Biomedical Research (Tokyo) 36 (3) 219-224, 2015

Nicotianamine is a novel angiotensin-converting enzyme 2 inhibitor in soy-bean

Saori TAKAHASHI1, Taku YOSHIYA

2, Kumiko YOSHIZAWA-KUMAGAYE2, and Toshihiro SUGIYAMA

3

1 Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan; 2 Peptide Institute, Inc., 7-2-9 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan; and 3 Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan

(Received 9 February 2015; and accepted 7 March 2015)

ABSTRACTAngiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase which is highly homologous to angiotensin-converting enzyme (ACE). ACE2 produces vasodilator peptides angiotensin 1-7 from angiotensin II. In the present study, we synthesized various internally quenched fluorogenic (IQF) substrates (fluorophore-Xaa-Pro-quencher) based on the cleavage site of angiotensin II introducing N-terminal fluorophore N-methylanthranilic acid (Nma) and C-terminal quencher N ε-2,4-dinitrophenyl-lysine [Lys(Dnp)]. The synthesized mixed substrates “Nma-Xaa-Pro-Lys(Dnp)” were hydrolyzed by recombinant human (rh) ACE2. The amount of each product was determined by liquid chromatography mass spectrometry (LC-MS) with fluorescence detection and it was found that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2. The Km, kcat, and kcat/Km values of Nma-His-Pro-Lys(Dnp) on rhACE2 were determined to be 23.3 μM, 167 s−1, and 7.17 μM−1 s−1, respectively. Using the rhACE2 and the newly developed IQF substrate, we found rhACE2 inhibitory activity in soybean and isolated the active compound soybean ACE2 inhibitor (ACE2iSB). The physicochemical data on the isolated ACE2iSB were identical to those of nicoti-anamine. ACE2iSB strongly inhibited rhACE2 activity with an IC50 value of 84 nM. This is the first demonstration of an ACE2 inhibitor from foodstuffs.

The renin-angiotensin system (RAS) is one of the most important blood pressure control system in mammals. Renin catalyzes the liberation of inactive decapeptide angiotensin I from its plasma substrate angiotensinogen. The produced angiotensin I is acti-vated by angiotensin-converting enzyme (ACE). ACE is a membrane-bound and zinc- and chloride-dependent peptidyl dipeptidase that cleaves C-termi-nus dipeptide from angiotensin I to produce active octapeptide angiotensin II. Angiotensin II is the ma-jor vasoactive peptide in the RAS, acting as a po-tent vasoconstrictor through its receptor AT1R (1, 14, 15). Angiotensin II also activates the release of

aldosterone from the adrenal cortex. This also causes high blood pressure. Thus, control of RAS is the major target for cardiovascular disease therapies.　In 2000, human homologue of ACE, referred to as “ACE2”, was identified and showed to be an es-sential regulator of cardiac function (4, 19). ACE2 consists of 805 amino acids and is a type I trans-membrane glycoprotein with a single extracellular catalytic motif, His-Glu-Met-Gly-His. The expres-sion of ACE2 mRNA in human is highly restricted to the heart, kidney, and testis (4, 19). ACE2 is a carboxypeptidase that catalyzes liberation of vasodi-lator peptide, angiotensin 1-7, from angiotensin II (Fig. 1). Thus, ACE2 is responsible for counterbal-ancing the potent vasoconstrictor effects of angio-tensin II (23). On the other hand, ACE2 has been demonstrated to be a functional receptor for the coronavirus (CoV) that causes severe acute respirato-ry syndrome (SARS) (12, 13, 21). Moreover, ACE2

Address correspondence to: Saori Takahashi Ph.D., Akita Research Institute of Food and Brewing, 4-26, Sanuki Arayamachi, Akita 010-1623, JapanTel: +81-18-888-2000 (Ext. 222), Fax: +81-18-888-2008E-mail: saori@arif.pref.akita.jp

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tested Nma-Phe-His-Lys(Dnp) for the determination of rhACE2 activity. However Nma-Phe-His-Lys(Dnp) was poor substrate for rhACE2 (data not shown). Thus, in the present study, we designed novel IQF substrates for rhACE2 based on the amino acid se-quence of angiotensin II because angiotensin II is more susceptible than angiotensin I by rhACE2 hy-drolysis (19) (Fig. 1). Initially, we prepared mixed substrates “Nma-Xaa-Pro-Lys(Dnp)” containing 19 kinds of naturally occurring amino acids except for Cys at P2 position (Xaa). Substrates were divided into two groups (A and B groups) because Leu and Ile have the same molecular weight and cannot be distinguished by liquid chromatography mass spec-trometry (LC-MS). Group A substrates contains Ile or 9 amino acids (Ala, Pro, Gln, Asp, Thr, Met, Phe, Arg, His). Group B substrates contains Leu or 9 amino acids (Gly, Val, Asn, Glu, Ser, Trp, Tyr, Lys, His). Both groups contain Nma-His-Pro-Lys(Dnp) as an internal standard. Hydrolysis of substrates by rhACE2 was as follows. The reaction mixture con-tained 450 μL of 0.1 M HEPES, pH 7.5, 0.3 M NaCl, 50 μg mixed substrates, 0.01% Triton X-100, 0.02% NaN3, 50 μL of rhACE2 solution (50 ng/mL) in a total volume of 500 μL. The reaction mixtures were incubated at 37°C for 0–120 min. After the incuba-tion, reactions were terminated by heating (100°C for 5 min), and then the amounts of fluorescence products (Nma-Xaa-Pro) and residual substrates (data not shown) were determined by LC-MS with fluo-rescence detection according to the previous report (18). Production of Nma-Xaa-Pro from both group A and B substrates was shown in Fig. 2. The fluo-rescence intensity of the products was normalized to the internal standard, Nma-His-Pro-Lys(Dnp). At 30 min point, the order of production in the group-A substrates was Nma-His-Pro (100%), Nma-Met-Pro (56.1%), Nma-Arg-Pro (52.9%), Nma-Phe-Pro (41.9%), Nma-Gln-Pro (27.1%), and Nma-Ala-Pro (8.45%). The production of Nma-Asp-Pro, Nma-Thr-

has been shown to be essential for SARS-CoV in-fection in vitro (10). Recently, Hashimoto et al. (5) identified that ACE2 is a key regulator of dietary amino acid homeostasis in colitis. As mentioned above, ACE2 has various physiological functions. Thus, ACE2-specific substrates and inhibitors should provide more information about the physiological roles of ACE2.　In the present study, we developed novel internal-ly quenched fluorogenic (IQF) substrate for ACE2, Nma-His-Pro-Lys(Dnp). Using Nma-His-Pro-Lys(Dnp) and ACE2, we screened ACE2 inhibitory activity in various foodstuffs, found that soybean contained strong ACE2 inhibitory activity, and iso-lated the active compound “soybean ACE2 inhibitor” (ACE2iSB). The isolated ACE2iSB was suggested to be identical to nicotianamine by direct compari-son with standard compound.　The recombinant human (rh) ACE2 and rhACE were obtained from Calbiochem (San Diego CA, USA; Lot: D00130319) and R&D Systems (Minne-apolis, MN, USA; Lot: FQJ0207011), respectively. Authentic nicotianamine was from Santa Cruz Bio-technology (Dallas, TX, USA; Lot: D0314). Bio-gel P-4 and Sephadex G-15 were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), and GE Healthcare (Buckinghamshire, UK), respective-ly. The Km and kcat values were determined using Lineweaver-Burk plots. Nma-His-Pro-Lys(Dnp) and group-A and B substrates (described below) were synthesized by the solid phase method with an ABI 430A peptide synthesizer using the Boc strategy. The protected peptide resins were treated with hy-drogen fluoride to give crude products, which were subsequently purified by HPLC. ACE2 inhibition as-says using IQF substrates were as follows. The re-action mixture contained 40 μL of 0.1 M HEPES, pH 7.5, 0.3 M NaCl, 20 μM IQF substrate, 0.01% Trion X-100, 0.02% NaN3, 5 μL of inhibitor solu-tion or buffer, and 5 μL of rhACE2 solution (50 ng/mL) in a total volume of 50 μL. The reaction mix-ture was incubated at 37°C for 30 min and then the reaction was terminated by adding 0.2 mL of 0.1 M sodium borate buffer, pH 10.5. The increase in fluo-rescence intensity was measured at an emission wavelength at 440 nm upon excitation at 340 nm. The concentration of the inhibitor that is required for 50% inhibition of the rhACE2 activity was de-fined as the IC50 value.　Previously, we developed sensitive IQF substrate for rhACE based on the amino acid sequence of an-giotensin I, Nma-Phe-His-Lys(Dnp) (17). As ACE2 converts angiotensin I to angiotensin 1-9 (4), we

Fig. 1　Structures of angiotensin II, angiotensin 1-7, and IQF substrates. IQF substrates were synthesized as de-scribed in the text. *, scissile peptide bond; #, possible scis-sile peptide bond.

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　The effects of pH and NaCl concentration on rhACE2 activity were also determined using Nma-His-Pro-Lys(Dnp). The rhACE2 activity had strong pH dependence, especially under acidic conditions. The enzyme had only 15% activity at pH 6.0 to that at optimal pH (pH 7.5). On the other hand, rhACE2 maintained substantial activity within the range of pH 7.0–pH 8.5 (Fig. 3A). The rhACE2 activity was enhanced by the addition of NaCl. The hydrolytic activity was optimal in the presence of 0.25 M– 0.5 M NaCl (Fig. 3B).　We also determined the kinetic parameters of rhACE2 for IQF substrate. The reaction mixture contained 45 μL of 0.1 M HEPES, pH 7.5, containing 0.3 M NaCl, 5–80 μM Nma-His-Pro-Lys(Dnp), 0.01%

Pro, Nma-Ile-Pro, and Nma-Pro-Pro could not be de-tected (Fig. 2A). In the case of group B substrates, the order of production was Nma-His-Pro (100%), Nma-Asn-Pro (51.7%), Nma-Tyr-Pro (43.5%), Nma-Lys-Pro (26.7%), Nma-Trp-Pro (23.8%), Nma-Leu-Pro(19.0%), and Nma-Ser-Pro (5.0%). Production of Nma-Glu-Pro, Nma-Val-Pro, and Nma-Gly-Pro could not be detected (Fig. 2B). Moreover, the amount of residual substrate, Nma-His-Pro-Lys(Dnp) was much less than those other Nma-Xaa-Pro-Lys(Dnp) sub-strates (data not shown). These results clearly indi-cate that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2.

Fig. 2　Production of Nma-Xaa-Pro from Nma-Xaa-Pro-Lys(Dnp) by rhACE2. Group A and B substrates were incu-bated with rhACE2 for 30 and 60 min. Then the products were quantified by LC-MS. (A) Products of group A sub-strates. Xaa = □, His; ○, Met; △, Arg; ■, Phe; ●, Gln; ▲, Ala; ◆, Asp, Thr, Ile, Pro. Substrates containing Asp, Thr, Ile or Pro at the P2 position (Xaa) were not hydrolyzed by rhACE2. (B) Products of group B substrates. Xaa = □, His; ○, Asn; △, Tyr; ■, Lys; ●, Trp; ▲, Leu; *, Ser; ◆, Glu, Val, Gly. Substrates containing Glu, Val or Gly at P2 position (Xaa) were not hydrolyzed by rhACE2.

Fig. 3　pH dependent activity of rhACE2 (A) and effects of NaCl on rhACE2 (B). (A) The rhACE2-catalyzed IQF sub-strate hydrolysis reactions were performed with 5 ng/mL of rhACE2 and 20 μM Nma-His-Pro-Lys(Dnp) in 0.1 M HEPES, pH 6.0–8.5, 0.3 M NaCl, 0.01% Triton X-100, and 0.02% NaN3. (B) The rhACE2 catalyzed IQF substrate hydrolysis reactions were performed with 5 ng/mL rhACE2 and 20 μM Nma-His-Pro-Lys(Dnp) in 0.1 M HEPES, pH 7.5, 0 to 2.0 M NaCl, 0.01% Triton X-100, and 0.02% NaN3. Each result is the mean value for triplicate determinations.

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inhibitory activity were pooled and lyophilized. The lyophilized matter (9.0 mg) was used in the identifi-cation of ACE2iSB. The isolated ACE2iSB gave a protonated ion peak, (M+H)+ = 304.1 by electron spray ionization mass spectrometry (ESI-MS), which was identical to the theoretical value of nicoti-anamine. Additionally, 1H-NMR spectrum, HPLC re-tention times on octa decyl silyl (ODS) and TSKgel amide-80 (Tosoh, Tokyo), and amino acid analysis on cation-exchange columns of the isolated ACE2iSB were all identical to those of the authentic nicoti-anamine (Fig. 4). Nicotianamine is a major ACE in-hibitory compound in soy sauce (9) and vegetables (6, 11). It is an intermediate compound in biosyn-thetic pathway of mugineic acids and ubiquitous in higher plant (16). However, because of the delay of development of a rhACE2 assay method, the screen-ing of rhACE2 inhibitory activity of nicotianamine has not been studied. The present results clearly in-dicate that nicotianamine is the rhACE2 inhibitory compound in soybean.　Fig. 5 shows dose-dependent inhibition of rhACE2

Trion X-100, 0.02% NaN3, and 5 μL of rhACE2 so-lution (25 ng/mL) in a total volume of 50 μL. The reaction mixture was incubated at 37°C for 30 min and then the reaction was terminated by adding 0.2 mL of 0.1 M sodium borate buffer, pH 10.5. The increase in fluorescence intensity was measured at an emission wavelength at 440 nm upon excitation at 340 nm. The Km, kcat, and kcat/Km values of rhACE2 for Nma-His-Pro-Lys(Dnp) were 23.3 μM, 167 s−1, and 7.17 μM−1 s−1, respectively. In previous studies, caspase-1 substrate, (7-methoxycumarin-4-yl) acetyl (MCA)-Tyr-Val-Ala-Asp-Pro-Lys(Dnp) or MCA-Ala-Pro-Lys(Dnp), was used for rhACE2 as-say (7, 20, 22), the latter being a better substrate than the former. The rhACE2 hydrolyzed MCA-Ala-Pro-Lys(Dnp) with Km, kcat, and kcat/Km values of 147 μM, 114 s−1, and 0.77 μM−1 s−1, respectively (20). The calculated kcat/Km value for Nma-His-Pro-Lys(Dnp) was one order magnitude higher than that for MCA-Ala-Pro-Lys(Dnp) (20). These results clearly indicate that newly developed Nma-His-Pro-Lys(Dnp) is an excellent substrate for rhACE2.　Using rhACE2 and the IQF substrate, we screened various foodstuffs and found that soybean contains strong rhACE2 inhibitory activity. Then, we isolated the active compound “soybean rhACE2 inhibitor” (ACE2iSB). Soybean (100 g) was soaked 1.5 L of distilled water at room temperature overnight, and heated at 105°C for 30 min. The sample was ho-mogenized in food processor and centrifuged at 10,000 × g for 30 min. The supernatant (200 mL) was applied to a Sep-Pak Vac C18 35cc (Waters, Bedford, MA) that had been equilibrated with dis-tilled water. The column was washed with 100 mL of distilled water. This step was repeated several times. Then the flow-through fractions were pooled and lyophilized. The lyophilized sample (9.45 g) was dissolved in 60 mL of 10% ethanol and applied to a Bio-Gel P-4 (5.5 × 65 cm) column equilibrated with 10% ethanol. The column was eluted with 10% ethanol, and fractions containing rhACE2 inhibitory activity were pooled and lyophilized. The lyophi-lized sample (250 mg) was dissolved in 20 mL of 10% ethanol and applied to a Sephadex G-15 (5.5 × 65 cm) column equilibrated with 10% etha-nol. The column was eluted with 10% ethanol, and fractions exhibiting rhACE2 inhibitory activity were pooled and lyophilized. The lyophilized sample (64.8 mg) was dissolved in 5 mL of 10% ethanol containing 10 mg/mL of NaCl and applied to a Bio-Gel P-4 (2.5 × 90 cm) column equilibrated with 10% ethanol. Fractions of 4 mL each were collected and monitored at 210 nm. Fractions exhibiting rhACE2

Fig. 4　Chemical structure of nicotianamine (N-[N-(3-ami-no-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidine-2-carboxylic acid) (A) and 1H-NMR spectra of nicotianamine (upper panel) and ACE2iSB (lower panel) in D2O contain-ing 0.3% CF3COOD (B).

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and rhACE by ACE2iSB and nicotianamine. ACE2iSB inhibited both rhACE2 and rhACE with IC50 values of 84 nM and 62 nM, respectively (Fig. 5A). The authentic nicotianamine also inhibited rhACE2 and rhACE with IC50 values of 76 nM and 59 nM, re-spectively (Fig. 5B). The IC50 values of ACE2iSB and nicotianamine for rhACE2 and rhACE were in-distinguishable. These results also support the asser-tion that ACE2iSB is nicotianamine.　Recently, various peptide and synthetic ACE2 in-hibitors have been developed (2, 3, 7, 8). The ACE2 inhibitor N-(2-aminoethyl)-1-aziridine-ethanamine discovered by structure-based studies is effective in blocking the SARS coronavirus spike protein-mediat-ed cell fusion (2). On the other hand, synthetic ACE2 inhibitor, GL1001 has anti-inflammatory activity in the mouse digestive tract (3). The isolated novel rhACE2 inhibitor from soybean, nicotianamine, is a beneficial compound for in vivo studies because of its origin in a foodstuff. Nicotianamine should be a useful tool for elucidation of physiological function of ACE2. Detailed inhibition kinetic studies of nico-tianamine on ACE2 are underway.

Acknowledgments

This study was supported in part by research grant from Central Miso Research Institute (Chuo-ku, To-kyo). We thank Ms. Asami Sutoh for her technical assistance.

REFERENCES

Fig. 5　Dose-dependent inhibition of rhACE2 (closed circles) and rhACE (open circles) by ACE2iSB (A) and nicotianamine (B). The rhACE2 or rhACE was incubated with indicated amounts of ACE2iSB and nicotianamine. The rhACE2 inhibitory ac-tivity was measured described in the text. Each result is the mean value for triplicate determinations.

1. Bumpus FM, Catt KJ, Chiu AT, DeGasparo M, Goodfriend T, Husain A, Peach MJ, Taylor DG, Jr and Timmermans PBMWM (1991) Nomenclature for angiotensin receptors. A

report of the nomenclature committee of the council for high blood pressure research. Hypertension 17, 720–721.

2. Byrnes JJ, Gross S, Ellard C, Connolly K, Donahue S and Picarella D (2009) Effects of ACE2 inhibitor GL1001 on acute dextran sodium sulfate-induced colitis in mice. Inflamm Res 58, 819–827.

3. Diz DI, Garcia-Espinosa MA, Gegick S, Tommasi EN, Ferrario CM, Tallant EA, Chappell MC and Gallagher PE (2008) Injection of angiotensin-converting enzyme 2 inhibi-tor MLN4760 into nucleus tracts solitarii reduce baroreceptor reflex sensitivity for heart rate control in rats. Exp Physiol 93, 694–700.

4. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE and Acton S (2000) A novel angiotensin-con-verting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res 87, e1–e9.

5. Hashimoto T, Perlot T, Rehman A, Trichereau J, Ishiguro H, Paolino M, Sigl V, Hanada T, Hanada R, Lipinski S, Wild B, Camargo SMR, Singer D, Richter A, Kuba K, Fukamizu A, Schreiber S, Clevers H, Verrey F, Rosenstiel P and Penninger JM (2012) ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation. Nature 487, 477–481.

6. Hayashi A and Kimoto K (2007) Nicotianamine preferentially inhibits angiotensin I-converting enzyme. J Nutr Sci Vitamin-ol 53, 331–336.

7. Huang L, Sexton DJ, Skogerson K, Devlin M, Smith R, Sanya I, Parry T, Kent R, Enright J, Wu Q, Conley G, DeOliveira D, Morganelli L, Ducar M, Wescott CR and Ladner RC (2003) Novel peptide inhibitors of angiotensin-converting enzyme 2. J Biol Chem 278, 15532–15540.

8. Huentelman MJ, Zubcevic J, Prada JAH, Xiao X, Dimitrov DS, Raizada MK and Ostrov DA (2004) Structure-based dis-covery of a novel angiotensin-converting enzyme 2 inhibitor. Hypertension 44, 903–906.

9. Kinoshita E, Yamakoshi J and Kikuchi M (1993) Purification and identification of an angiotensin I-converting enzyme in-hibitor from soy sauce. Biosci Biotechnol Biochem 57, 1107–1110.

10. Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, Huan Y, Yang P, Zhang Y, Deng W, Bao L, Zhang B, Liu G, Wang Z, Chappell M, Liu Y, Zheng D, Leibbrandt A, Wada T, Slutsky

S. Takahashi et al.224

AS, Liu D, Qin C, Jiang C and Penninger JM (2005) A cru-cial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med 11, 875–879.

11. Kuroda K, Ishihara K and Masuoka N (2013) Characteriza-tion of nicotianamine isolated from soybeans. J Food Res 2, 49–54.

12. Li F, Li W, Frazan M and Harrison SC (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864–1868.

13. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H and Farzan M (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454.

14. Murphy TJ, Alexander RW, Griendling KK, Runge MS and Bernstein KE (1991) Isolation of a cDNA encoding the vas-cular type-1 angiotensin II receptor. Nature 351, 233–236.

15. Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y and Inagami T (1991) Cloning and expression of a complementary DNA encoding a bovine adrenal angio-tensin II type-1 receptor. Nature 351, 230–233.

16. Takahashi M, Terada Y, Nakai I, Nakanishi H, Yoshimura E, Mori S and Nishizawa NK (2003) Role of nicotianamine in the intracellular delivery of metals and plant reproductive de-velopment. Plant Cell 15, 1263–1280.

17. Takahashi S, Ono H, Gotoh T, Yoshizawa-Kumagae K and

Sugiyama T (2011) Novel internally quenched fluorogenic substrates for angiotensin I-converting enzyme and carboxy-peptidase Y. Biomed Res (Tokyo) 32, 407–411.

18. Tanskul S, Oda K, Oyama H, Noparatnaraporn, Tsunemi M and Takada K (2003) Substrate specificity of alkaline serine proteinase isolated from photosynthetic bacterium, Rubriviax gelatinosus KDDS1. Biochem Biophys Res Commun 309, 547–551.

19. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G and Tuner AJ (2000) A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem 275, 33238–33243.

20. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A and Tummino P (2002) Hydrolysis of biologi-cal peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277, 14838–14843.

21. Wong SK, Li W, Moore MJ, Choe H and Farzan M (2004) A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279, 3197–3201.

22. Wysocki J, Ye M, Soler MJ, Gurley SB, Xiao HD, Bernstein KE, Coffman TM, Chen S and Batlle D (2006) ACE and ACE2 activity in diabetic mice. Diabetes 55, 2132–2139.

23. Yagil Y and Yagil C (2003) Hypothesis. ACE2 modulates blood pressure in the mammalian organism. Hypertension 41, 871–873.