). To examine the effects of B38-CAP in vivo, we ﬁrst injected B38- Ang 1–9, Ang II, and Ang 1–7 peptidesweredetectableat 10min CAP into mice and measured plasma enzymatic activity as after mixing Ang I with B38-CAP (Fig. 1j). Ang 1–7production indicative of the plasma B38-CAP levels by using a newly NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 3 Marker BS-CAP BA-CAP B38-CAP Relative activity Arbitrary units Arbitrary units Arbitrary units Phe Ang 1–7 Ang II Arbitrary units Arbitrary units Arbitrary units Phe Phe Ang 1–7 Ang 1–7 Ang 1–7 Ang 1–9 Ang I Arbitrary units Amino acids (nmol) Peptides (nmol) Phe Ang 1–7 Ang 1–9 Ang II Ang I ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z 1–7 levels in the plasma of these mice. Ang 1–7 levels in the Table 2 Hydrolysis of ACE2-substrate peptides by B38-CAP. plasma were signiﬁcantly upregulated in both acute and chronic experimental settings (Fig. 2k, l). On the other hand, when we co- Substrate ACE2* B38-CAP Sequence treated the Ang II-injected mice with B38-CAP and A779, an Angiotensin I ++ DRVYIHPFH↓L antagonist for Mas/Ang 1–7 receptor, the suppressive effects of Angiotensin 1–9 − + DRVYIHPF↓H B38-CAP on Ang II-induced elevation of BP were not affected Angiotensin II ++ DRVYIHP↓F (Fig. 2m and Supplementary Fig. 9a–d). Furthermore, co- Angiotensin 1–7 −− DRVYIHP treatment of Ang 1–7 and Ang II did not downregulate Ang II- Apelin-13 ++ QRPRLSHKGPMP↓F induced elevation of BP (Supplementary Fig. 9e–h). Thus, the Apelin-36 ++ …QRPRLSHKGPMP↓F 9 hypotensive effects of B38-CAP are primarily mediated through des-Arg -bradykinin++ RPPGFSP↓F downregulation of Ang II levels. Lys-des-Arg - ++ KRPPGFSP↓F As chronic infusion of Ang II induces cardiac hypertrophy and bradykinin β-Casomorphin ++ YPFVEP↓I ﬁbrosis, we examined the hearts of mice chronically treated with −1 Dynorphin A 1–13 ++ YGGFLRRIRPKL↓K high-dose Ang II (1.5 mg kg per day) with or without B38-CAP −1 Ghrelin ++ …ESKKPPAKLQP↓R (3 mg kg per day) for 2 weeks. B38-CAP suppressed Ang II- Neurotensin 1–8 ++ pE-LYENKP↓R induced cardiac hypertrophy and increase of heart weight (HW) as measured with HW-to-body weight ratios (HW/BW) or HW Summary of cleavability of peptides by B38-CAP. “+” indicates that it is cleaved with ACE2 or B38-CAP, whereas “−” means it is not cleaved. HPLC analyses for the metabolites of each to tibia length (HW/TL) (Fig. 3a–c and Supplementary Table 2). peptide after B38-CAP treatment are shown in Supplementary Fig. 4a. *Cleavability of the Consistently, Ang II-induced wall thickening of the hearts was peptides by ACE2 is from ref. . signiﬁcantly downregulated by B38-CAP treatment as shown by echocardiography (Fig. 3d, e and Supplementary Table 2). In developed B38-CAP-speciﬁc substrate Nma-Leu-Pro-Lys(Dnp). addition, mild decrease of cardiac contractility in Ang II-infused −1 In 1 h after intraperitoneal (i.p.) injection of B38-CAP (2 mg kg mice were prevented by B38-CAP treatment as determined by % i.p.), the concentration of B38-CAP in plasma was markedly fractional shortening (%FS) (Fig. 3f and Supplementary Table 2). increased and peaked (Fig. 2a). The plasma B38-CAP levels Moreover, B38-CAP signiﬁcantly prevented Ang II-induced gradually decreased to almost baseline at 8 h but it was still cardiac ﬁbrosis (Fig. 3g, h) and upregulation of ﬁbrotic genes detectable in the plasma through 12 h (Fig. 2a). Estimated initial expression (Collagen 8a (Col8a1), Periostin (Postn), and TGF-β2 half-life in systemic circulation was 3.5 h, which is almost similar (Tgfb2)) (Fig. 3i–k). Similar results were obtained when B38-CAP 25,26 to 1.8–8.5 h of rhACE2 . We next examined whether B38- was daily i.p. injected for 4 weeks to Ang II-infused mice CAP treatment affects Ang II-induced elevation of BP. We ﬁrst (Supplementary Fig. 7f–n and Supplementary Table 3). These measured the BP in the carotid artery in anesthetized mice using results demonstrate that B38-CAP suppresses Ang II-induced a transducer catheter (Fig. 2b). Intraperitoneal injection of Ang II hypertension, cardiac hypertrophy, and ﬁbrosis. −1 (0.2 mg kg i.p.) induced acute elevation of arterial BP in wild- −1 type mice, whereas pretreatment of B38-CAP (2 mg kg i.p.) signiﬁcantly suppressed the Ang II-induced elevation of arterial B38-CAP mitigates pressure overload-induced heart failure. pressure (Fig. 2c and Supplementary Fig. 5a–c). We next We further treated the mice under pressure overload cardiac stress addressed the effects of B38-CAP on hypertension induced induced by transverse aortic constriction (TAC) with continuous −1 by chronic Ang II treatment. Although continuous infusion of per day), which was initiated infusion of B38-CAP (2 mg kg Ang II with an osmotic mini-pump elevated BP as measured in immediately after TAC surgery (Fig. 4a). After 2 weeks of TAC, conscious mice, daily i.p. injection of B38-CAP downregulated the HW (HW/BW or HW/TL) was also signiﬁcantly decreased in elevation of BP at days 8, 14, and 28 (Supplementary Fig. 6a–e the B38-CAP-treated group as compared with vehicle-treated and Supplementary Fig. 7a–e). Furthermore, we tested whether controls (Fig. 4b–d). In addition, pulmonary congestion was continuous infusion of B38-CAP suppresses Ang II-induced suppressed by B38-CAP as determined by lung weight to BW ratio hypertension (Fig. 2d). When B38-CAP was infused sub- (LW/BW) and LW/TL ratio (Fig. 4e, f). Echocardiography also cutaneously using an osmotic mini-pump, B38-CAP was detect- showed that wall thickening was signiﬁcantly downregulated by able in the plasma of mice for 14 days (Supplementary Fig. 8a, b). B38-CAP treatment (Fig. 4g–i and Supplementary Table 4). In Although it had been reported that an immune response is addition, although %FS was signiﬁcantly decreased in the vehicle associated with the chronic infusion of rhACE2 resulting in the treatment group, %FS was preserved in B38-CAP-treated mice degradation of rhACE2 , this was not observed for B38-CAP; (Fig. 4g–l). Similarly, B38-CAP treatment signiﬁcantly suppressed there were no antibodies against B38-CAP detectable in the the increased expression of mRNA associated with cardiac serum of mice infused with B38-CAP for 2 weeks (Supplementary hypertrophy, such as ANF (atrial natriuretic factor), BNP (brain Fig. 8c). Implantation of B38-CAP-ﬁlled osmotic mini-pumps natriuretic peptide), and β-myhc (β-myosin heavy chain, Myh7) in signiﬁcantly suppressed Ang II-induced hypertension in con- the TAC mice (Fig. 5c–e). scious mice (Fig. 2e–g) without affecting the heart rate (Fig. 2h). Histological analysis further revealed that B38-CAP treatment These results indicate that B38-CAP antagonizes the vasopressor reduced the area of cardiac ﬁbrosis in the interstitial space and effect of Ang II. perivascular region in the hearts of TAC mice (Fig. 5a, b). We further examined the effects of B38-CAP treatment on Ang Consistently, although the expression of the pro-ﬁbrotic genes II levels in the blood. In the acute experiment with i.p. injection of Col8a1, Postn, and Tgfb2 were increased in the hearts of vehicle- Ang II (Fig. 2b), pretreatment of B38-CAP markedly down- treated mice with TAC, B38-CAP markedly downregulated the regulated a massive increase of plasma Ang II levels at 5 min after expression of those pro-ﬁbrotic genes (Fig. 5f–h). These results Ang II injection (Fig. 2i). Consistently, in the chronic experiment indicate that exogenous B38-CAP treatment protects mice from with continuous infusion of Ang II (Fig. 2d), continuous infusion pressure overload-induced cardiac dysfunction, hypertrophy, and of B38-CAP with additional osmotic pump signiﬁcantly ﬁbrosis. Furthermore, we examined whether any potential toxic decreased Ang II levels in the plasma at day 14 (Fig. 2j). As effects of B38-CAP on the liver and kidneys, and both serum Ang 1–7 is known to exert beneﬁcial effects in the cardiovascular markers of liver injury and kidney dysfunction, were not affected systems through Mas/Ang 1–7 receptor , we also measured Ang by B38-CAP, as measured with aspartate transaminase (AST) or 4 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE ab c Ang II + vehicle Ang II + B38-CAP B38-CAP i .p. Ang II i .p. or vehicle i .p. or vehicle i .p. <0.0001 –90 –10 0 5 10 20 30 (min) Invasive BP measurements 02468 10 12 Baseline 5 10 15 20 Ang II measurements Hours after B38-CAP Minutes after Ang II i .p. injection i .p . injection de fh <0.0001 <0.0001 g <0.0001 <0.0001 <0.0001 <0.0001 190 130 150 800 110 130 Continuous B38-CAP infusion 90 110 Continuous Ang II infusion 130 400 70 90 0 2 4 6 8 10 12 14 (days) 50 70 BP, echo, Ang II 70 30 50 0 B38-CAP: – + – + B38-CAP: –+ – + measurements B38-CAP: – + – + B38-CAP: – + – + Vehicle Ang II Vehicle Ang II Vehicle Ang II Vehicle Ang II ik 0.0001 0.0011jl m 0.0004 0.0009 0.0040 1400 800 400 0.0007 0.0004 170 n.s. 0.0262 <0.0001 0.0082 1200 250 600 300 800 100 400 50 200 100 0 0 0 0 70 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: –+ – + B38-CAP: –+ – + Ang II: – –– – + ++ + Vehicle Ang II B38-CAP: Vehicle Ang II Vehicle Ang II Vehicle Ang II – + – + –– + + A779: – – + +–+ – + Fig. 2 Effects of B38-CAP on plasma angiotensin II levels and blood pressure in mice. a Plasma B38-CAP levels in mice after intraperitoneal injection −1 of B38-CAP (2 mg kg ). B38-CAP activity was measured with the B38-CAP substrate Nma-Leu-Pro-Lys(Dnp) (n = 4, 6, 8, 6, 6, and 6 for 0, 1, 2, 4, 8, and −1 12 h, respectively). b, c Invasive measurements of arterial blood pressure (BP). Experimental protocol (b); mice pretreated with B38-CAP (2 mg kg i.p.) −1 had liquid-ﬁlled catheter inserted into carotid artery for BP measurements and Ang II (0.2 mg kg i.p.) was injected and BP measured. Systolic arterial pressure is shown (c). (n = 6 mice per group). d–h Blood pressure measurements with conscious mice. Experimental protocol (d); mice were treated with −1 −1 −1 −1 continuous infusion of vehicle, Ang II (1.5 mg kg per day), B38-CAP (3 mg kg per day), or Ang II (1.5 mg kg per day) plus B38-CAP (3 mg kg per day), and BP was measured by tail-cuff system after 2 weeks. Systolic (e), diastolic (f), and mean (g) BP and heart rate (h) are shown for mice treated vehicle+ vehicle (n = 10), vehicle+ B38-CAP (n = 11), Ang II + vehicle (n = 11), and Ang II + B38-CAP (n = 11). i–l Measurements of Ang II and Ang 1–7in the plasma of mice. The plasma was obtained from the mice treated acutely (i, k) and chronically (j, l) with Ang II, in the cohort of b, c, and d–h, respectively. Ang II (i, j) and Ang 1–7(k, l) levels were measured with ELISA. m BP measurements in conscious mice. The mice pretreated with B38-CAP −1 −1 (2 mg/kg i.p.) at 90 min before acquisition of baseline BP were treated with Ang II (0.2 mg kg i.p.), with or without A779 (0.2 mg kg i.p.). BP was measured every 5 min by tail-cuff system (Supplementary Fig. 9a–d) and the BP at 5 min after the last injection is shown (n = 6 mice per group). All values are means ± SEM. c, e–m, Two-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. alanine transaminase (ALT) and blood urea nitrogen (BUN) or dysfunction with signiﬁcant decrease of %FS (Fig. 7f, g). The Creatinine (Cr), respectively (Fig. 6a–d). In addition, the decrease osmotic mini-pumps containing B38-CAP or vehicle were −1 of BW due to TAC heart failure was prevented by B38-CAP implanted at this time point and B38-CAP (2 mg kg per day) treatment (Supplementary Table 4). These results suggest that was infused for 2 weeks. Treatment with B38-CAP signiﬁcantly B38-CAP does not exhibit overt side effects in mice for at least increased %FS as compared with that in vehicle-treated mice or in 2 weeks after treatment. the mice before treatment (Fig. 7g and Table 3), indicating that B38-CAP improved cardiac dysfunction. In addition, cardiac hypertrophy was signiﬁcantly suppressed by B38-CAP treatment B38-CAP improves established hypertension and heart failure. (Fig. 7h–j) and pulmonary congestion was also improved by B38- To determine therapeutic effects of B38-CAP in established dis- CAP (Fig. 4k, l). Consistently, B38-CAP treatment signiﬁcantly eases, we ﬁrst examined whether B38-CAP treatment improve downregulated increased expression of mRNA associated with the established hypertension in the mice, which have received Ang II pathology of cardiac hypertrophy (BNP and β-myhc) and ﬁbrosis infusion prior to the treatment (Fig. 7a). When BP was elevated (Col8a1, Postn, and Tgfb2) (Fig. 7m–q). Furthermore, we chal- after 7 days of Ang II infusion, daily i.p. injection of B38-CAP lenged B38-CAP to severe cardiac dysfunction in C57BL/6N was initiated (Fig. 7a). B38-CAP treatment signiﬁcantly down- mice under TAC, in which the mice exhibit profound decline of regulated Ang II-induced increase of BP to the levels in vehicle- cardiac contractility at 2 weeks after TAC (Fig. 4l and Supple- treated control mice (Fig. 7b–e). Thus, B38-CAP ameliorated mentary Fig. 10a–c). When B38-CAP treatment was started at established hypertension. Next, we investigated the effects of 2 weeks after TAC, B38-CAP markedly suppressed progression of B38-CAP on established cardiac dysfunction. At 5 weeks after heart failure in C57BL/6N mice (Supplementary Fig. 10b–o and TAC surgery, the mice (C57BL/6J background) exhibits cardiac NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 5 Plasma Ang II (pg/ml) Plasma B38-CAP (ng/ml) Plasma Ang II (pg/mL) Systolic BP (mmHg) Plasma Ang 1–7 (pg/ml) Diastolic BP (mmHg) Plasma Ang 1–7 (pg/ml) Systolic arterial pressure (mmHg) Mean BP (mmHg) Systolic BP (mmHg) Heart rate (bpm) ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ab c <0.0001 <0.0001 Vehicle Ang II <0.0001 <0.0001 Vehicle B38-CAP Vehicle B38-CAP 7 4 3 B38-CAP: –+ – + B38-CAP: – ++ – Vehicle Ang II Vehicle Ang II de fg Vehicle Ang II + vehicle Ang II + B38-CAP 1.2 1.2 <0.0001 <0.0001 <0.0001 <0.0001 0.0131 0.0343 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 20 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Vehicle Ang II Vehicle Ang II Vehicle Ang II hi <0.0001 0.0207 j k 0.0005 0.0454 4 8 2.0 <0.0001 0.0712 <0.0001 0.0002 3 6 1.5 2 4 1.0 1 2 0 0 0 0.5 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Vehicle Ang II Vehicle Ang II Vehicle Ang II Vehicle Ang II Fig. 3 Effects of B38-CAP on angioteinsin II-induced cardiac hypertrophy and ﬁbrosis. a–c Cardiac hypertrophy in the mice chronically co-treated with −1 −1 Ang II (1.5 mg kg per day) and B38-CAP (3 mg kg per day) in the cohort of Fig. 2d–h. Macroscopic heart images (a), heart weight to body weight ratio (HW/BW) (b), and heart weight to tibia length ratio (HW/TL) (c) in mice treated vehicle+ vehicle (n = 10), vehicle + B38-CAP (n= 11), Ang II + vehicle (n = 11), and Ang II+ B38-CAP (n = 11). Bars indicate 2 mm. d–f Echocardiography parameters of left ventricular end-diastolic posterior wall thickness (PWD) (d), end-diastolic interventricular septal wall thickness (IVSD) (e), and %fractional shortening (%FS) (f) in the mouse hearts. Complete echocardiography data are shown in Supplementary Table 2. g, h Histology of hearts. Masson’s trichrome staining (g); bars indicate 2 mm and 100 μmin the upper panels and lower panels, respectively. Quantiﬁcation of ﬁbrosis in the hearts (h) of mice treated with vehicle+ vehicle (n = 5), vehicle+ B38- CAP (n = 5), Ang II + vehicle (n = 7), and Ang II + B38-CAP (n = 8). i–k qRT-PCR analysis of pro-ﬁbrotic gene expressions in the hearts of mice treated with vehicle+ vehicle (n = 5), vehicle+ B38-CAP (n = 5), Ang II + vehicle (n = 7), and Ang II + B38-CAP (n = 8); mRNA levels of Collagen 8a (Col8a1) (f), Periostin (Postn)(g), and TGF-β (Tgfb2)(h) normalized with Gapdh. All values are means ± SEM. b–f, h–k Two-way ANOVA with Sidak’s multiple- comparisons test. Numbers above square brackets show signiﬁcant P-values. Supplementary Table 5), albeit mainly through decrease of LV similar proteolytic activity to rhACE2, there seems a difference in dimensions rather than wall thickness. These results indicate substrate speciﬁcity between two enzymes. B38-CAP is likely to that B38-CAP exerts therapeutic effects in established cardiac have more broad speciﬁcity for proteolytic effects on peptides, dysfunction. because B38-CAP converted Ang I and Ang 1–9 into Ang 1–7, whereas ACE2 does not cleave Ang 1–9. The superimposition of ACE2 and BS-CAP structures indicated that the positions of Discussion substrate-binding amino acid residues and metal-binding residues In this study, we elucidated that bacteria-derived carbox- are matched. Although the ACE2 substrate Nma-His-Pro-Lys ypeptidases have ACE2-like enzymatic activity and showed that (Dnp) was cleaved by B38-CAP with the same potency as ACE2, B38-CAP cleaves Ang II and Ang I to Ang 1–7 and down- we found that the Nma-Leu-Pro-Lys(Dnp) was catalyzed speci- regulates Ang II levels in mice. We demonstrated that Ang II- ﬁcally by B38-CAP but not by ACE2 (not shown), suggesting that induced hypertension, cardiac hypertrophy, and ﬁbrosis were the S2-subsite of B38-CAP is more hydrophobic than ACE2. The suppressed by B38-CAP treatment. We further showed that B38- difference in substrate speciﬁcity of B38-CAP and ACE2 should CAP improves cardiac dysfunction, hypertrophy, and ﬁbrosis be further elucidated by our ongoing analysis for crystal structure induced by pressure overload in mice. of B38-CAP. Among three bacterial carboxypeptidases we tested, only B38- Although the hypotensive action of B38-CAP is mediated CAP showed dependence of proteolytic activity on anion con- −1 mainly through Ang II downregulation, B38-CAP (2 ~ 3 mg kg centration, which is characteristic of ACE2 activity . B38-CAP 6,19 per day) exhibits a more potent therapeutic effect in TAC- also showed pH optimum of 7.5 equivalent to rhACE2 . induced heart failure than in Ang II-induced heart failure (e.g., In addition, IC of MLN-4760 was also equivalent between 87.9% inhibition of cardiac hypertrophy (HW/BW increase) in rhACE2 and B38-CAP. Although B38-CAP exhibited quite 6 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications PWD (mm) %area of fibrosis IVSD (mm) Col8a1/Gapdh %FS Postn/Gapdh HW/BW (mg/g) Tgfb2/Gapdh HW/TL (mg/mm) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE ac b d Sham TAC <0.0001 <0.0001 <0.0001 <0.0001 Vehicle B38-CAP Vehicle B38-CAP Continuous B38-CAP infusion 8 0 7 14 (days) 6 TAC Echo, sampling B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC ef 0.0167 0.0105 g 0.0162 0.0089 Sham TAC 20 20 Vehicle B38-CAP Vehicle B38-CAP 15 15 0 0 B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC h ij k l <0.0001 <0.0001 1.2 1.2 4 4.5 70 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0003 4.0 1.0 1.0 3 3.5 0.8 0.8 2 40 3.0 0.6 0.6 1 2.5 0.4 0.4 0 2.0 10 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC Sham TAC Sham TAC Sham TAC Fig. 4 B38-CAP mitigates pressure overload (TAC)-induced cardiac dysfunction and hypertrophy. a Experimental protocol. The mice were subjected to −1 the surgery of transverse aortic constriction (TAC) surgery and then continuous infusion of B38-CAP (2 mg kg per day) was initiated. b–f B38-CAP suppressed cardiac hypertrophy. Representative photograph (b) of the hearts of mice under TAC. Bars indicate 2 mm. HW/BW (c), HW/TL (d), lung weight to body weight ratio (LW/BW) (e), and lung weight to tibia length ratio (LW/TL) (f) in the mice treated with sham + vehicle (n = 7), sham+ B38- CAP (n= 5), TAC + vehicle (n = 9), and TAC + B38-CAP (n = 8). g–l Echocardiography measurements. Representative M-mode echocardiography images (g), measurements of IVSD (h), PWD (i), LVESD (j), LVEDD (k), and %FS (l) are shown. Complete echocardiography data are shown in Supplementary Table 4. All values are means ± SEM. c–f, h–l One-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. TAC model vs. 53.2% inhibition in Ang II-infusion model). On therapeutic strategy in cardiovascular disease and other Ang II- the other hand, in a previous study, rhACE2 showed similar anti- related diseases, e.g. ARDS. On the other hand, although mass hypertrophic effects in both TAC and Ang II-infusion models . production of rhACE2 as a protein drug costs due to requirement The difference may be explained by the slight difference of sub- of mammalian cell expression systems, B38-CAP is easily pre- strate speciﬁcity of B38-CAP and ACE2. Although ACE2 con- pared with E. coli expression system and is cost effective. Ther- verts Ang I to Ang 1–9 inefﬁciently and requires ACE for further apeutic efﬁcacy and less toxicity of B38-CAP in mouse heart conversion of Ang 1–9 to Ang 1–7 , B38-CAP targets all the Ang failure models would warrant further investigation of B38-CAP or I, Ang 1–9, and Ang II peptides as a mono-carboxypeptidase. other microbial carboxypeptidases in disease models. Further- Thus, the conversion of Ang I and Ang II to Ang 1–7 by B38- more, human ACE2-like enzyme in bacteria might pave the way CAP may contribute to more efﬁcient down-modulation of RAS to a new strategy to engineer evolution of bacterial proteins for in the TAC heart failure. In addition, Ang 1–7 is a vasoprotective better designing and preparations of recombinant protein drugs. peptide which acts through its cognate Mas receptor, and also has anti-ﬁbrotic and cardioprotective functions in heart failure . Methods Thus, B38-CAP-mediated degradation of Ang I into Ang 1–7 Searching for bacteria-derived ACE2-like enzymes. Sequence comparison was would be beneﬁcial in enhancing Ang 1–7 generation for treating performed using BLAST and MEROPS (http://merops.sanger.ac.uk/) tools avail- failing hearts. Furthermore, as ACE2 and B38-CAP target other able online. Similarity search and superposition of a 3D structure of proteins was performed by Molecular Operating Environment (MOE 2016.08; Chemical Com- biological peptides than angiotensin peptides, subtle differences puting Group, Inc., Montreal, QC, Canada). Multiple sequence alignment between in substrate speciﬁcity may license B38-CAP to degrade such enzyme sequences was performed using the CLUSTALW tool available online. peptides in a different manner or even to have a new peptide Phylogenetic tree drawing was performed using NJplot program (http://doua.prabi. substrate different from ACE2 targets. fr/software/njplot). In addition to the currently used drugs to inhibit Ang II gen- eration or signaling, such as ACE inhibitors or Angiotensin Recombinant proteins. Genomic DNAs were isolated from Paenibacillus sp. receptor blockers, direct down-modulation of Ang II levels by 22 B38 , B. subtilis subsp. subtilis NBRC 13719, and B. amyloliquefaciens NBRC 3022 rhACE2 protein is one of the promising candidates for new (Supplementary Table 1). Expression plasmids, which encode B38-CAP, BS-CAP, NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 7 IVSD (mm) LW/BW (mg/g) PWD (mm) LW/TL (mg/mm) LVESD (mm) LVEDD (mm) HW/BW (mg/g) %FS HW/TL (mg/mm) ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z a b Sham TAC <0.0001 <0.0001 Vehicle B38-CAP Vehicle B38-CAP B38-CAP: –+ – + Vehicle TAC cd e 0.0012 0.0319 <0.0001 <0.0001 0.0003 <0.0001 15 120 10 3 5 40 0 0 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC Sham TAC fg <0.0001 <0.0001 <0.0001 <0.0001 h <0.0001 <0.0001 8 4 6 3 4 2 2 1 0 0 0 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC Sham TAC Fig. 5 B38-CAP suppresses TAC-induced cardiac ﬁbrosis. a, b Histology. The hearts of B38-CAP or vehicle-treated mice under TAC were stained with Masson’s trichrome. Bars indicate 1 mm (upper) or 100 μm (lower). c–h qRT-PCR analysis for the expression of heart failure genes and pro-ﬁbrosis genes; mRNA levels of atrial natriuretic factor (ANF)(c), B-type natriuretic peptide (BNP)(d), β-myosin heavy chain (β-myhc)(e), Collagen 8a (Col8a1)(f), Periostin (Postn)(g), and TGF-β (Tgfb2)(h) in the hearts of mice treated with sham+ vehicle (n = 7), sham+ B38-CAP (n= 5), TAC + vehicle (n = 9), and TAC + B38-CAP (n= 8). All values are means ± SEM. b–h Two-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. ab c d 150 50 40 0.6 0.0103 100 0.4 0.2 0 0 0 0.0 B38-CAP–+ – + B38-CAP–+ – + B38-CAP–+ – + B38-CAP –+ – + Sham TAC Sham TAC Sham TAC Sham TAC Fig. 6 No overt toxic effects of B38-CAP on liver and kidney. a, b Liver function test with measurements of aspartate aminotransferase (AST) (a) and alanine aminotransferase (ALT) (b) in the blood. c, d Kidney function assessment with measurements of BUN (c) and Creatinine (Cr) (d) in the blood. sham+ vehicle (n = 7), sham+ B38-CAP (n = 5), TAC + vehicle (n = 9), and TAC + B38-CAP (n = 8). All values are means ± SEM. Two-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. or BA-CAP, were constructed by PCR. PCR products were ligated into a XbaI- and (2.6 × 60 cm; GE Healthcare) (Supplementary Fig. 2a). To exclude potential con- XhoI-double-digested pET28a plasmid, and recombinant proteins were generated tamination of endotoxin, the eluates were further passed through a Polymyxin B by isopropyl β-D-thiogalactopyranoside (IPTG) induction of E. coli. Cells were column. For preparation of recombinant human ACE2 in Sf9 insect cells, human collected and the cell lysate prepared and centrifuged at 13,000 × g for 15 min. The ACE2 cDNA was inserted into the XbaI and KpnI sites of pFastBac1 vector resulting supernatant was subjected to ammonium sulfate precipitation, anion- (Invitrogen) and the generated recombinant bacmid DNA was transfected into Sf9 exchange chromatography with a Q-Sepharose Fast Flow column (1.6 × 10 cm; GE cells using Cellfectin (Invitrogen) to construct recombinant baculovirus encoding Healthcare), and gel ﬁltration chromatography with a Superdex 75 pg column human ACE2. Sf9 cells were infected with the recombinant baculovirus at a 8 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications AST (U/l) ANF/Gapdh Col8a1/Gapdh ALT (U/l) Postn/Gapdh BNP/Gapdh BUN (mg/dl) -myhc/Gapdh Tgfb2/Gapdh %area of fibrosis Cr (mg/dl) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE Ang II + vehicle Ang II + vehicle Ang II + vehicle Ang II + vehicle ab c d e Daily B38-CAP Ang II + B38-CAP Ang II + B38-CAP Ang II + B38-CAP Ang II + B38-CAP i .p.injection 800 160 Vehicle + vehicle Vehicle + vehicle Vehicle + vehicle Vehicle + vehicle No treatment Ang II infusion <0.0001 0.0003 <0.0001 100 <0.0001 120 80 –7 0 2 4 6 (days) BP measurements 60 200 0246 0246 0246 0246 Days after treatment Days after treatment Days after treatment Days after treatment fg <0.0001 h ij TAC 0.0001 0.0250 Sham 60 10 <0.0001 0.0129 <0.0001 + vehicle Vehicle B38-CAP B38-CAP 0.0010 No treatment infusion 8 –5 0 2 (weeks) TAC Echo Echo, C57BL/6J sampling mice TAC: – + + TAC: – + + Weeks: 0 2 02 2 0 B38-CAP: – – + B38-CAP: – – + Vehicle Vehicle B38-CAP + sham TAC kl m 0.0011 0.0191n 0.0048 0.0137 o p 0.0027 0.0029q 0.0029 0.0003 8 3 8 4 4 2.0 <0.0001 <0.0001 0.0044 0.0118 0.0039 0.0137 6 3 3 1.5 6 4 2 2 1.0 2 1 1 0.5 0.0 3 0 0 0 0 TAC:–+ + TAC:–+ + TAC:–+ + TAC:–+ + TAC:–+ + TAC:–+ + TAC: – + + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + Fig. 7 Therapeutic effects of B38-CAP on established hypertension and cardiac dysfunction. a–e Therapeutic effects of B38-CAP on established hypertension. Experimental protocol (a); Ang II infusion (1 mg/kg/day) was initiated at 7 days before treatment. The mice were injected with B38-CAP (2 mg/kg i.p.) or vehicle twice a day and blood pressure was measured by tail-cuff system at 2 h after injection. Systolic (b), diastolic (c), and mean (d)BP and heart rate (e) in the mice treated Ang II+ vehicle (n= 7), Ang II+ B38-CAP (n= 7), and vehicle+ vehicle (n= 5). f–l Therapeutic effects of B38-CAP on established cardiac dysfunction. Experimental protocol (f); the C57BL/6J mice had TAC surgery at 5 weeks before treatment and B38-CAP (2 mg/kg/day) or vehicle was continuously infused with osmotic mini-pumps. Echocardiography parameters of %fractional shortening (%FS) (g) in the mice treated with sham+ vehicle (n= 5), TAC + vehicle (n= 6), and TAC+ B38-CAP (n= 6). Representative photographs of the hearts of mice under TAC (h). Bars indicate 2 mm. HW/BW (i), HW/TL (j), LW/BW (k), and LW/TL (l) are in the mice treated with sham+ vehicle (n= 5), TAC+ vehicle (n= 6), and TAC+ B38- CAP (n= 6). m–q qRT-PCR analysis for the expression of heart failure genes and pro-ﬁbrosis genes in the hearts (n= 5 mice per group). All values are means ± SEM. b–e Two-way ANOVA with Sidak’s multiple-comparisons test. g One-way ANOVA with Sidak’s multiple-comparisons test for comparison of groups. Two-tailed paired t-test between before and after treatment of the same group. i–q One-way ANOVA with Sidak’s multiple-comparisons test. Numbers next to square brackets show signiﬁcant P-values. Table 3 Echocardiographic parameters in the mice with established cardiac dysfunction treated with B38-CAP for 2 weeks. Sham + vehicle Sham+ vehicle TAC + vehicle TAC + vehicle TAC + B38-CAP TAC + B38-CAP Before treatment After treatment Before treatment After treatment Before treatment After treatment N 55 6 6 6 6 Age (weeks) 15 17 15 17 15 17 BW (g) 23.14 ± 1.24 25.82 ± 1.03 23.12 ± 0.87 25.92 ± 0.74 23.55 ±± 0.83 24.30 ± 1.16 HR (bpm) 594 ± 49 631 ± 68 551 ± 51 559 ± 49 570 ± 65 644 ± 61 ### †† FS (%) 51.78 ± 4.09 51.91 ± 1.56 29.49 ± 2.79*** 27.20 ± 1.43 29.06 ± 2.36*** 36.38 ± 2.03 ### †† EF (%) 76.70 ± 3.70 83.89 ± 1.27 57.44 ± 4.5*** 53.56 ± 2.28 56.70 ± 3.54*** 67.03 ± 2.86 ## † LVESD (mm) 1.54 ± 0.15 1.54 ± 0.13 2.43 ± 0.38*** 2.75 ± 0.14 2.49 ± 0.23*** 2.15 ± 0.17 LVEDD (mm) 3.20 ± 0.08 3.21 ± 0.22 3.44 ± 0.45 3.77 ± 0.17 3.51 ± 0.34 3.38 ± 0.24 IVSD (mm) 0.74 ± 0.04 0.79 ± 0.03 1.05 ± 0.17* 1.04 ± 0.05 1.09 ± 0.05*** 0.99 ± 0.07 PWD (mm) 0.78 ± 0.06 0.89 ± 0.13 1.11 ± 0.12*** 0.99 ± 0.05 1.03 ± 0.06*** 1.08 ± 0.09 Results are presented as mean ± SEM. One-way ANOVA plus Sidak’s multiple-comparisons test was used to detect signiﬁcance. BW body weight, FS left ventricular fractional shortening, EF left ventricular ejection fraction, HR heart rate, IVSD end-diastolic interventricular septal wall thickness, LVEDD left ventricular end-diastolic diameter, LVESD left ventricular end-systolic diameter, PWD left ventricular end-diastolic posterior wall. *P < 0.05 vs. sham + vehicle before treatment; **P < 0.001 vs. sham + vehicle before treatment; ***P < 0.0001 vs. sham + vehicle before treatment; #P < 0.05 vs. TAC + vehicle after treatment; ##P < 0.001 vs. TAC+ vehicle after treatment; ###P < 0.0001 vs. TAC+ vehicle after treatment. Two-tailed paired t-test was used to detect signiﬁcance. †P < 0.05 vs. TAC+ B38-CAP before treatment; ††P < 0.001 vs. TAC+ B38-CAP before treatment. NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 9 LW/BW (mg/g) LW/TL (mg/mm) Systolic BP (mmHg) %FS BNP/Gapdh Diastolic BP (mmHg) -myhc/Gapdh Col8a1/Gapdh Mean BP (mmHg) HW/BW (mg/g) Postn/Gapdh HW/TL (mg/mm) Heart rate (bpm) Tgfb2/Gapdh ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z multiplicity of infection (MOI) of 1 pfu/cell and then cultured in SF-900 II serum- (Fig. 7a–e) or %FS for the experiments of TAC-induced cardiac dysfunction free medium (Invitrogen) using 250 mL shaker ﬂasks at 100 r.p.m. at 28 °C for 72 h. (Fig. 7f–q and Supplementary Fig. 10). The treatments and measurements were rhACE2 was puriﬁed with Proﬁnity IMAC Ni-charged resin (Bio-Rad), eluted with performed using a double-blind method. 250 mM imidazole, and further dialyzed against phosphate-buffered saline (PBS). Invasive measurements of arterial blood pressure. Acute hemodynamic 26,29 In vitro ACE2 activity measurements. For determination of kinetic constants, the experiment was performed with 10-week-old male C57BL/6J mice . Ninety K and k values for B38-CAP and recombinant human ACE2 (Calbiochem) minutes before hemodynamic measurements, mice were pretreated with an i.p. m cat −1 using Nma-His-Pro-Lys(Dnp) as ACE2 substrate were determined by injection of either B38-CAP (2 mg kg i.p.) or sterile PBS as vehicle. Mice Michaelis–Menten model using GraphPad Prism Version 6.01 (La Jolla, CA, were anesthetized with isoﬂurane (1–1.5%) and body temperature was maintained 19,28 USA) . The IC values of ACE2 inhibitor MLN-4760 (EMD, Millipore) for at 37–38 °C using a heating pad throughout the experiments. The mouse was recombinant human ACE2 and B38-CAP were measured as follows. The reaction securely restrained in a supine position and mechanically ventilated with a tracheal −1 mixture contained 40 μl of 0.1 M HEPES pH 7.5, containing 0.3 M NaCl, 0.01% cannulation (peak inﬂation pressure of 10 cm H O and 160 breaths min ; Ven- Triton X-100, 0.02% NaN ,5 μl MLN-4760 solution, and 5 μl of recombinant telite, Harvard Apparatus). A tapered PE-50 catheter ﬁlled with heparinized saline −1 human ACE2 or B38-CAP in a total volume of 50 μl. The reaction mixture was (20 units ml ) was inserted into the right carotid artery to record arterial BP via a incubated at 37 °C for 30 min and then the reaction was terminated by adding pressure transducer. After 5–10 min of stabilization period, systolic, diastolic, and 0.2 ml of 0.1 M sodium borate buffer pH 10.5. The ﬂuorescein intensity was mean BPs were obtained (Power Lab data acquisition system, AD Instruments) and measured spectrophotometrically at an emission wavelength 440 nm upon exci- heart rate was calculated using LabChart8 software (AD Instruments). At 90 min −1 tation wavelength 340 nm (Hitachi F-2500). The sample concentration required to after vehicle or B38-CAP injection, Ang II (0.2 mg kg i.p.) or vehicle was inhibit 50% of B38-CAP activity under the assay condition was taken as the IC injected. The changes in BP were analyzed for a subsequent 20 min time period. value. For plasma B38-CAP activity measurements, we developed a new B38-CAP- speciﬁc substrate Nma-Leu-Pro-Lys(Dnp) by screening the amino acids of P2 Transverse aortic constriction. Ten-week-old male C57BL/6N mice were sub- position in Nma-X-Pro-Lys(Dnp) substrates (not shown), and the K , k , and m cat jected to pressure overload by TAC . In one of the therapeutic experiments −1 k /K values using this substrate were determined to be 9.52 μM, 224 s , and cat m (Fig. 7), C57BL/6J mice were used. The same surgical TAC procedure results in −1 −1 23.5 s μM , respectively. Heparinized plasma was diluted with assay buffer and more severe dysfunction in C57BL/6N than in C57BL/6J mouse strains. Brieﬂy, the reaction mixture contained 45 μl of HEPES buffer pH 7.5, 0.3 M NaCl, 20 μM −1 mice were anesthetized via i.p. injection of ketamine (100 mg kg ) and xylazine Nma-Leu-Pro-Lys(Dnp), 0.01% Triton X-100, 0.02% NaN , and 5 μl diluted 3 −1 (20 mg kg ), and a longitudinal incision was made in the proximal portion of plasma or recombinant B38-CAP in a total volume of 50 μl. The reaction mixture sternum. The aortic arch was ligated with an overlying 27-gauge needle by 7-0 silk. was incubated at 37 °C for 60 min and then the reaction was terminated by adding The needle was immediately removed leaving a discrete region of constriction. The 0.2 ml of 0.1 M sodium borate buffer pH 10.5, and the ﬂuorescence intensity was sham-treated group underwent a similar procedure without ligation. Echocardio- measured. The enzyme concentration in plasma was calculated based on the graphy was performed at indicated time points after TAC or sham surgery, and standard recombinant B38-CAP activity. mice were then killed by cervical dislocation. Hydrolysis of angiotensin peptides by B38-CAP. Each reaction mixture Echocardiography and blood pressure measurements. Echocardiographic (185.3 µl) was formulated as 1 mg/ml of angiotensin peptides in 15.4 mM HEPES measurements were performed as previously described . Brieﬂy, conscious mice (pH 7.5), 184 mM NaCl, and 2 µg of recombinant B38-CAP, and reactions were were gently grabbed in hand or held in the apparatus, echocardiography was incubated at 37 °C. The reaction was terminated by the addition of 14.7 µl of 0.5 M performed using Vevo770 equipped with a 30 MHz linear transducer (Visual EDTA. The reaction mixture (20 µl) was analyzed by reverse-phase HPLC (TSKgel Sonics). The %FS was calculated as follows: %FS = [(LVEDD – LVESD)/ Super-ODS, 0.46 × 5, or 10 cm, Tosoh Corporation, Tokyo, Japan) and eluted with LVEDD] × 100. M-mode images were obtained for measurement of wall thickness a linear gradient of 0–100% acetonitrile in 0.05% triﬂuoroacetic acid (TFA). and chamber dimensions with the use of the leading-edge convention adapted by Quantiﬁcation was achieved using the peak area of the standard angiotensins I, II, the American Society of Echocardiography. For BP measurements, conscious mice and 1–7 (Peptide Institute, Inc.) and Ang 1–9 (Wako, Osaka, Japan). were warmed at 10 min before measurements through during measurements. BP was measured by a programmable sphygmomanometer (BP-200, Softron, Japan) LC-MS quantiﬁcation of amino acids (Leu, Phe, and His). Samples (100 µL) were using the tail-cuff method after 5 days of daily training . diluted with 9.2 M perchloric acid (4.3 µL), then centrifuged at 13,000 × g for 15 min. A 5 µl aliquot was injected for a Shimadzu LC-MS system (LCMS2020, Pharmacological intervention. When we treated the mice with B38-CAP, we Kyoto, Japan) equipped with an electrospray ion source with nebulizer gas examined two routes of administration; daily i.p. injection and subcutaneous con- −1 −1 1.5 L min , drying gas 15 L min , desolvation line temperature 250 °C, and heat tinuous infusion with osmotic mini-pumps (Alzet model 1002, Alza Corp.). The block temperature 200 °C. Chromatographic separations were performed with an dosage and route of B38-CAP and Ang II (Sigma-Aldrich) treatments in the Intrada Amino Acid column (3 × 100 mm, Imtakt, Kyoto, Japan). Leu and Phe experiments are described in each ﬁgure legends. In prevention experiments, were analyzed with the mobile phase consisting of (mobile phase A) acetonitrile : treatment with B38-CAP was initiated at the same time as implantation of Ang II- tetrahydrofuran (THF) : 25 mM ammonium formate : formic acid 10 : 80 : 10 : ﬁlled osmotic mini-pumps or completion of TAC surgery . For co-infusion of Ang 0.4 [v/v] and (mobile phase B) acetonitrile : 100 mM ammonium formate 20 : 80 at II and B38-CAP into the subcutaneous of mice for 2 weeks (Fig. 2d–h and Fig. 3), −1 0.4 mL min . The initial mobile-phase composition was 20% B maintained for Ang II and B38-CAP were loaded into individual pumps and the mice were 4 min, which was gradually increased to 100% B in 7 min, and then maintained at implanted with the two pumps. When Ang II was infused for 4 weeks (Supple- 100% B for 3 min and back to the initial condition of 20% B in 6 min for re- mentary Fig. 7), Ang II-ﬁlled osmotic mini-pumps (Alzet model 1002) were replaced equilibration. Furthermore, His was analyzed with the mobile phase consisting of with a new one at 2 weeks after implantation. Two or 4 weeks after treatment, BP (mobile phase A) acetonitrile : water : formic acid 85 : 15 : 0.3 [v/v] and (mobile measurement and echocardiography were performed. In the chronic experiments −1 phase B) 100 mM ammonium formate at 0.4 mL min . The initial mobile-phase with i.p. injection of B38-CAP, BP was measured at 2 h after i.p. injection (Sup- composition was 55% B maintained for 4 min, which was gradually increased to plementary Figs. 6 and 7a–e). In therapeutic experiments (Fig. 7 and Supplementary 100% B in 8 min, and then maintained at 100% B for 5 min and back to the initial Fig. 10), treatment with B38-CAP was initiated after the establishment of hyper- condition of 55% B in 6 min for re-equilibration. Single-ion monitoring in negative tension or pressure overload-induced cardiac dysfunction. To examine the effects of mode with m/z 132, 156, and 166, representing Leu, His, and Phe, respectively. A779 on B38-CAP hypotensive action (Fig. 2m and Supplementary Fig. 9a–d), Under these conditions, Leu and Phe were eluted at 7.93 and 7.16 min, respectively, −1 conscious mice were pre-injected with B38-CAP (2.0 mg kg i.p.) or vehicle at and His was eluted at 12.56 min. Quantiﬁcation was achieved using the peak area of −1 90 min before measurements. Just before injection of Ang II (0.2 mg kg i.p.), A779 the standard amino acids mixture, type H (Wako, Osaka, Japan). −1 (0.2 mg kg i.p.) or its combination, baseline data of BP was obtained, and then BP was measured every 5 min after injection by a tail-cuff method. Mice. C57BL/6N or C57BL/6J wild-type male mice were purchased from CLEA Japan, Inc. and maintained at the animal facilities of Akita University Graduate Histology. Heart tissues were ﬁxed with 4% formalin and embedded in parafﬁn. School of Medicine or Research Institute of National Cerebral and Cardiovascular Five-μm-thick sections were prepared and stained with hematoxylin and eosin or Center. All animal experiments conformed to the Guide for the Care and Use of Masson’s trichrome stain. For measurement of cardiac ﬁbrosis area, the high- Laboratory Animals, Eighth Edition, updated by the US National Research Council resolution images (×100 magniﬁcation) of the heart sections stained with Masson’s Committee in 2011, and approvals of the experiments were granted by the ethics trichrome were taken using a BIOREVO microscope (BZ9000; Keyence) and review board of Akita University or Research Institute of National Cerebral and ﬁbrosis area was quantiﬁed using the Image-Pro software (Media Cybernetics). Cardiovascular Center. Randomization was performed by using random numbers. In prevention experiments (Figs. 2–6 and Supplementary Figs. 5–9), the mice were assigned by stratiﬁed randomization based on BW. In therapeutic experiments Quantitative real-time PCR. RNA was extracted using TRIzol reagent (Invitro- (Fig. 7 and Supplementary Fig. 10), the mice were assigned by stratiﬁed rando- gen) and cDNA synthesized using the PrimeScript RT reagent kit (TAKARA). mization based on systolic BP for the experiments of Ang II-induced hypertension Sequences of the forward and reverse primers of the genes studied are shown in 10 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE Supplementary Table 6. Real-time PCR was run in 96-well plates using a SYBR 9. Kuba, K., Imai, Y. & Penninger, J. M. Multiple functions of angiotensin- Premix ExTaq II (TAKARA) according to the instructions of the manufacturer. converting enzyme 2 and its relevance in cardiovascular diseases. Circ. J. 77, Relative gene expression levels were quantiﬁed by using the Thermal Cycler Dice 301–308 (2013). 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When a comparison is done for groups with two factor levels, two-way angiotensin-system. Int. J. Hypertens. 2012, 428950 (2012). ANOVA with Sidak’s multiple-comparisons test were used. P < 0.05 was con- 19. Takahashi, S., Yoshiya, T., Yoshizawa-Kumagaye, K. & Sugiyama, T. sidered signiﬁcant. Power analysis was performed on the results of initial animal Nicotianamine is a novel angiotensin-converting enzyme 2 inhibitor in experiments by statistical software R. The value of Cohen’s f was calculated with soybean. Biomed. Res. 36, 219–224 (2015). between-groups variance and within-subgroup variance, the effect size of each 20. Towler, P. et al. ACE2 X-ray structures reveal a large hinge-bending motion experiment data was calculated under P < 0.05 and Power 0.8, and the minimal important for inhibitor binding and catalysis. J. Biol. Chem. 279, 17996–18007 number of animals was determined. (2004). 21. Natesh, R., Schwager, S. L., Sturrock, E. D. & Acharya, K. R. Crystal structure Reporting summary. Further information on research design is available in of the human angiotensin-converting enzyme-lisinopril complex. Nature 421, the Nature Research Reporting Summary linked to this article. 551–554 (2003). 22. Takahashi, S. et al. Paenidase, a novel D-aspartyl endopeptidase from Paenibacillus sp. B38: puriﬁcation and substrate speciﬁcity. J. Biochem. 139, Data availability 197–202 (2006). The data that support the ﬁndings of this study are available from the corresponding 23. Nirasawa, S., Nakahara, K. & Takahashi, S. Cloning and characterization of author upon reasonable request. Source data used to generate Figs. 1–7 and the novel d-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38. J. Supplementary Figs. 3, 5–10 are provided in the Source Data ﬁle. The accession codes of Biochem. 164, 103–112 (2018). DNA or amino acid sequences are listed in Supplementary Table 1. An uncropped image 24. Lee, M. M. et al. Insight into the substrate length restriction of M32 of SDS-PAGE analysis is shown in Supplementary Fig. 11. carboxypeptidases: characterization of two distinct subfamilies. Proteins 77, 647–657 (2009). Received: 26 October 2018; Accepted: 10 February 2020; 25. Liu, P. et al. Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation. Kidney Int. 94, 114–125 (2018). 26. Wysocki, J. et al. Targeting the degradation of angiotensin II with recombinant angiotensin-converting enzyme 2: prevention of angiotensin II- dependent hypertension. Hypertension 55,90–98 (2010). 27. Santos, R. A. S. et al. The ACE2/angiotensin-(1-7)/MAS axis of the renin- References angiotensin system: focus on angiotensin-(1-7). Physiol. Rev. 98, 505–553 1. Corvol, P., Jeunemaitre, X., Charru, A., Kotelevtsev, Y. & Soubrier, F. Role of (2018). the renin-angiotensin system in blood pressure regulation and in human 28. 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The CCR4-NOT deadenylase complex controls Atg7- carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ. Res. dependent cell death and heart function. Sci. Signal. 11, https://doi.org/ 87,E1–E9 (2000). 10.1126/scisignal.aan3638 (2018). 5. Tipnis, S. R. et al. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J. Biol. Chem. 275, 33238–33243 (2000). 6. Vickers, C. et al. Hydrolysis of biological peptides by human angiotensin- Acknowledgements converting enzyme-related carboxypeptidase. J. Biol. Chem. 277, 14838–14843 We thank all members of our laboratories for technical assistance and helpful discus- (2002). sions, and we are grateful to Mrs M. Momma and Dr K. Hiwatashi for assistance in protein preparation and analysis, and to Mrs C. Inoue and Mr T. Takeda for initial 7. Crackower, M. A. et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828 (2002). animal experiments. K.K. is supported by the Kaken [17H04028] from Japanese Ministry 8. Liao, F. T., Chang, C. Y., Su, M. T. & Kuo, W. C. Necessity of angiotensin- of Science, the Takeda Science Foundation, Uehara Memorial Foundation and Daiichi converting enzyme-related gene for cardiac functions and longevity of Sankyo Foundation. Y.I. is supported by the Kaken [17H06179], T.S. is supported by the Drosophila melanogaster assessed by optical coherence tomography. J. Kaken [18K15879], and T.Y. is supported by the Kaken [18K15038] from Japanese Biomed. Opt. 19, 011014 (2014). Ministry of Science. NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z Author contributions Reprints and permission information is available at http://www.nature.com/reprints S.N., Y.I., S.T., and K.K. conceived the study. T.M., S.N., T.S., T.I., S.Y., T.G., Y.N., J.M.P., H.W., Y.I., S.T., and K.K. designed the methodology and experiments. T.M., S.N., T.S., Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in T. Yamaguchi, M.H., T.I., K.N., T. Yoshihashi, R.O., S.Y., M.N., S.K., T. Yoshiya, K.Y-K., published maps and institutional afﬁliations. S.M., S.T., and K.K. conducted experiments and/or analyzed data. K.K., T.M., S.N., and T.S. wrote the manuscript with input from all authors. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Competing interests adaptation, distribution and reproduction in any medium or format, as long as you give The authors declare no competing interests. appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party Additional information material in this article are included in the article’s Creative Commons licence, unless Supplementary information is available for this paper at https://doi.org/10.1038/s41467- indicated otherwise in a credit line to the material. If material is not included in the 020-14867-z. article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from Correspondence and requests for materials should be addressed to S.N. or K.K. the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. Peer review information Nature Communications thanks Eric Lazartigues, and other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. © The Author(s) 2020 12 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals http://www.deepdyve.com/lp/springer-journals/b38-cap-is-a-bacteria-derived-ace2-like-enzyme-that-suppresses-abr0Aw4qN0

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eISSN: 2041-1723
DOI: 10.1038/s41467-020-14867-z
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Abstract

ARTICLE https://doi.org/10.1038/s41467-020-14867-z OPEN B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction 1,13 2,13 1,3,13 1 4 Takafumi Minato , Satoru Nirasawa , Teruki Sato , Tomokazu Yamaguchi , Midori Hoshizaki , 5 2 2 1 6 1 Tadakatsu Inagaki , Kazuhiko Nakahara , Tadashi Yoshihashi , Ryo Ozawa , Saki Yokota , Miyuki Natsui , 7 8 8 9 6 Souichi Koyota , Taku Yoshiya , Kumiko Yoshizawa-Kumagaye , Satoru Motoyama , Takeshi Gotoh , 5 10,11 3 4 12 Yoshikazu Nakaoka , Josef M. Penninger , Hiroyuki Watanabe , Yumiko Imai , Saori Takahashi & 1,13 Keiji Kuba Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1–7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angio- tensin II-induced hypertension, cardiac hypertrophy, and ﬁbrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial ﬁbrosis, and car- diac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carbox- ypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure. 1 2 Department of Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. 3 4 Department of Cardiovascular Medicine, Akita University Graduate School of Medicine, Akita, Japan. Laboratory of Regulation of Intractable Infectious Diseases, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Department of Vascular Physiology, Research Institute National Cerebral and Cardiovascular Center, 6-1 Kishibe Shinmachi, Suita, Osaka 564-8565, Japan. Department of Materials Science, Applied Chemistry Course, Graduate School of Engineering Science, Akita University, 1-1 Tegatagakuen-machi, Akita 010-8502, Japan. Molecular Medicine Laboratory, Bioscience Education and Research Support Center, Akita University, 1-1-1 Hondo, Akita 010-8543, Japan. Peptide Institute, Inc., 7-2-9 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Department of Surgery, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. 10 11 IMBA -Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Campus Vienna BioCenter, Vienna 1030, Austria. Department of Medical Genetics, Life Science Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan. These authors contributed equally: Takafumi Minato, Satoru Nirasawa, Teruki Sato, Keiji Kuba. email: stnirasa@affrc.go.jp; kuba@med.akita-u.ac.jp NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 1 1234567890():,; ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z he renin–angiotensin system (RAS) has an essential role in searched published crystal structures of M32 carboxypeptidase in maintaining blood pressure (BP) homeostasis, as well as the database of MEROPS (http://merops.sanger.ac.uk/) and found 1–3 Tﬂuid and salt balance . When the RAS is activated, that three microbial M32 carboxypeptidases are reported for their angiotensin-converting enzyme (ACE) cleaves the C-terminus of three-dimensional (3D) structures, including BS-CAP (or BsuCP) deca-peptide angiotensin I (Ang I or Ang 1–10) to generate a (B. subtilis), Taq (Thermus aquaticus), and Pfu (P. furiosus). vasopressor octa-peptide angiotensin II (Ang II or Ang 1–8). Although BS-CAP, Taq, or Pfu showed low sequence identity to 4,5 ACE2 was discovered as a human homolog of ACE in 2000 . ACE2, we examined whether BS-CAP, Taq, or Pfu has structural ACE2 is a negative regulator of the RAS, which catalyzes the similarity to human ACE2 by using the program of Molecular conversion of Ang II to angiotensin 1–7 (Ang 1–7) and down- Operating Environment (Chemical Computing Group, Canada). 4–6 regulates Ang II levels, thereby counterbalancing ACE activity . The program detected only BS-CAP as a protein structurally Physiological function of ACE2 was initially identiﬁed as a related to ACE2 and the 3D structures of both proteins are mostly regulator of heart function and BP, and ACER, a ﬂy homolog of merged (Fig. 1a). Importantly, the position of key amino acids ACE2, was shown to be essential for heart morphogenesis and constituting the catalytic site (His-Glu-X-X-His motif) and 7,8 cardiac functions in ﬂies . Although activation of the RAS and substrate-binding region (Arg273/348, His345/234, His505/408, generation of Ang II worsen cardiovascular pathologies, such as and Tyr515/420 in ACE2/BS-CAP) were almost identical between cardiac ﬁbrosis and pathological hypertrophy in heart failure, the both proteins, implicating that BS-CAP may have similar sub- enzymatic activity of ACE2 exhibits a protective role in cardio- strate preference to ACE2 (Fig. 1a, Supplementary Fig. 1, and 9,10 vascular diseases . ACE2 also has protective roles to improve Supplementary Table 1). Indeed, a previous study on BS-CAP the pathologies in acute respiratory distress syndrome (ARDS)/ structure had predicted that there may be structural similarity acute lung injury and diabetic nephropathy, in which Ang II is between ACE2 and BS-CAP . We further searched for bacterial 11–13 overproduced or its signaling enhanced . Loss of ACE2 can be proteins that exhibit high sequence identity to BS-CAP in the detrimental, as it leads to progression of cardiac, renal, and pul- BLAST and found that BA-CAP, a carboxypeptidase derived from 11,14,15 monary pathologies . Treatment with recombinant human Bacillus amyloliquefaciens, is homologous to BS-CAP (Fig. 1b; ACE2 protein (rhACE2), which is devoid of its membrane- Supplementary Fig. 1; Supplementary Table 1). Furthermore, we anchored domain thus soluble, has been demonstrated to exhibit found that our recently identiﬁed bacterial strain, Paenibacillus beneﬁcial effects in various animal models including heart failure, sp. B38, also has a similar M32 carboxypeptidase to BS-CAP with 11,13,16 acute lung injury, and diabetic nephropathy, and so forth . high sequence identity (Fig. 1b, Supplementary Fig. 1, and Sup- rhACE2 is currently tested in the clinic to treat ARDS patients . plementary Table 1). We termed this Paenibacillus sp. B38- Despite its beneﬁcial effects, rhACE2 is a glycosylated protein derived M32 carboxypeptidase as B38-CAP hereafter. Despite and thus its preparation requires time- and cost-consuming pro- evolutionally distant relationship to ACE2 (Fig. 1b), these bac- tein expression system with mammalian or insect cells, which terial enzymes are likely homologs of ACE2 with divergent may not be advantageous in drug development and medical evolution. 6,18–20 economy . We prepared recombinant proteins of BS-CAP, BA-CAP, and Both ACE2 and ACE proteins belong to the M2 family of zinc- B38-CAP in the Escherichia coli protein expression system (Fig. 1c) binding metallopeptidases containing the HEXXH metal- and all of the proteins were highly expressed and soluble in E. coli coordinating motif, although the biological activities of these and easily puriﬁed with anion-exchange and gel ﬁltration two enzymes are different; ACE2 functions as a mono-carbox- chromatography (Supplementary Fig. 2a). Indeed, the production 2,4,5 ypeptidase, whereas ACE is a dipeptidyl-carboxypeptidase . of recombinant B38-CAP in E. coli (16.8 mg protein yield per Structural analyses had revealed signiﬁcant homology between culture volume (L)) was more efﬁcient in terms of the recovered ACE and a carboxypeptidase from the hyperthermophilic protein amount compared with the production of His-tagged archaeon Pfu (Pyrococcus furiosus), which is a member of the rhACE2 in baculovirus-Sf9 insect cells (5.42 mg protein yield per M32 family of carboxypeptidases belonging to the family of culture volume (L)). Moreover, the time for culture and metallopeptidases with the HEXXH active-site motif . The two puriﬁcation of B38-CAP (2 days) was shorter than that of rhACE2 enzymes share little amino acid sequence identity, yet they exhibit (6 days, not including baculovirus preparation) (Supplementary similarities in core structure and the active-site regions .In Fig. 2a–d). We ﬁrst tested whether these enzymes have ACE2-like addition, a structural similarity within the active-site region proteolytic activity to hydrolyze the ﬂuorogenic peptide Nma-His- between ACE2 and the M32 carboxypeptidase from the bacter- Pro-Lys(Dnp), which we had previously developed as a speciﬁc 21 19 ium Bacillus subtilis has been reported , suggesting that the ACE2 substrate . As a result, all the enzymes were revealed to functions might be conserved. We had previously cloned a D- catalyze the hydrolysis of the ACE2 substrate Nma-His-Pro-Lys aspartyl endopeptidase (paenidase I) from Paenibacillus sp. B38, a (Dnp) (Fig. 1d and Table 1). When we incubated Ang II peptide 22,23 new substrain of B. subtilis . Paenidase I cleaves D-α-Asp- with BS-CAP, BA-CAP, or B38-CAP in vitro, all of the enzymes containing amyloid-β peptide, which is detected in Alzheimer’s converted Ang II to Ang 1–7 (Fig. 1e and Supplementary Fig. 3a). disease, suggesting a potential application as a therapeutic . On the other hand, the dependency of ACE2-like enzymatic In this study, we show that B38-CAP, a Paenibacillus sp. B38- activity on anion (Cl ) concentration is much higher in B38-CAP derived carboxypeptidase, is an ACE2-like enzyme, which cleaves than in BS-CAP and BA-CAP (Fig. 1d), suggesting that B38-CAP both Ang I and Ang II to Ang 1–7. We show that recombinant is the most potent in ACE2-like activity under physiological B38-CAP protein downregulates Ang II levels in mice and conditions of mammals. Analysis for kinetic constants with the antagonizes Ang II-induced hypertension, pathological cardiac ﬂuorogenic ACE2 substrate revealed that B38-CAP has the same hypertrophy, and myocardial ﬁbrosis. We also show beneﬁcial potency as rhACE2 protein (Table 1 and Supplementary Fig. 3b). effects of B38-CAP on the pathology of pressure overload- Consistently, the IC values of ACE2 inhibitor (MLN-4760) for induced heart failure in mice without overt toxicities. B38-CAP and human ACE2 were almost equivalent (Table 1 and Supplementary Fig. 3c). In addition, the dependence of B38-CAP proteolytic activity on pH and temperature was also similar to that 6,19 Results of ACE2 (Supplementary Fig. 3d, e). Moreover, when various Identiﬁcation of B38-CAP as an ACE2-like enzyme. To address ACE2-substrate biological peptides were treated with B38-CAP, all whether there are any ACE2-like proteins in bacteria, we ﬁrst the peptides tested were C-terminally cleaved by B38-CAP (Table 2 2 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE a b 0.252 B38-CAP His Glu 0.195 0.090 BA-CAP 0.150 His 0.092 BS-CAP Zn Tyr 0.092 Human ACE2 0.221 His 0.047 Glu Rat ACE2 Arg 0.126 0.037 0.049 Mouse ACE2 0.05 0.328 His Fly ACER ACE2 f i 0 min BS-CAP Ang 1–9 40 min 90 min 600 100 c e Ang II + vehicle (kDa) 0246 4 68 10 Retention time (min) 0 Ang 1–7 Ang I Ang 1–9 Ang II Ang 1–7 Ang II + ACE2 h 0 102030405060708090 Ang I Ang II + B38-CAP B38-CAP Leu His Phe 600 600 BS-CAP 4 200 20 BA-CAP 0 0246 0 0.5 1 1.5 2 Retention time (min) Retention time (min) NaCl (M) 0 102030405060708090 Vehicle; B38-CAP Minutes after reaction initiation Fig. 1 B38-CAP, a bacteria-derived carboxypeptidase, is Angiotensin-converting enzyme 2 (ACE2)-like enzyme. a Crystal structures of BS-CAP and human ACE2 proteins. Inset: metal-coordinating residues (red) and substrate-binding residues (black) are shown. b Phylogenetic tree of ACE2 and bacterial ACE2-like carboxypeptidases. c SDS-PAGE analysis of recombinant proteins of BS-CAP, BA-CAP, and B38-CAP. d Dependence of ACE2-like proteolytic activity of BS-CAP, BA-CAP, and B38-CAP on anion concentration. ACE2 activity was measured with hydrolysis rate of the ﬂuorogenic ACE2 substrate Nma-His-Pro-Lys(Dnp). e–h HPLC analysis of B38-CAP-treated angiotensin peptides. Ang II (e), Ang 1–9(f), Ang 1–7(g), or Ang I (h) (5 nmol each) was incubated with vehicle, recombinant B38-CAP protein, or recombinant ACE2 protein (5 μg each) for 90 min, then subjected to HPLC analysis. i, j Kinetic analysis for hydrolysis of Ang I with B38-CAP. HPLC analysis of angiotensin peptides generated after incubating Ang I with B38-CAP (i, j, upper panel). Amino acids in the same samples were quantiﬁed with LC-MS system (j, lower panel). Experiments were repeated more than three times and representative chromatography charts are shown. j Values are means ± SEM. n = 3 independent experiments. increased and ﬁnally reached the same levels of the initial Ang I Table 1 Kinetic constants for hydrolysis of ACE2 substrate amount, whereas Ang I was undetectable at 90 min (Fig. 1i, j). On by B38-CAP and IC of MLN-4760, an ACE2 inhibitor. the other hand, Ang 1–9 and Ang II exhibited a minor peak at 20 min and 60 min, respectively, and both peptides became −1 −1 −1 K (μM) k (s ) k /K (s μM )IC (pM) m cat cat m 50 undetectable at the end of the reaction period (Fig. 1j). Consistent B38-CAP 23.3 ± 1.70 188 ± 6.87 8.07 ± 0.295 710 ± 173 with peptide kinetics, the amino acids leucine (Leu), histidine ACE2 23.8 ± 1.91 168 ± 6.18 7.08 ± 0.260 340 ± 50.1 (His), and phenylalanine (Phe) were generated in the same order as mono-carboxyl proteolysis of Ang I, Ang 1–9, and Ang II, respectively (Fig. 1j), indicating that the conversion of Ang I to Ang 1–7 by B38-CAP is mediated through three steps of mono- and Supplementary Fig. 4a). For non-ACE2-substrate peptides carboxyl proteolysis. Therefore, B38-CAP has an ACE2-like Ang 1–7 and angiotensin 1–9(Ang 1–9), however, B38-CAP did activity, which converts both Ang II and Ang I peptides to Ang cleave Ang 1–9, whereas it did not affect Ang 1–7 (Fig. 1f, g and 1–7invitro. Table 2). Consistently, B38-CAP converted Ang I to Ang 1–7 (Fig. 1h), which is distinct from ACE2 conversion of Ang I to Ang 1–9 . To address how B38-CAP converts Ang I to Ang 1–7, we B38-CAP suppresses Ang II-induced cardiovascular pathology. conducted kinetic analysis (Fig. 1i, j and Supplementary Fig. 4b). To examine the effects of B38-CAP in vivo, we ﬁrst injected B38- Ang 1–9, Ang II, and Ang 1–7 peptidesweredetectableat 10min CAP into mice and measured plasma enzymatic activity as after mixing Ang I with B38-CAP (Fig. 1j). Ang 1–7production indicative of the plasma B38-CAP levels by using a newly NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 3 Marker BS-CAP BA-CAP B38-CAP Relative activity Arbitrary units Arbitrary units Arbitrary units Phe Ang 1–7 Ang II Arbitrary units Arbitrary units Arbitrary units Phe Phe Ang 1–7 Ang 1–7 Ang 1–7 Ang 1–9 Ang I Arbitrary units Amino acids (nmol) Peptides (nmol) Phe Ang 1–7 Ang 1–9 Ang II Ang I ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z 1–7 levels in the plasma of these mice. Ang 1–7 levels in the Table 2 Hydrolysis of ACE2-substrate peptides by B38-CAP. plasma were signiﬁcantly upregulated in both acute and chronic experimental settings (Fig. 2k, l). On the other hand, when we co- Substrate ACE2* B38-CAP Sequence treated the Ang II-injected mice with B38-CAP and A779, an Angiotensin I ++ DRVYIHPFH↓L antagonist for Mas/Ang 1–7 receptor, the suppressive effects of Angiotensin 1–9 − + DRVYIHPF↓H B38-CAP on Ang II-induced elevation of BP were not affected Angiotensin II ++ DRVYIHP↓F (Fig. 2m and Supplementary Fig. 9a–d). Furthermore, co- Angiotensin 1–7 −− DRVYIHP treatment of Ang 1–7 and Ang II did not downregulate Ang II- Apelin-13 ++ QRPRLSHKGPMP↓F induced elevation of BP (Supplementary Fig. 9e–h). Thus, the Apelin-36 ++ …QRPRLSHKGPMP↓F 9 hypotensive effects of B38-CAP are primarily mediated through des-Arg -bradykinin++ RPPGFSP↓F downregulation of Ang II levels. Lys-des-Arg - ++ KRPPGFSP↓F As chronic infusion of Ang II induces cardiac hypertrophy and bradykinin β-Casomorphin ++ YPFVEP↓I ﬁbrosis, we examined the hearts of mice chronically treated with −1 Dynorphin A 1–13 ++ YGGFLRRIRPKL↓K high-dose Ang II (1.5 mg kg per day) with or without B38-CAP −1 Ghrelin ++ …ESKKPPAKLQP↓R (3 mg kg per day) for 2 weeks. B38-CAP suppressed Ang II- Neurotensin 1–8 ++ pE-LYENKP↓R induced cardiac hypertrophy and increase of heart weight (HW) as measured with HW-to-body weight ratios (HW/BW) or HW Summary of cleavability of peptides by B38-CAP. “+” indicates that it is cleaved with ACE2 or B38-CAP, whereas “−” means it is not cleaved. HPLC analyses for the metabolites of each to tibia length (HW/TL) (Fig. 3a–c and Supplementary Table 2). peptide after B38-CAP treatment are shown in Supplementary Fig. 4a. *Cleavability of the Consistently, Ang II-induced wall thickening of the hearts was peptides by ACE2 is from ref. . signiﬁcantly downregulated by B38-CAP treatment as shown by echocardiography (Fig. 3d, e and Supplementary Table 2). In developed B38-CAP-speciﬁc substrate Nma-Leu-Pro-Lys(Dnp). addition, mild decrease of cardiac contractility in Ang II-infused −1 In 1 h after intraperitoneal (i.p.) injection of B38-CAP (2 mg kg mice were prevented by B38-CAP treatment as determined by % i.p.), the concentration of B38-CAP in plasma was markedly fractional shortening (%FS) (Fig. 3f and Supplementary Table 2). increased and peaked (Fig. 2a). The plasma B38-CAP levels Moreover, B38-CAP signiﬁcantly prevented Ang II-induced gradually decreased to almost baseline at 8 h but it was still cardiac ﬁbrosis (Fig. 3g, h) and upregulation of ﬁbrotic genes detectable in the plasma through 12 h (Fig. 2a). Estimated initial expression (Collagen 8a (Col8a1), Periostin (Postn), and TGF-β2 half-life in systemic circulation was 3.5 h, which is almost similar (Tgfb2)) (Fig. 3i–k). Similar results were obtained when B38-CAP 25,26 to 1.8–8.5 h of rhACE2 . We next examined whether B38- was daily i.p. injected for 4 weeks to Ang II-infused mice CAP treatment affects Ang II-induced elevation of BP. We ﬁrst (Supplementary Fig. 7f–n and Supplementary Table 3). These measured the BP in the carotid artery in anesthetized mice using results demonstrate that B38-CAP suppresses Ang II-induced a transducer catheter (Fig. 2b). Intraperitoneal injection of Ang II hypertension, cardiac hypertrophy, and ﬁbrosis. −1 (0.2 mg kg i.p.) induced acute elevation of arterial BP in wild- −1 type mice, whereas pretreatment of B38-CAP (2 mg kg i.p.) signiﬁcantly suppressed the Ang II-induced elevation of arterial B38-CAP mitigates pressure overload-induced heart failure. pressure (Fig. 2c and Supplementary Fig. 5a–c). We next We further treated the mice under pressure overload cardiac stress addressed the effects of B38-CAP on hypertension induced induced by transverse aortic constriction (TAC) with continuous −1 by chronic Ang II treatment. Although continuous infusion of per day), which was initiated infusion of B38-CAP (2 mg kg Ang II with an osmotic mini-pump elevated BP as measured in immediately after TAC surgery (Fig. 4a). After 2 weeks of TAC, conscious mice, daily i.p. injection of B38-CAP downregulated the HW (HW/BW or HW/TL) was also signiﬁcantly decreased in elevation of BP at days 8, 14, and 28 (Supplementary Fig. 6a–e the B38-CAP-treated group as compared with vehicle-treated and Supplementary Fig. 7a–e). Furthermore, we tested whether controls (Fig. 4b–d). In addition, pulmonary congestion was continuous infusion of B38-CAP suppresses Ang II-induced suppressed by B38-CAP as determined by lung weight to BW ratio hypertension (Fig. 2d). When B38-CAP was infused sub- (LW/BW) and LW/TL ratio (Fig. 4e, f). Echocardiography also cutaneously using an osmotic mini-pump, B38-CAP was detect- showed that wall thickening was signiﬁcantly downregulated by able in the plasma of mice for 14 days (Supplementary Fig. 8a, b). B38-CAP treatment (Fig. 4g–i and Supplementary Table 4). In Although it had been reported that an immune response is addition, although %FS was signiﬁcantly decreased in the vehicle associated with the chronic infusion of rhACE2 resulting in the treatment group, %FS was preserved in B38-CAP-treated mice degradation of rhACE2 , this was not observed for B38-CAP; (Fig. 4g–l). Similarly, B38-CAP treatment signiﬁcantly suppressed there were no antibodies against B38-CAP detectable in the the increased expression of mRNA associated with cardiac serum of mice infused with B38-CAP for 2 weeks (Supplementary hypertrophy, such as ANF (atrial natriuretic factor), BNP (brain Fig. 8c). Implantation of B38-CAP-ﬁlled osmotic mini-pumps natriuretic peptide), and β-myhc (β-myosin heavy chain, Myh7) in signiﬁcantly suppressed Ang II-induced hypertension in con- the TAC mice (Fig. 5c–e). scious mice (Fig. 2e–g) without affecting the heart rate (Fig. 2h). Histological analysis further revealed that B38-CAP treatment These results indicate that B38-CAP antagonizes the vasopressor reduced the area of cardiac ﬁbrosis in the interstitial space and effect of Ang II. perivascular region in the hearts of TAC mice (Fig. 5a, b). We further examined the effects of B38-CAP treatment on Ang Consistently, although the expression of the pro-ﬁbrotic genes II levels in the blood. In the acute experiment with i.p. injection of Col8a1, Postn, and Tgfb2 were increased in the hearts of vehicle- Ang II (Fig. 2b), pretreatment of B38-CAP markedly down- treated mice with TAC, B38-CAP markedly downregulated the regulated a massive increase of plasma Ang II levels at 5 min after expression of those pro-ﬁbrotic genes (Fig. 5f–h). These results Ang II injection (Fig. 2i). Consistently, in the chronic experiment indicate that exogenous B38-CAP treatment protects mice from with continuous infusion of Ang II (Fig. 2d), continuous infusion pressure overload-induced cardiac dysfunction, hypertrophy, and of B38-CAP with additional osmotic pump signiﬁcantly ﬁbrosis. Furthermore, we examined whether any potential toxic decreased Ang II levels in the plasma at day 14 (Fig. 2j). As effects of B38-CAP on the liver and kidneys, and both serum Ang 1–7 is known to exert beneﬁcial effects in the cardiovascular markers of liver injury and kidney dysfunction, were not affected systems through Mas/Ang 1–7 receptor , we also measured Ang by B38-CAP, as measured with aspartate transaminase (AST) or 4 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE ab c Ang II + vehicle Ang II + B38-CAP B38-CAP i .p. Ang II i .p. or vehicle i .p. or vehicle i .p. <0.0001 –90 –10 0 5 10 20 30 (min) Invasive BP measurements 02468 10 12 Baseline 5 10 15 20 Ang II measurements Hours after B38-CAP Minutes after Ang II i .p. injection i .p . injection de fh <0.0001 <0.0001 g <0.0001 <0.0001 <0.0001 <0.0001 190 130 150 800 110 130 Continuous B38-CAP infusion 90 110 Continuous Ang II infusion 130 400 70 90 0 2 4 6 8 10 12 14 (days) 50 70 BP, echo, Ang II 70 30 50 0 B38-CAP: – + – + B38-CAP: –+ – + measurements B38-CAP: – + – + B38-CAP: – + – + Vehicle Ang II Vehicle Ang II Vehicle Ang II Vehicle Ang II ik 0.0001 0.0011jl m 0.0004 0.0009 0.0040 1400 800 400 0.0007 0.0004 170 n.s. 0.0262 <0.0001 0.0082 1200 250 600 300 800 100 400 50 200 100 0 0 0 0 70 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: –+ – + B38-CAP: –+ – + Ang II: – –– – + ++ + Vehicle Ang II B38-CAP: Vehicle Ang II Vehicle Ang II Vehicle Ang II – + – + –– + + A779: – – + +–+ – + Fig. 2 Effects of B38-CAP on plasma angiotensin II levels and blood pressure in mice. a Plasma B38-CAP levels in mice after intraperitoneal injection −1 of B38-CAP (2 mg kg ). B38-CAP activity was measured with the B38-CAP substrate Nma-Leu-Pro-Lys(Dnp) (n = 4, 6, 8, 6, 6, and 6 for 0, 1, 2, 4, 8, and −1 12 h, respectively). b, c Invasive measurements of arterial blood pressure (BP). Experimental protocol (b); mice pretreated with B38-CAP (2 mg kg i.p.) −1 had liquid-ﬁlled catheter inserted into carotid artery for BP measurements and Ang II (0.2 mg kg i.p.) was injected and BP measured. Systolic arterial pressure is shown (c). (n = 6 mice per group). d–h Blood pressure measurements with conscious mice. Experimental protocol (d); mice were treated with −1 −1 −1 −1 continuous infusion of vehicle, Ang II (1.5 mg kg per day), B38-CAP (3 mg kg per day), or Ang II (1.5 mg kg per day) plus B38-CAP (3 mg kg per day), and BP was measured by tail-cuff system after 2 weeks. Systolic (e), diastolic (f), and mean (g) BP and heart rate (h) are shown for mice treated vehicle+ vehicle (n = 10), vehicle+ B38-CAP (n = 11), Ang II + vehicle (n = 11), and Ang II + B38-CAP (n = 11). i–l Measurements of Ang II and Ang 1–7in the plasma of mice. The plasma was obtained from the mice treated acutely (i, k) and chronically (j, l) with Ang II, in the cohort of b, c, and d–h, respectively. Ang II (i, j) and Ang 1–7(k, l) levels were measured with ELISA. m BP measurements in conscious mice. The mice pretreated with B38-CAP −1 −1 (2 mg/kg i.p.) at 90 min before acquisition of baseline BP were treated with Ang II (0.2 mg kg i.p.), with or without A779 (0.2 mg kg i.p.). BP was measured every 5 min by tail-cuff system (Supplementary Fig. 9a–d) and the BP at 5 min after the last injection is shown (n = 6 mice per group). All values are means ± SEM. c, e–m, Two-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. alanine transaminase (ALT) and blood urea nitrogen (BUN) or dysfunction with signiﬁcant decrease of %FS (Fig. 7f, g). The Creatinine (Cr), respectively (Fig. 6a–d). In addition, the decrease osmotic mini-pumps containing B38-CAP or vehicle were −1 of BW due to TAC heart failure was prevented by B38-CAP implanted at this time point and B38-CAP (2 mg kg per day) treatment (Supplementary Table 4). These results suggest that was infused for 2 weeks. Treatment with B38-CAP signiﬁcantly B38-CAP does not exhibit overt side effects in mice for at least increased %FS as compared with that in vehicle-treated mice or in 2 weeks after treatment. the mice before treatment (Fig. 7g and Table 3), indicating that B38-CAP improved cardiac dysfunction. In addition, cardiac hypertrophy was signiﬁcantly suppressed by B38-CAP treatment B38-CAP improves established hypertension and heart failure. (Fig. 7h–j) and pulmonary congestion was also improved by B38- To determine therapeutic effects of B38-CAP in established dis- CAP (Fig. 4k, l). Consistently, B38-CAP treatment signiﬁcantly eases, we ﬁrst examined whether B38-CAP treatment improve downregulated increased expression of mRNA associated with the established hypertension in the mice, which have received Ang II pathology of cardiac hypertrophy (BNP and β-myhc) and ﬁbrosis infusion prior to the treatment (Fig. 7a). When BP was elevated (Col8a1, Postn, and Tgfb2) (Fig. 7m–q). Furthermore, we chal- after 7 days of Ang II infusion, daily i.p. injection of B38-CAP lenged B38-CAP to severe cardiac dysfunction in C57BL/6N was initiated (Fig. 7a). B38-CAP treatment signiﬁcantly down- mice under TAC, in which the mice exhibit profound decline of regulated Ang II-induced increase of BP to the levels in vehicle- cardiac contractility at 2 weeks after TAC (Fig. 4l and Supple- treated control mice (Fig. 7b–e). Thus, B38-CAP ameliorated mentary Fig. 10a–c). When B38-CAP treatment was started at established hypertension. Next, we investigated the effects of 2 weeks after TAC, B38-CAP markedly suppressed progression of B38-CAP on established cardiac dysfunction. At 5 weeks after heart failure in C57BL/6N mice (Supplementary Fig. 10b–o and TAC surgery, the mice (C57BL/6J background) exhibits cardiac NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 5 Plasma Ang II (pg/ml) Plasma B38-CAP (ng/ml) Plasma Ang II (pg/mL) Systolic BP (mmHg) Plasma Ang 1–7 (pg/ml) Diastolic BP (mmHg) Plasma Ang 1–7 (pg/ml) Systolic arterial pressure (mmHg) Mean BP (mmHg) Systolic BP (mmHg) Heart rate (bpm) ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ab c <0.0001 <0.0001 Vehicle Ang II <0.0001 <0.0001 Vehicle B38-CAP Vehicle B38-CAP 7 4 3 B38-CAP: –+ – + B38-CAP: – ++ – Vehicle Ang II Vehicle Ang II de fg Vehicle Ang II + vehicle Ang II + B38-CAP 1.2 1.2 <0.0001 <0.0001 <0.0001 <0.0001 0.0131 0.0343 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 20 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Vehicle Ang II Vehicle Ang II Vehicle Ang II hi <0.0001 0.0207 j k 0.0005 0.0454 4 8 2.0 <0.0001 0.0712 <0.0001 0.0002 3 6 1.5 2 4 1.0 1 2 0 0 0 0.5 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Vehicle Ang II Vehicle Ang II Vehicle Ang II Vehicle Ang II Fig. 3 Effects of B38-CAP on angioteinsin II-induced cardiac hypertrophy and ﬁbrosis. a–c Cardiac hypertrophy in the mice chronically co-treated with −1 −1 Ang II (1.5 mg kg per day) and B38-CAP (3 mg kg per day) in the cohort of Fig. 2d–h. Macroscopic heart images (a), heart weight to body weight ratio (HW/BW) (b), and heart weight to tibia length ratio (HW/TL) (c) in mice treated vehicle+ vehicle (n = 10), vehicle + B38-CAP (n= 11), Ang II + vehicle (n = 11), and Ang II+ B38-CAP (n = 11). Bars indicate 2 mm. d–f Echocardiography parameters of left ventricular end-diastolic posterior wall thickness (PWD) (d), end-diastolic interventricular septal wall thickness (IVSD) (e), and %fractional shortening (%FS) (f) in the mouse hearts. Complete echocardiography data are shown in Supplementary Table 2. g, h Histology of hearts. Masson’s trichrome staining (g); bars indicate 2 mm and 100 μmin the upper panels and lower panels, respectively. Quantiﬁcation of ﬁbrosis in the hearts (h) of mice treated with vehicle+ vehicle (n = 5), vehicle+ B38- CAP (n = 5), Ang II + vehicle (n = 7), and Ang II + B38-CAP (n = 8). i–k qRT-PCR analysis of pro-ﬁbrotic gene expressions in the hearts of mice treated with vehicle+ vehicle (n = 5), vehicle+ B38-CAP (n = 5), Ang II + vehicle (n = 7), and Ang II + B38-CAP (n = 8); mRNA levels of Collagen 8a (Col8a1) (f), Periostin (Postn)(g), and TGF-β (Tgfb2)(h) normalized with Gapdh. All values are means ± SEM. b–f, h–k Two-way ANOVA with Sidak’s multiple- comparisons test. Numbers above square brackets show signiﬁcant P-values. Supplementary Table 5), albeit mainly through decrease of LV similar proteolytic activity to rhACE2, there seems a difference in dimensions rather than wall thickness. These results indicate substrate speciﬁcity between two enzymes. B38-CAP is likely to that B38-CAP exerts therapeutic effects in established cardiac have more broad speciﬁcity for proteolytic effects on peptides, dysfunction. because B38-CAP converted Ang I and Ang 1–9 into Ang 1–7, whereas ACE2 does not cleave Ang 1–9. The superimposition of ACE2 and BS-CAP structures indicated that the positions of Discussion substrate-binding amino acid residues and metal-binding residues In this study, we elucidated that bacteria-derived carbox- are matched. Although the ACE2 substrate Nma-His-Pro-Lys ypeptidases have ACE2-like enzymatic activity and showed that (Dnp) was cleaved by B38-CAP with the same potency as ACE2, B38-CAP cleaves Ang II and Ang I to Ang 1–7 and down- we found that the Nma-Leu-Pro-Lys(Dnp) was catalyzed speci- regulates Ang II levels in mice. We demonstrated that Ang II- ﬁcally by B38-CAP but not by ACE2 (not shown), suggesting that induced hypertension, cardiac hypertrophy, and ﬁbrosis were the S2-subsite of B38-CAP is more hydrophobic than ACE2. The suppressed by B38-CAP treatment. We further showed that B38- difference in substrate speciﬁcity of B38-CAP and ACE2 should CAP improves cardiac dysfunction, hypertrophy, and ﬁbrosis be further elucidated by our ongoing analysis for crystal structure induced by pressure overload in mice. of B38-CAP. Among three bacterial carboxypeptidases we tested, only B38- Although the hypotensive action of B38-CAP is mediated CAP showed dependence of proteolytic activity on anion con- −1 mainly through Ang II downregulation, B38-CAP (2 ~ 3 mg kg centration, which is characteristic of ACE2 activity . B38-CAP 6,19 per day) exhibits a more potent therapeutic effect in TAC- also showed pH optimum of 7.5 equivalent to rhACE2 . induced heart failure than in Ang II-induced heart failure (e.g., In addition, IC of MLN-4760 was also equivalent between 87.9% inhibition of cardiac hypertrophy (HW/BW increase) in rhACE2 and B38-CAP. Although B38-CAP exhibited quite 6 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications PWD (mm) %area of fibrosis IVSD (mm) Col8a1/Gapdh %FS Postn/Gapdh HW/BW (mg/g) Tgfb2/Gapdh HW/TL (mg/mm) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE ac b d Sham TAC <0.0001 <0.0001 <0.0001 <0.0001 Vehicle B38-CAP Vehicle B38-CAP Continuous B38-CAP infusion 8 0 7 14 (days) 6 TAC Echo, sampling B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC ef 0.0167 0.0105 g 0.0162 0.0089 Sham TAC 20 20 Vehicle B38-CAP Vehicle B38-CAP 15 15 0 0 B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC h ij k l <0.0001 <0.0001 1.2 1.2 4 4.5 70 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0003 4.0 1.0 1.0 3 3.5 0.8 0.8 2 40 3.0 0.6 0.6 1 2.5 0.4 0.4 0 2.0 10 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC Sham TAC Sham TAC Sham TAC Fig. 4 B38-CAP mitigates pressure overload (TAC)-induced cardiac dysfunction and hypertrophy. a Experimental protocol. The mice were subjected to −1 the surgery of transverse aortic constriction (TAC) surgery and then continuous infusion of B38-CAP (2 mg kg per day) was initiated. b–f B38-CAP suppressed cardiac hypertrophy. Representative photograph (b) of the hearts of mice under TAC. Bars indicate 2 mm. HW/BW (c), HW/TL (d), lung weight to body weight ratio (LW/BW) (e), and lung weight to tibia length ratio (LW/TL) (f) in the mice treated with sham + vehicle (n = 7), sham+ B38- CAP (n= 5), TAC + vehicle (n = 9), and TAC + B38-CAP (n = 8). g–l Echocardiography measurements. Representative M-mode echocardiography images (g), measurements of IVSD (h), PWD (i), LVESD (j), LVEDD (k), and %FS (l) are shown. Complete echocardiography data are shown in Supplementary Table 4. All values are means ± SEM. c–f, h–l One-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. TAC model vs. 53.2% inhibition in Ang II-infusion model). On therapeutic strategy in cardiovascular disease and other Ang II- the other hand, in a previous study, rhACE2 showed similar anti- related diseases, e.g. ARDS. On the other hand, although mass hypertrophic effects in both TAC and Ang II-infusion models . production of rhACE2 as a protein drug costs due to requirement The difference may be explained by the slight difference of sub- of mammalian cell expression systems, B38-CAP is easily pre- strate speciﬁcity of B38-CAP and ACE2. Although ACE2 con- pared with E. coli expression system and is cost effective. Ther- verts Ang I to Ang 1–9 inefﬁciently and requires ACE for further apeutic efﬁcacy and less toxicity of B38-CAP in mouse heart conversion of Ang 1–9 to Ang 1–7 , B38-CAP targets all the Ang failure models would warrant further investigation of B38-CAP or I, Ang 1–9, and Ang II peptides as a mono-carboxypeptidase. other microbial carboxypeptidases in disease models. Further- Thus, the conversion of Ang I and Ang II to Ang 1–7 by B38- more, human ACE2-like enzyme in bacteria might pave the way CAP may contribute to more efﬁcient down-modulation of RAS to a new strategy to engineer evolution of bacterial proteins for in the TAC heart failure. In addition, Ang 1–7 is a vasoprotective better designing and preparations of recombinant protein drugs. peptide which acts through its cognate Mas receptor, and also has anti-ﬁbrotic and cardioprotective functions in heart failure . Methods Thus, B38-CAP-mediated degradation of Ang I into Ang 1–7 Searching for bacteria-derived ACE2-like enzymes. Sequence comparison was would be beneﬁcial in enhancing Ang 1–7 generation for treating performed using BLAST and MEROPS (http://merops.sanger.ac.uk/) tools avail- failing hearts. Furthermore, as ACE2 and B38-CAP target other able online. Similarity search and superposition of a 3D structure of proteins was performed by Molecular Operating Environment (MOE 2016.08; Chemical Com- biological peptides than angiotensin peptides, subtle differences puting Group, Inc., Montreal, QC, Canada). Multiple sequence alignment between in substrate speciﬁcity may license B38-CAP to degrade such enzyme sequences was performed using the CLUSTALW tool available online. peptides in a different manner or even to have a new peptide Phylogenetic tree drawing was performed using NJplot program (http://doua.prabi. substrate different from ACE2 targets. fr/software/njplot). In addition to the currently used drugs to inhibit Ang II gen- eration or signaling, such as ACE inhibitors or Angiotensin Recombinant proteins. Genomic DNAs were isolated from Paenibacillus sp. receptor blockers, direct down-modulation of Ang II levels by 22 B38 , B. subtilis subsp. subtilis NBRC 13719, and B. amyloliquefaciens NBRC 3022 rhACE2 protein is one of the promising candidates for new (Supplementary Table 1). Expression plasmids, which encode B38-CAP, BS-CAP, NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 7 IVSD (mm) LW/BW (mg/g) PWD (mm) LW/TL (mg/mm) LVESD (mm) LVEDD (mm) HW/BW (mg/g) %FS HW/TL (mg/mm) ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z a b Sham TAC <0.0001 <0.0001 Vehicle B38-CAP Vehicle B38-CAP B38-CAP: –+ – + Vehicle TAC cd e 0.0012 0.0319 <0.0001 <0.0001 0.0003 <0.0001 15 120 10 3 5 40 0 0 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC Sham TAC fg <0.0001 <0.0001 <0.0001 <0.0001 h <0.0001 <0.0001 8 4 6 3 4 2 2 1 0 0 0 B38-CAP: – + – + B38-CAP: – + – + B38-CAP: – + – + Sham TAC Sham TAC Sham TAC Fig. 5 B38-CAP suppresses TAC-induced cardiac ﬁbrosis. a, b Histology. The hearts of B38-CAP or vehicle-treated mice under TAC were stained with Masson’s trichrome. Bars indicate 1 mm (upper) or 100 μm (lower). c–h qRT-PCR analysis for the expression of heart failure genes and pro-ﬁbrosis genes; mRNA levels of atrial natriuretic factor (ANF)(c), B-type natriuretic peptide (BNP)(d), β-myosin heavy chain (β-myhc)(e), Collagen 8a (Col8a1)(f), Periostin (Postn)(g), and TGF-β (Tgfb2)(h) in the hearts of mice treated with sham+ vehicle (n = 7), sham+ B38-CAP (n= 5), TAC + vehicle (n = 9), and TAC + B38-CAP (n= 8). All values are means ± SEM. b–h Two-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. ab c d 150 50 40 0.6 0.0103 100 0.4 0.2 0 0 0 0.0 B38-CAP–+ – + B38-CAP–+ – + B38-CAP–+ – + B38-CAP –+ – + Sham TAC Sham TAC Sham TAC Sham TAC Fig. 6 No overt toxic effects of B38-CAP on liver and kidney. a, b Liver function test with measurements of aspartate aminotransferase (AST) (a) and alanine aminotransferase (ALT) (b) in the blood. c, d Kidney function assessment with measurements of BUN (c) and Creatinine (Cr) (d) in the blood. sham+ vehicle (n = 7), sham+ B38-CAP (n = 5), TAC + vehicle (n = 9), and TAC + B38-CAP (n = 8). All values are means ± SEM. Two-way ANOVA with Sidak’s multiple-comparisons test. Numbers above square brackets show signiﬁcant P-values. or BA-CAP, were constructed by PCR. PCR products were ligated into a XbaI- and (2.6 × 60 cm; GE Healthcare) (Supplementary Fig. 2a). To exclude potential con- XhoI-double-digested pET28a plasmid, and recombinant proteins were generated tamination of endotoxin, the eluates were further passed through a Polymyxin B by isopropyl β-D-thiogalactopyranoside (IPTG) induction of E. coli. Cells were column. For preparation of recombinant human ACE2 in Sf9 insect cells, human collected and the cell lysate prepared and centrifuged at 13,000 × g for 15 min. The ACE2 cDNA was inserted into the XbaI and KpnI sites of pFastBac1 vector resulting supernatant was subjected to ammonium sulfate precipitation, anion- (Invitrogen) and the generated recombinant bacmid DNA was transfected into Sf9 exchange chromatography with a Q-Sepharose Fast Flow column (1.6 × 10 cm; GE cells using Cellfectin (Invitrogen) to construct recombinant baculovirus encoding Healthcare), and gel ﬁltration chromatography with a Superdex 75 pg column human ACE2. Sf9 cells were infected with the recombinant baculovirus at a 8 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications AST (U/l) ANF/Gapdh Col8a1/Gapdh ALT (U/l) Postn/Gapdh BNP/Gapdh BUN (mg/dl) -myhc/Gapdh Tgfb2/Gapdh %area of fibrosis Cr (mg/dl) NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE Ang II + vehicle Ang II + vehicle Ang II + vehicle Ang II + vehicle ab c d e Daily B38-CAP Ang II + B38-CAP Ang II + B38-CAP Ang II + B38-CAP Ang II + B38-CAP i .p.injection 800 160 Vehicle + vehicle Vehicle + vehicle Vehicle + vehicle Vehicle + vehicle No treatment Ang II infusion <0.0001 0.0003 <0.0001 100 <0.0001 120 80 –7 0 2 4 6 (days) BP measurements 60 200 0246 0246 0246 0246 Days after treatment Days after treatment Days after treatment Days after treatment fg <0.0001 h ij TAC 0.0001 0.0250 Sham 60 10 <0.0001 0.0129 <0.0001 + vehicle Vehicle B38-CAP B38-CAP 0.0010 No treatment infusion 8 –5 0 2 (weeks) TAC Echo Echo, C57BL/6J sampling mice TAC: – + + TAC: – + + Weeks: 0 2 02 2 0 B38-CAP: – – + B38-CAP: – – + Vehicle Vehicle B38-CAP + sham TAC kl m 0.0011 0.0191n 0.0048 0.0137 o p 0.0027 0.0029q 0.0029 0.0003 8 3 8 4 4 2.0 <0.0001 <0.0001 0.0044 0.0118 0.0039 0.0137 6 3 3 1.5 6 4 2 2 1.0 2 1 1 0.5 0.0 3 0 0 0 0 TAC:–+ + TAC:–+ + TAC:–+ + TAC:–+ + TAC:–+ + TAC:–+ + TAC: – + + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + B38-CAP: – – + Fig. 7 Therapeutic effects of B38-CAP on established hypertension and cardiac dysfunction. a–e Therapeutic effects of B38-CAP on established hypertension. Experimental protocol (a); Ang II infusion (1 mg/kg/day) was initiated at 7 days before treatment. The mice were injected with B38-CAP (2 mg/kg i.p.) or vehicle twice a day and blood pressure was measured by tail-cuff system at 2 h after injection. Systolic (b), diastolic (c), and mean (d)BP and heart rate (e) in the mice treated Ang II+ vehicle (n= 7), Ang II+ B38-CAP (n= 7), and vehicle+ vehicle (n= 5). f–l Therapeutic effects of B38-CAP on established cardiac dysfunction. Experimental protocol (f); the C57BL/6J mice had TAC surgery at 5 weeks before treatment and B38-CAP (2 mg/kg/day) or vehicle was continuously infused with osmotic mini-pumps. Echocardiography parameters of %fractional shortening (%FS) (g) in the mice treated with sham+ vehicle (n= 5), TAC + vehicle (n= 6), and TAC+ B38-CAP (n= 6). Representative photographs of the hearts of mice under TAC (h). Bars indicate 2 mm. HW/BW (i), HW/TL (j), LW/BW (k), and LW/TL (l) are in the mice treated with sham+ vehicle (n= 5), TAC+ vehicle (n= 6), and TAC+ B38- CAP (n= 6). m–q qRT-PCR analysis for the expression of heart failure genes and pro-ﬁbrosis genes in the hearts (n= 5 mice per group). All values are means ± SEM. b–e Two-way ANOVA with Sidak’s multiple-comparisons test. g One-way ANOVA with Sidak’s multiple-comparisons test for comparison of groups. Two-tailed paired t-test between before and after treatment of the same group. i–q One-way ANOVA with Sidak’s multiple-comparisons test. Numbers next to square brackets show signiﬁcant P-values. Table 3 Echocardiographic parameters in the mice with established cardiac dysfunction treated with B38-CAP for 2 weeks. Sham + vehicle Sham+ vehicle TAC + vehicle TAC + vehicle TAC + B38-CAP TAC + B38-CAP Before treatment After treatment Before treatment After treatment Before treatment After treatment N 55 6 6 6 6 Age (weeks) 15 17 15 17 15 17 BW (g) 23.14 ± 1.24 25.82 ± 1.03 23.12 ± 0.87 25.92 ± 0.74 23.55 ±± 0.83 24.30 ± 1.16 HR (bpm) 594 ± 49 631 ± 68 551 ± 51 559 ± 49 570 ± 65 644 ± 61 ### †† FS (%) 51.78 ± 4.09 51.91 ± 1.56 29.49 ± 2.79*** 27.20 ± 1.43 29.06 ± 2.36*** 36.38 ± 2.03 ### †† EF (%) 76.70 ± 3.70 83.89 ± 1.27 57.44 ± 4.5*** 53.56 ± 2.28 56.70 ± 3.54*** 67.03 ± 2.86 ## † LVESD (mm) 1.54 ± 0.15 1.54 ± 0.13 2.43 ± 0.38*** 2.75 ± 0.14 2.49 ± 0.23*** 2.15 ± 0.17 LVEDD (mm) 3.20 ± 0.08 3.21 ± 0.22 3.44 ± 0.45 3.77 ± 0.17 3.51 ± 0.34 3.38 ± 0.24 IVSD (mm) 0.74 ± 0.04 0.79 ± 0.03 1.05 ± 0.17* 1.04 ± 0.05 1.09 ± 0.05*** 0.99 ± 0.07 PWD (mm) 0.78 ± 0.06 0.89 ± 0.13 1.11 ± 0.12*** 0.99 ± 0.05 1.03 ± 0.06*** 1.08 ± 0.09 Results are presented as mean ± SEM. One-way ANOVA plus Sidak’s multiple-comparisons test was used to detect signiﬁcance. BW body weight, FS left ventricular fractional shortening, EF left ventricular ejection fraction, HR heart rate, IVSD end-diastolic interventricular septal wall thickness, LVEDD left ventricular end-diastolic diameter, LVESD left ventricular end-systolic diameter, PWD left ventricular end-diastolic posterior wall. *P < 0.05 vs. sham + vehicle before treatment; **P < 0.001 vs. sham + vehicle before treatment; ***P < 0.0001 vs. sham + vehicle before treatment; #P < 0.05 vs. TAC + vehicle after treatment; ##P < 0.001 vs. TAC+ vehicle after treatment; ###P < 0.0001 vs. TAC+ vehicle after treatment. Two-tailed paired t-test was used to detect signiﬁcance. †P < 0.05 vs. TAC+ B38-CAP before treatment; ††P < 0.001 vs. TAC+ B38-CAP before treatment. NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 9 LW/BW (mg/g) LW/TL (mg/mm) Systolic BP (mmHg) %FS BNP/Gapdh Diastolic BP (mmHg) -myhc/Gapdh Col8a1/Gapdh Mean BP (mmHg) HW/BW (mg/g) Postn/Gapdh HW/TL (mg/mm) Heart rate (bpm) Tgfb2/Gapdh ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z multiplicity of infection (MOI) of 1 pfu/cell and then cultured in SF-900 II serum- (Fig. 7a–e) or %FS for the experiments of TAC-induced cardiac dysfunction free medium (Invitrogen) using 250 mL shaker ﬂasks at 100 r.p.m. at 28 °C for 72 h. (Fig. 7f–q and Supplementary Fig. 10). The treatments and measurements were rhACE2 was puriﬁed with Proﬁnity IMAC Ni-charged resin (Bio-Rad), eluted with performed using a double-blind method. 250 mM imidazole, and further dialyzed against phosphate-buffered saline (PBS). Invasive measurements of arterial blood pressure. Acute hemodynamic 26,29 In vitro ACE2 activity measurements. For determination of kinetic constants, the experiment was performed with 10-week-old male C57BL/6J mice . Ninety K and k values for B38-CAP and recombinant human ACE2 (Calbiochem) minutes before hemodynamic measurements, mice were pretreated with an i.p. m cat −1 using Nma-His-Pro-Lys(Dnp) as ACE2 substrate were determined by injection of either B38-CAP (2 mg kg i.p.) or sterile PBS as vehicle. Mice Michaelis–Menten model using GraphPad Prism Version 6.01 (La Jolla, CA, were anesthetized with isoﬂurane (1–1.5%) and body temperature was maintained 19,28 USA) . The IC values of ACE2 inhibitor MLN-4760 (EMD, Millipore) for at 37–38 °C using a heating pad throughout the experiments. The mouse was recombinant human ACE2 and B38-CAP were measured as follows. The reaction securely restrained in a supine position and mechanically ventilated with a tracheal −1 mixture contained 40 μl of 0.1 M HEPES pH 7.5, containing 0.3 M NaCl, 0.01% cannulation (peak inﬂation pressure of 10 cm H O and 160 breaths min ; Ven- Triton X-100, 0.02% NaN ,5 μl MLN-4760 solution, and 5 μl of recombinant telite, Harvard Apparatus). A tapered PE-50 catheter ﬁlled with heparinized saline −1 human ACE2 or B38-CAP in a total volume of 50 μl. The reaction mixture was (20 units ml ) was inserted into the right carotid artery to record arterial BP via a incubated at 37 °C for 30 min and then the reaction was terminated by adding pressure transducer. After 5–10 min of stabilization period, systolic, diastolic, and 0.2 ml of 0.1 M sodium borate buffer pH 10.5. The ﬂuorescein intensity was mean BPs were obtained (Power Lab data acquisition system, AD Instruments) and measured spectrophotometrically at an emission wavelength 440 nm upon exci- heart rate was calculated using LabChart8 software (AD Instruments). At 90 min −1 tation wavelength 340 nm (Hitachi F-2500). The sample concentration required to after vehicle or B38-CAP injection, Ang II (0.2 mg kg i.p.) or vehicle was inhibit 50% of B38-CAP activity under the assay condition was taken as the IC injected. The changes in BP were analyzed for a subsequent 20 min time period. value. For plasma B38-CAP activity measurements, we developed a new B38-CAP- speciﬁc substrate Nma-Leu-Pro-Lys(Dnp) by screening the amino acids of P2 Transverse aortic constriction. Ten-week-old male C57BL/6N mice were sub- position in Nma-X-Pro-Lys(Dnp) substrates (not shown), and the K , k , and m cat jected to pressure overload by TAC . In one of the therapeutic experiments −1 k /K values using this substrate were determined to be 9.52 μM, 224 s , and cat m (Fig. 7), C57BL/6J mice were used. The same surgical TAC procedure results in −1 −1 23.5 s μM , respectively. Heparinized plasma was diluted with assay buffer and more severe dysfunction in C57BL/6N than in C57BL/6J mouse strains. Brieﬂy, the reaction mixture contained 45 μl of HEPES buffer pH 7.5, 0.3 M NaCl, 20 μM −1 mice were anesthetized via i.p. injection of ketamine (100 mg kg ) and xylazine Nma-Leu-Pro-Lys(Dnp), 0.01% Triton X-100, 0.02% NaN , and 5 μl diluted 3 −1 (20 mg kg ), and a longitudinal incision was made in the proximal portion of plasma or recombinant B38-CAP in a total volume of 50 μl. The reaction mixture sternum. The aortic arch was ligated with an overlying 27-gauge needle by 7-0 silk. was incubated at 37 °C for 60 min and then the reaction was terminated by adding The needle was immediately removed leaving a discrete region of constriction. The 0.2 ml of 0.1 M sodium borate buffer pH 10.5, and the ﬂuorescence intensity was sham-treated group underwent a similar procedure without ligation. Echocardio- measured. The enzyme concentration in plasma was calculated based on the graphy was performed at indicated time points after TAC or sham surgery, and standard recombinant B38-CAP activity. mice were then killed by cervical dislocation. Hydrolysis of angiotensin peptides by B38-CAP. Each reaction mixture Echocardiography and blood pressure measurements. Echocardiographic (185.3 µl) was formulated as 1 mg/ml of angiotensin peptides in 15.4 mM HEPES measurements were performed as previously described . Brieﬂy, conscious mice (pH 7.5), 184 mM NaCl, and 2 µg of recombinant B38-CAP, and reactions were were gently grabbed in hand or held in the apparatus, echocardiography was incubated at 37 °C. The reaction was terminated by the addition of 14.7 µl of 0.5 M performed using Vevo770 equipped with a 30 MHz linear transducer (Visual EDTA. The reaction mixture (20 µl) was analyzed by reverse-phase HPLC (TSKgel Sonics). The %FS was calculated as follows: %FS = [(LVEDD – LVESD)/ Super-ODS, 0.46 × 5, or 10 cm, Tosoh Corporation, Tokyo, Japan) and eluted with LVEDD] × 100. M-mode images were obtained for measurement of wall thickness a linear gradient of 0–100% acetonitrile in 0.05% triﬂuoroacetic acid (TFA). and chamber dimensions with the use of the leading-edge convention adapted by Quantiﬁcation was achieved using the peak area of the standard angiotensins I, II, the American Society of Echocardiography. For BP measurements, conscious mice and 1–7 (Peptide Institute, Inc.) and Ang 1–9 (Wako, Osaka, Japan). were warmed at 10 min before measurements through during measurements. BP was measured by a programmable sphygmomanometer (BP-200, Softron, Japan) LC-MS quantiﬁcation of amino acids (Leu, Phe, and His). Samples (100 µL) were using the tail-cuff method after 5 days of daily training . diluted with 9.2 M perchloric acid (4.3 µL), then centrifuged at 13,000 × g for 15 min. A 5 µl aliquot was injected for a Shimadzu LC-MS system (LCMS2020, Pharmacological intervention. When we treated the mice with B38-CAP, we Kyoto, Japan) equipped with an electrospray ion source with nebulizer gas examined two routes of administration; daily i.p. injection and subcutaneous con- −1 −1 1.5 L min , drying gas 15 L min , desolvation line temperature 250 °C, and heat tinuous infusion with osmotic mini-pumps (Alzet model 1002, Alza Corp.). The block temperature 200 °C. Chromatographic separations were performed with an dosage and route of B38-CAP and Ang II (Sigma-Aldrich) treatments in the Intrada Amino Acid column (3 × 100 mm, Imtakt, Kyoto, Japan). Leu and Phe experiments are described in each ﬁgure legends. In prevention experiments, were analyzed with the mobile phase consisting of (mobile phase A) acetonitrile : treatment with B38-CAP was initiated at the same time as implantation of Ang II- tetrahydrofuran (THF) : 25 mM ammonium formate : formic acid 10 : 80 : 10 : ﬁlled osmotic mini-pumps or completion of TAC surgery . For co-infusion of Ang 0.4 [v/v] and (mobile phase B) acetonitrile : 100 mM ammonium formate 20 : 80 at II and B38-CAP into the subcutaneous of mice for 2 weeks (Fig. 2d–h and Fig. 3), −1 0.4 mL min . The initial mobile-phase composition was 20% B maintained for Ang II and B38-CAP were loaded into individual pumps and the mice were 4 min, which was gradually increased to 100% B in 7 min, and then maintained at implanted with the two pumps. When Ang II was infused for 4 weeks (Supple- 100% B for 3 min and back to the initial condition of 20% B in 6 min for re- mentary Fig. 7), Ang II-ﬁlled osmotic mini-pumps (Alzet model 1002) were replaced equilibration. Furthermore, His was analyzed with the mobile phase consisting of with a new one at 2 weeks after implantation. Two or 4 weeks after treatment, BP (mobile phase A) acetonitrile : water : formic acid 85 : 15 : 0.3 [v/v] and (mobile measurement and echocardiography were performed. In the chronic experiments −1 phase B) 100 mM ammonium formate at 0.4 mL min . The initial mobile-phase with i.p. injection of B38-CAP, BP was measured at 2 h after i.p. injection (Sup- composition was 55% B maintained for 4 min, which was gradually increased to plementary Figs. 6 and 7a–e). In therapeutic experiments (Fig. 7 and Supplementary 100% B in 8 min, and then maintained at 100% B for 5 min and back to the initial Fig. 10), treatment with B38-CAP was initiated after the establishment of hyper- condition of 55% B in 6 min for re-equilibration. Single-ion monitoring in negative tension or pressure overload-induced cardiac dysfunction. To examine the effects of mode with m/z 132, 156, and 166, representing Leu, His, and Phe, respectively. A779 on B38-CAP hypotensive action (Fig. 2m and Supplementary Fig. 9a–d), Under these conditions, Leu and Phe were eluted at 7.93 and 7.16 min, respectively, −1 conscious mice were pre-injected with B38-CAP (2.0 mg kg i.p.) or vehicle at and His was eluted at 12.56 min. Quantiﬁcation was achieved using the peak area of −1 90 min before measurements. Just before injection of Ang II (0.2 mg kg i.p.), A779 the standard amino acids mixture, type H (Wako, Osaka, Japan). −1 (0.2 mg kg i.p.) or its combination, baseline data of BP was obtained, and then BP was measured every 5 min after injection by a tail-cuff method. Mice. C57BL/6N or C57BL/6J wild-type male mice were purchased from CLEA Japan, Inc. and maintained at the animal facilities of Akita University Graduate Histology. Heart tissues were ﬁxed with 4% formalin and embedded in parafﬁn. School of Medicine or Research Institute of National Cerebral and Cardiovascular Five-μm-thick sections were prepared and stained with hematoxylin and eosin or Center. All animal experiments conformed to the Guide for the Care and Use of Masson’s trichrome stain. For measurement of cardiac ﬁbrosis area, the high- Laboratory Animals, Eighth Edition, updated by the US National Research Council resolution images (×100 magniﬁcation) of the heart sections stained with Masson’s Committee in 2011, and approvals of the experiments were granted by the ethics trichrome were taken using a BIOREVO microscope (BZ9000; Keyence) and review board of Akita University or Research Institute of National Cerebral and ﬁbrosis area was quantiﬁed using the Image-Pro software (Media Cybernetics). Cardiovascular Center. Randomization was performed by using random numbers. In prevention experiments (Figs. 2–6 and Supplementary Figs. 5–9), the mice were assigned by stratiﬁed randomization based on BW. In therapeutic experiments Quantitative real-time PCR. RNA was extracted using TRIzol reagent (Invitro- (Fig. 7 and Supplementary Fig. 10), the mice were assigned by stratiﬁed rando- gen) and cDNA synthesized using the PrimeScript RT reagent kit (TAKARA). mization based on systolic BP for the experiments of Ang II-induced hypertension Sequences of the forward and reverse primers of the genes studied are shown in 10 NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z ARTICLE Supplementary Table 6. Real-time PCR was run in 96-well plates using a SYBR 9. Kuba, K., Imai, Y. & Penninger, J. M. Multiple functions of angiotensin- Premix ExTaq II (TAKARA) according to the instructions of the manufacturer. converting enzyme 2 and its relevance in cardiovascular diseases. Circ. J. 77, Relative gene expression levels were quantiﬁed by using the Thermal Cycler Dice 301–308 (2013). 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When a comparison is done for groups with two factor levels, two-way angiotensin-system. Int. J. Hypertens. 2012, 428950 (2012). ANOVA with Sidak’s multiple-comparisons test were used. P < 0.05 was con- 19. Takahashi, S., Yoshiya, T., Yoshizawa-Kumagaye, K. & Sugiyama, T. sidered signiﬁcant. Power analysis was performed on the results of initial animal Nicotianamine is a novel angiotensin-converting enzyme 2 inhibitor in experiments by statistical software R. The value of Cohen’s f was calculated with soybean. Biomed. Res. 36, 219–224 (2015). between-groups variance and within-subgroup variance, the effect size of each 20. Towler, P. et al. ACE2 X-ray structures reveal a large hinge-bending motion experiment data was calculated under P < 0.05 and Power 0.8, and the minimal important for inhibitor binding and catalysis. J. Biol. Chem. 279, 17996–18007 number of animals was determined. (2004). 21. Natesh, R., Schwager, S. L., Sturrock, E. D. & Acharya, K. R. 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Insight into the substrate length restriction of M32 of SDS-PAGE analysis is shown in Supplementary Fig. 11. carboxypeptidases: characterization of two distinct subfamilies. Proteins 77, 647–657 (2009). Received: 26 October 2018; Accepted: 10 February 2020; 25. Liu, P. et al. Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation. Kidney Int. 94, 114–125 (2018). 26. Wysocki, J. et al. Targeting the degradation of angiotensin II with recombinant angiotensin-converting enzyme 2: prevention of angiotensin II- dependent hypertension. Hypertension 55,90–98 (2010). 27. Santos, R. A. S. et al. The ACE2/angiotensin-(1-7)/MAS axis of the renin- References angiotensin system: focus on angiotensin-(1-7). Physiol. Rev. 98, 505–553 1. Corvol, P., Jeunemaitre, X., Charru, A., Kotelevtsev, Y. & Soubrier, F. Role of (2018). the renin-angiotensin system in blood pressure regulation and in human 28. 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The CCR4-NOT deadenylase complex controls Atg7- carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ. Res. dependent cell death and heart function. Sci. Signal. 11, https://doi.org/ 87,E1–E9 (2000). 10.1126/scisignal.aan3638 (2018). 5. Tipnis, S. R. et al. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J. Biol. Chem. 275, 33238–33243 (2000). 6. Vickers, C. et al. Hydrolysis of biological peptides by human angiotensin- Acknowledgements converting enzyme-related carboxypeptidase. J. Biol. Chem. 277, 14838–14843 We thank all members of our laboratories for technical assistance and helpful discus- (2002). sions, and we are grateful to Mrs M. Momma and Dr K. Hiwatashi for assistance in protein preparation and analysis, and to Mrs C. Inoue and Mr T. Takeda for initial 7. Crackower, M. A. et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828 (2002). animal experiments. K.K. is supported by the Kaken [17H04028] from Japanese Ministry 8. Liao, F. T., Chang, C. Y., Su, M. T. & Kuo, W. C. Necessity of angiotensin- of Science, the Takeda Science Foundation, Uehara Memorial Foundation and Daiichi converting enzyme-related gene for cardiac functions and longevity of Sankyo Foundation. Y.I. is supported by the Kaken [17H06179], T.S. is supported by the Drosophila melanogaster assessed by optical coherence tomography. J. Kaken [18K15879], and T.Y. is supported by the Kaken [18K15038] from Japanese Biomed. Opt. 19, 011014 (2014). Ministry of Science. NATURE COMMUNICATIONS | (2020) 11:1058 | https://doi.org/10.1038/s41467-020-14867-z | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14867-z Author contributions Reprints and permission information is available at http://www.nature.com/reprints S.N., Y.I., S.T., and K.K. conceived the study. T.M., S.N., T.S., T.I., S.Y., T.G., Y.N., J.M.P., H.W., Y.I., S.T., and K.K. designed the methodology and experiments. T.M., S.N., T.S., Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in T. Yamaguchi, M.H., T.I., K.N., T. Yoshihashi, R.O., S.Y., M.N., S.K., T. Yoshiya, K.Y-K., published maps and institutional afﬁliations. S.M., S.T., and K.K. conducted experiments and/or analyzed data. K.K., T.M., S.N., and T.S. wrote the manuscript with input from all authors. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Competing interests adaptation, distribution and reproduction in any medium or format, as long as you give The authors declare no competing interests. appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party Additional information material in this article are included in the article’s Creative Commons licence, unless Supplementary information is available for this paper at https://doi.org/10.1038/s41467- indicated otherwise in a credit line to the material. If material is not included in the 020-14867-z. article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from Correspondence and requests for materials should be addressed to S.N. or K.K. the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. Peer review information Nature Communications thanks Eric Lazartigues, and other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

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B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

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