a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to...

A Novel Angiotensin-Converting Enzyme–RelatedCarboxypeptidase (ACE2) Converts Angiotensin I

to Angiotensin 1-9Mary Donoghue, Frank Hsieh, Elizabeth Baronas, Kevin Godbout, Michael Gosselin,Nancy Stagliano, Michael Donovan, Betty Woolf, Keith Robison, Raju Jeyaseelan,

Roger E. Breitbart, Susan Acton

Abstract—ACE2, the first known human homologue of angiotensin-converting enzyme (ACE), was identified from59 sequencing of a human heart failure ventricle cDNA library. ACE2 has an apparent signal peptide, a singlemetalloprotease active site, and a transmembrane domain. The metalloprotease catalytic domains of ACE2 andACE are 42% identical, and comparison of the genomic structures indicates that the two genes arose throughduplication. In contrast to the more ubiquitous ACE, ACE2 transcripts are found only in heart, kidney, and testisof 23 human tissues examined. Immunohistochemistry shows ACE2 protein predominantly in the endothelium ofcoronary and intrarenal vessels and in renal tubular epithelium. Active ACE2 enzyme is secreted from transfectedcells by cleavage N-terminal to the transmembrane domain. Recombinant ACE2 hydrolyzes the carboxy terminalleucine from angiotensin I to generate angiotensin 1-9, which is converted to smaller angiotensin peptides by ACEin vitro and by cardiomyocytes in culture. ACE2 can also cleave des-Arg bradykinin and neurotensin but notbradykinin or 15 other vasoactive and hormonal peptides tested. ACE2 is not inhibited by lisinopril or captopril.The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest anessential role for ACE2 in the local renin-angiotensin system of the heart and kidney. The full text of this articleis available at http://www.circresaha.org.(Circ Res. 2000;87:e1-e9.)

Key Words: angiotensin-converting enzymen ACE2 n angiotensinn renin-angiotensin system

Angiotensin-converting enzyme (ACE) is a pivotal com-ponent of the renin-angiotensin system (RAS), mediat-

ing numerous systemic and local effects in the cardiovascularsystem. ACE is produced in the endothelium of somatictissues as a transmembrane protein containing two activedomains, both of which are inhibited by ACE inhibitors.1

Analysis of the genomic sequence suggests that the twoactive domains appear to have come from a common ances-tor.2 ACE is also produced in the testis via an alternatepromoter, generating a shorter testis-specific form with onlyone active site.2 Both the somatic endothelial ACE and thetesticular form can be cleaved to generate soluble activeenzymes. ACE removes the carboxy terminal dipeptide fromthe decapeptide angiotensin I (Ang I) to generate angiotensinII (Ang II), a potent vasoconstrictor, and degrades bradykinin,a vasodilator. Thus, ACE activity serves to increase systemicvascular tone, and pharmacological ACE inhibition is effec-tive in the treatment of hypertension.

In addition to the systemic RAS, a local RAS is knownto operate within certain tissues (for review, see Reference3). Cardiac myocytes express Ang II receptors and undergohypertrophy in response to Ang II in vitro. Ang II also

induces cardiac fibroblast proliferation and collagen pro-duction. In vivo, ACE inhibition reverses cardiac myocytehypertrophy and fibrosis associated with ventricular re-modeling and heart failure, presumably by reducing localgeneration of Ang II. The tissue RAS has additionalcomplexity, however. At least one other enzyme, a heartchymase that is secreted by mast cells, is capable ofconverting Ang I to Ang II.4 Furthermore, several otherAng I cleavage products have been found, for example,Ang1-9, Ang1-7, Ang III,2– 8 and Ang IV.3– 8 AlthoughAng1-7 has been shown to have physiological effects suchas diuresis and vasodilation (for review, see Reference 5),in general the activities of these and other angiotensinpeptides are not well understood.

In the present study, we report the first human homologue ofACE, identified in an ongoing search for novel genes related toheart failure. ACE-related carboxypeptidase (ACE2), like ACE,is a membrane-associated and secreted enzyme expressed pre-dominantly on endothelium, but unlike ACE, it is highlyrestricted in humans to heart, kidney, and testis. ACE2 catalyzesthe cleavage of Ang I to Ang1-9 and may have a unique role inthe local RAS in heart and kidney.

Received July 25, 2000; revision received August 8, 2000; accepted August 8, 2000.From Millennium Pharmaceuticals, Inc, Cambridge, Mass. Current address for M.G. is Praecis Pharmaceuticals, Cambridge, Mass.Correspondence to Susan Acton, Millennium Pharmaceuticals, Inc, 75 Sidney St, Cambridge, MA 02139. E-mail [email protected]© 2000 American Heart Association, Inc.

Circulation Researchis available at http://www.circresaha.org

1

UltraRapid Communication

by guest on February 12, 2016http://circres.ahajournals.org/Downloaded from

http://circres.ahajournals.org/

Materials and MethodscDNA Library Construction, Sequencing,Extension, and Phylogenetic AnalysisA human cardiac left ventricle cDNA library was prepared fromthe explant of a heart transplant recipient, a 43-year-old white

woman with idiopathic dilated cardiomyopathy (sample obtainedfrom G. Sandusky and N. Bowling, Eli Lilly & Co, Indianapolis,Ind), using standard directional cloning techniques. Nineteenthousand clones were selected at random for high throughput 59end automated sequencing. Homology searching was done usingTBLASTN and Hidden Markov models from PFAM (see www.

Figure 1. Comparison of ACE2 and ACE protein sequences. The amino acid sequences of human ACE2, rat-secreted ACE2 (Rat-sACE2), human testicular ACE (human T-ACE), murine testicular ACE (murine T-ACE), rabbit testicular ACE (rabbit T-ACE), and Dro-sophila melanogaster ACE are aligned. Residues that do not match the consensus are shaded.

2 Circulation Research September 1, 2000



sanger.ac.uk/Software/Pfam/). Rapid amplification of cDNA ends(RACE) was done using the Marathon cDNA amplification kit(Clontech), the 59primer 59-GSP-ACE2 (CAC AGG TTC CACCAC CCC AAC TAT CTC), and heart mRNA from which theoriginal cDNA library was generated. The sequence can be foundat GenBank No. AF291820. A similar sequence was submitted toGenBank (No. AF241254) by independent investigators shortlybefore submission of this manuscript. For sequence alignment andphylogenetic tree analysis, the CLUSTAL V method was used to

group sequences into clusters by examining the distances betweenall pairs.6

Expression Vector ConstructionFull-length human ACE2 expression vector (pACE2) was generatedby ligating the 1.4-kbEcoRI/AflIII fragment of a 59cDNA extensionclone (nucleotides 1 to 1381, see GenBank No. AF291820) and the1.9-kbAflIII/NotI fragment of the original library clone (nucleotides1381 to 3325, see GenBank No. AF291820) into theEcoRI/NotI sites

Donoghue et al ACE2, an ACE-Related Carboxypeptidase 3



of pcDNA3.1 (Invitrogen). An expression vector for secreted humanACE2 (psACE2) was generated by replacing the 39end of full-lengthhuman ACE2 with a polymerase chain reaction fragment containinga stop codon inserted after the serine immediately preceding thepredicted transmembrane domain (amino acid 740).

Northern Blot AnalysisA 636-bp probe of the 39untranslated region of human ACE2 cDNAcontaining no homology to ACE (MscI/KpnI fragment, nucleotides2583 to 3219, GenBank No. AF291820) was labeled with32P usingthe Multiprime labeling system (Amersham) and hybridized tohuman multiple tissue Northern blots (Clontech) overnight at 65°Cin Nylon Wash (128 mmol/L Na2HPO4 z 7H2O, 14 mmol/L EDTA,0.2% Triton X-100, 14% SDS, pH 7.2). The blots were then washedthree times for 30 minutes each at 65°C in 0.53Nylon Wash.(Independent experiments on different Northern blots yielded iden-tical results.) ABstXI fragment of testicular ACE comprising basepairs 578 to 1736 (100% homologous to endothelial ACE) washybridized to the identical blots above after stripping ACE2 signal bywashing with boiling 0.5% SDS. Testicular ACE cDNA was thegenerous gift of the James Riordan Laboratory, Harvard MedicalSchool, Boston, Mass.

Cell Culture and TransfectionsChinese hamster ovary (CHO) K1 cells were maintained in serum-free Ultra CHO medium (Biowhittaker) at 37°C in a humidified 5%CO2 incubator. For transfections, cells were seeded at 13106

cells/100-mm dish on day 0 and transfected with Lipofectamine(Gibco BRL) on day 1 as per the manufacturer’s instructions.Briefly, 10 mg of DNA was combined with 40mL of Lipofectaminein Opti-MEM medium (Gibco BRL) and the mixture was incubatedon the cells for 5 to 6 hours. Ultra CHO medium was then added tothe cells and they were incubated overnight at 37°C. On day 2, themedium was changed and on day 4 the conditioned media werecollected. For some experiments, the conditioned media were con-centrated in Centriplus 30 concentrators (Amicon) in 10 mmol/LTris-Cl, pH 7.0. After the conditioned media were harvested fromcells, the cells were washed twice with PBS, and 2 mL of lysis bufferwas added (50 mmol/L Tris-Cl, pH 7.5, 150 mmol/L NaCl, 0.02%NaN3, and 1% NP-40 with Complete protease inhibitors [BoehringerMannheim]). The cells were then incubated for 20 minutes on ice.The cells were scraped, transferred to microfuge tubes, and the nuclei

were pelleted 10 minutes in a microfuge. The remaining cell lysate wasused immediately or frozen at220°C. Primary rat neonatal ventricularcardiomyocytes were prepared using 1- to 3-day-old neonates accordingto published methods.7 The cells were plated in modified Eagle’smedium containing 5% FCS, 1% penicillin/streptomycin, and0.1 mmol/L bromodeoxyuridine for 24 hours and then switched toserum-free medium supplemented with insulin-transferrin-sodium se-lenite (Sigma) for an additional 24 hours. Synthetic sarcosyl-blockedpeptides (Ang1-9, Ang1-8, Ang1-7, and Ang1-5), which are resistant toN-terminal degradation by aminopeptidases, were then added individu-ally at concentrations of 1 to 10mmol/L for periods of up to 48 hours.Aliquots of conditioned media were collected at different time points foranalysis by mass spectrometry.

Antibodies, Immunohistochemistry, andImmunoblot AnalysisTwo rabbit polyclonal anti-ACE2 antibodies (anti-ACE251 and anti-ACE2489) were raised against synthetic peptides representing humanACE2 residues 51 to 69 (NTNITEENVQNMNNAGDKW) andresidues 489 to 508 (EPVPHDETYCDPASLFHVSN) (ResearchGenetics). These were affinity-purified on peptide columns andconcentrated according to standard methods. For immunohistochem-istry, frozen sections of human heart and kidney were cut at 6mmthickness, mounted on charged slides, fixed in cold acetone (Sigma),and immersed in 0.3% H2O2 in methanol for 10 minutes to blockendogenous peroxidase. A 1:2000 dilution of anti-ACE251 wasapplied in 5% BSA/PBS for 30 minutes at room temperature. Slideswere then incubated with an alkaline phosphatase–labeled polymer(DAKO) for 30 minutes at room temperature. After multiple PBSwashes, Fast Red substrate-chromogen solution was applied for 10minutes and the slides were then counterstained with hematoxylin.To determine the specificity of the antibody, peptide competitionwas performed on serial sections. ACE2 peptide 51 to 69 or anonspecific peptide (not shown) was incubated for 2 hours at roomtemperature at a 1:10 ratio (by weight) with anti-ACE251. Thesolution was then applied at the step of the primary antibody additionabove. For Western blot analysis, conditioned medium from trans-fected cells was loaded onto 10% to 20% precast SDS-polyacryl-amide gels (Bio-Rad), followed by separation at 100 V for 2 hoursand transferred to Hybond P using a semidry transfer system(Amersham). The blots were incubated in blocking buffer (5%nonfat milk, 0.1% Tween-20, in PBS) for 1 hour at room temperature




or overnight at 4°C. The blot was then incubated with anti-ACE2489

at a 1:5000 dilution for 30 minutes in PBS with 0.1% Tween-20, thenwashed 1 time for 15 minutes and three times for 5 minutes in PBScontaining 0.1% Tween-20. The blot was then incubated withdonkey anti-rabbit HRP (Amersham) in PBS with 0.1% Tween-20for 30 minutes, followed by washing as above. Signal was detected usingthe ECL Plus kit (Amersham). Antibody specificity was tested againstACE2 and testicular ACE produced by transfected CHO cells in animmunoblot analysis. None of the antibodies cross-reacted with ACE.

Purification of Recombinant ACE2 EnzymeConditioned media from CHO cells transfected with soluble humanACE2 (see above) were concentrated 10-fold in an Amicon 10Kmolecular weight cutoff stirred cell apparatus (Amicon), then de-salted by injection on a Pharmacia PC 3.2-mm/100-mm 800-mL-bedvolume Fast Desalting column (Pharmacia) in bis-Tris buffer(25 mmol/L bis-Tris-propane, 25 mmol/L Tris HCl, pH 6.5). Theresulting protein mixture was injected onto a Pharmacia PC 1.6-mm/50-mm 100-mL-bed volume MonoQ anion exchange column inbis-Tris buffer. The column was washed with bis-Tris buffer andthen eluted with a 0 to 250 mmol/L NaCl gradient over 20 columnvolumes. The ACE2-containing fractions, as determined by Westernblot, were pooled. (NH4)2SO4 was added to a 1 mol/L final concen-tration. This mixture was injected onto a Pharmacia PC 1.6-mm/50-mm 100-mL-bed volume Phenyl Superose HIC column in 1mol/L (NH4)2SO4 and 100 mmol/L NaPi, pH 7.0. The column waswashed with (NH4)2SO4/NaPi, pH 7.0, loading buffer and eluted witha 1 to 0.5 mol/L (NH4)2SO4 reverse gradient in 100 mmol/L NaPi, pH7.0, over 10 column volumes. ACE2-containing fractions, as deter-mined by Coomassie staining of a reduced SDS-PAGE gel, werepooled and desalted by injection on a Pharmacia PC 3.2-mm/100-mm 800-mL-bed volume Fast Desalting column in 100 mmol/LNaPi, pH 7.0. ACE2-containing fractions, as resolved by reducedSDS-PAGE, were used for assays. Conditioned medium from emptyvector–transfected cells was purified as above to produce theappropriate negative control.

Analysis of ACE and ACE2 Activity byMass SpectrometryEnzymatic reactions were performed in 15mL. To each tube at roomtemperature was added 10mL of buffer (10 mmol/L Tris, pH 7.0)with or without enzyme. Five microliters of purified Ang I (DRVYI-HPFHL) (Sigma) or other peptide substrates were added to each tubefor a final concentration of 5mmol/L. Lisinopril or captopril (Sigma)was added to some reactions at final concentrations of 6.6mmol/L.For reactions and control experiments, the tubes were incubated at37°C for 30 minutes. A portion (1mL) of each reaction wasquenched by the addition of 1mL of a low-pH MALDI matrixcompound (10 g/La-cyano-4 hydroxycinnamic acid in a 1:1 mixtureof acetonitrile and water). One microliter of the resulting solutionwas applied to the surface of a MALDI plate. The plate was thenair-dried and inserted into the sample introduction port of theVoyager Elite biospectrometry MALDI time-of-flight (TOF) massspectrometer (PerSeptive Biosystems). The resulting signal wasdigitized at a frequency of 1 GHz and accumulated for 64 scans.Purified conditioned medium from empty vector transfections wasused to control individual experiments for variability in extent ofsubstrate conversion to product. For tandem mass spectrometrysequencing, a hybrid quadrupole time-of-flight mass spectrometer(Q-TOF MS) (Micromass UK Limited) equipped with an orthogonalelectrospray source (Z-spray) was used. The quadrupole was set upto pass precursor ions of selectedm/z to the hexapole collision cell(Q2), and product ion spectra were acquired with the TOF analyzer.Argon was introducedinto the Q2 with a collision energy of 35 eV andcone energy of 25 V.

ResultsIdentification of ACE2A novel human cDNA homologous to ACE was identifiedamong 19 000 59end sequences obtained from a heart failure

ventricle cDNA library. Complete sequencing of the 2.3-kbpartial cDNA clone and a 1178-bp 59extension yielded an805-codon open reading frame encoding ACE2 (see Gen-Bank No. AF291820 and Figure 1 for amino acid sequence).The sequence includes an initiator methionine codon within aKozak consensus sequence,8 a putative signal sequence (ami-no acids 1 to 18), a potential transmembrane domain (aminoacids 740 to 763), and a potential metalloprotease zincbinding site (amino acids 374 to 378, HEMGH).

Comparison with ACE proteins indicated that ACE2 con-tains a single catalytic domain (amino acids 147 to 555) thatis 42% identical to each of the two catalytic domains inendothelial ACE.1,9 Human ACE2 is 33% identical overall tohuman testicular ACE, the alternatively spliced product of theACE gene that also contains a single catalytic site, althoughthere is considerable divergence in C-terminal regions (Fig-ure 1). Phylogenetic analysis indicated that the mammalianACE family members are more similar to each other than theyare toDrosophilaACE (Figure 2).

Comparison of the human ACE2 cDNA with previouslyunannotated human genomic sequences (GenBank No.AC003669, BAC GS-594A7) revealed an ACE2 gene com-prising 19 exons (not shown). Relative conservation ofexon/intron organization between the ACE2 and ACE cata-lytic domains2 further suggests that these two genes arose byduplication of a common ancestor (Table 1).

ACE2 Tissue DistributionNorthern blot analysis revealed a'3.4-kb ACE2 transcriptexpressed only in heart, kidney, and testis of 23 human tissues

Figure 2. Phylogenetic tree of ACE family members. The phylo-genetic relationships among the several ACE and ACE2sequences were determined by the Clustal V method and theNeighbor-joining method (see Materials and Methods). The phy-logenies are based on divergence found in pair distances (num-bers are based on substitution events).

TABLE 1. Comparison of the Exon Sizes Encoding the PutativeCatalytic Domain of ACE2 and the Catalytic Domains of ACE

ACE2 ACE

Domain 1 Domain 2

Bases, bp (Exon) Bases, bp (Exon) Bases, bp (Exon)

144 (5) 144 (4) 144 (17)

113 (6) plus 106 (7) 192 (5) 192 (18)

98 (8) 98 (6) 100 (19)

170 (9) 173 (7) 171 (20)

227 (10) 224 (8) 224 (21)

145 (11) 145 (9) 145 (22)

99 (12) 99 (10) 99 (23)

123 (13) 123 (11) 122 (24)




examined; a minor'8-kb band was also detected in kidney(Figure 3A). In contrast, a doublet of endothelial ACE mRNAwas detected in 16 of 23 tissues examined, and the much shortertesticular isoform was also detected in testis (Figure 3B).Immunohistochemical analysis of the ventricular myocardiumidentified ACE2 localized to the endothelium of most intramyo-cardial vessels including capillaries, venules, and medium-sizedcoronary arteries and arterioles. Immunostaining was apparent,but to a lesser extent, in vascular smooth muscle cells and focallyin the adventitia of some larger vessels (Figures 4A through 4F).There was no observable difference in the distribution orintensity of protein expression in sections from failing andnonfailing heart samples. In the kidney, ACE2 protein was againpresent throughout the endothelium and focally in rare smoothmuscle cells of medium-sized vessels. It was also identified inproximal tubule epithelial cells (Figures 4G and 4H).

Expression of Recombinant ACE2ACE is made as a transmembrane protein, some of which iscleaved posttranslationally to generate a secreted form in vivoand in cell culture.10–12 To determine whether ACE2 isprocessed in a similar fashion, CHO cells were transientlytransfected with expression plasmids containing either ACE2cDNA (pACE2) or no insert (pcDNA3.1). Conditioned me-dium and whole-cell lysates were compared in Westernanalyses using polyclonal antiserum raised to an ACE2peptide (amino acids 489 to 508; see Materials and Methods)present in both full-length and putative cleaved secretedforms (Figure 5). An approximately 90-kDa immunoreactiveband was present in the whole-cell lysate, and a slightlysmaller band was detected in the conditioned medium ofACE2-transfected cells indicating that full-length ACE2 isprocessed in CHO cells to generate a secreted form.

ACE2 Catalytic ActivityWe tested a variety of vasoactive and hormonal peptides ascandidate substrates using conditioned media from trans-fected CHO cells as a source of secreted recombinant ACE2.We compared ACE2 activity with that of ACE. The enzymeswere incubated with synthetically prepared peptides in vitro,and reaction products were analyzed by mass spectrometry.ACE converted the decapeptide Ang I (Ang1-10;m/z

Figure 3. Tissue-restricted expression of human ACE2. North-ern blots representing multiple human tissues were hybridizedwith probes from either ACE2 (A) or ACE (B) (see Materials andMethods). RNA standards (kb) are shown at left. sk. muscleindicates skeletal muscle; sm. intestine, small intestine; and pbl,peripheral blood leukocyte.

Figure 4. Cell-specific expression of ACE2 protein in heart andkidney. Human tissue sections were probed with antibody tohuman ACE2 (Ab ACE251) in the presence or absence of peptideantigen (see Materials and Methods). Arrows indicate endothelialcells; a, artery; and t, proximal tubule. A, C, and E, Three sec-tions of human left ventricle stained with Ab ACE251. B, D, andF, Corresponding serial sections incubated with Ab ACE251 inthe presence of competing peptide ACE251. G and H, Serialsections of human kidney incubated with Ab ACE251 in theabsence and presence, respectively, of competing peptideACE251.

Figure 5. Recombinant ACE2 protein is secreted by transfectedCHO cells. Conditioned media and lysates from cells trans-fected with full-length ACE2 or empty vector control were runon SDS-PAGE and immunoblotted using an antibody to humanACE2 (Ab ACE2489). Molecular mass standards are shown at left.




1296.68) to Ang II (Ang1-8;m/z 1046.54), as expected, bycleavage of the C-terminal His-Leu dipeptide (Figure 6).ACE2, in contrast, converted Ang I to a new species ofm/z1183.60, which when sequenced by tandem mass spectrom-etry proved to be the nonapeptide Ang1-9 (data not shown).Thus, ACE2, apparently a carboxypeptidase, quantitativelycleaved the C-terminal Leu from this substrate. No furthercleavage of Ang1-9 by ACE2 was evident on prolongedincubation (not shown). In similar experiments, ACE2 re-moved the C-terminal residue from three other vasoactivepeptides, neurotensin, kinetensin, and des-Arg bradykinin(Table 2). However, it failed to cleave bradykinin and 15other unrelated vasoactive and hormonal peptides, indicatingconsiderable specificity.

We tested the effects of ACE inhibitors on ACE2 activity invitro. Conversion of Ang I to Ang1-9 by ACE2 was not inhibited bylisinopril under conditions that completely inhibited the generationof Ang II by ACE (Figure 7). Identical results were obtained usinganother ACE inhibitor, captopril (not shown). Thus, despite theirhomologous catalytic domains, ACE2 and ACE are biochemicallyand pharmacologically distinct.

Metabolism of Ang1-9Ang1-9 is found in vivo and accumulates in animals treated withACE inhibitors.13,14On the basis of the present studies, Ang1-9may be generated in heart and kidney by endothelium-associatedACE2. To explore possible fates for this peptide, we tested itsconversion in vitro and in serum-free cell culture. Incubation ofAng1-9 with ACE in vitro generated Ang1-7 (m/z899.47) andAng1-5 (m/z 665.36), apparently by sequential cleavage ofC-terminal dipeptides (Figure 8). Conversion to Ang II (Ang1-8)was not detected. Incubation of Ang1-9 with primary rat neo-

natal cardiac myocytes, which produce ACE,15 also yieldedAng1-7 and Ang1-5, as well as Ang1-4 (m/z508.28) (Figure 9).Addition of lisinopril blocked only the generation of Ang1-5,indicating that in these cultures generation of Ang1-5 requiresACE, whereas generation of Ang1-7 and Ang1-4 do not. Othercardiomyocyte peptidases that might cleave Ang1-9 are un-known at present.

DiscussionThe present study identifies ACE2 as the first known humanhomologue of ACE, an enzyme that plays a central role invascular, renal, and myocardial physiology. The ACE2 andACE catalytic domains are 42% identical in amino acidsequence, and conservation of exon/intron organization fur-ther indicates that the two genes evolved from a commonancestor. Also like ACE, ACE2 is expressed in endotheliumand can be secreted in vitro through proteolytic cleavage of aputative membrane-bound form. In contrast to ACE, how-ever, ACE2 is highly tissue-specific: whereas ACE is ex-pressed ubiquitously in the vasculature, human ACE2 isrestricted to heart, kidney, and testis. In addition to endothe-lial expression, ACE2 is present in smooth muscle in somecoronary vessels and focally in tubular epithelium of thekidney. Thus, ACE2 is particularly well poised to participatein both cardiac and renal physiology.

Both ACE and ACE2 cleave Ang I, but their activities aredistinct. Whereas ACE is a dipeptidase, ACE2 removes the

Figure 6. ACE2 converts Ang I to Ang1-9. Ang I was incubatedwith ACE2, ACE, or no enzyme, as indicated, for 30 minutes at37°C, and the products were analyzed by mass spectrometry:no enzyme control (purified fraction from conditioned medium ofCHO cells transfected with empty vector) showing no conver-sion (A), purified porcine kidney ACE showing conversion toAng1-8 (Ang II) (B), and purified ACE2 showing conversion toAng1-9 (C).

Figure 7. ACE2 conversion of Ang I is not blocked by lisinopril.Ang I was incubated for 30 minutes at 37°C in the presence ofporcine kidney ACE (A), porcine kidney ACE1lisinopril (6.6 mmol/L)(B), purified ACE2 (C), and purified ACE21lisinopril (6.6 mmol/L) (D),and the products were analyzed by mass spectrometry.




single C-terminal Leu residue to generate Ang1-9. Ang1-9 hasbeen identified in vivo in rat and human plasma, but its functionis unknown.13,14Some studies have suggested that it may be anendogenous inhibitor of ACE.16 Our findings indicate thatAng1-9 is a competitive inhibitor of ACE because it is itself anACE substrate. Under conditions in which ACE is inhibited,

such as after long-term administration of ACE inhibitors in rats,Ang1-9 levels have been shown to be increased in plasma andkidney.13,14This increase in Ang1-9 steady-state levels could bedue to decreased catabolism of Ang1-9 by ACE. Conversely, theincreased levels of Ang1-9 could be due to increased productionby ACE2 as a result of increased availability of Ang I substrate.Together these results indicate that alternate pathways of Ang Imetabolism exist and that these pathways may be amplified inthe presence of ACE inhibitors.

Other investigators have found that Ang II can be generated inhuman renal extracts in a two-step process with Ang1-9 as anintermediate.14,17The enzymes responsible for this activity havenot been definitively determined, but cathepsin A has beenproposed.18 ACE2 is most highly expressed in kidney and maybe responsible for some or all of the Ang1-9 generated from AngI in these studies. ACE2 does not convert Ang1-9 further to AngII, at least under our in vitro conditions. Thus, ACE2 would notdirectly be responsible for Ang II generation in the presence ofACE inhibitors.

Other angiotensin peptides generated by the combined activ-ities of ACE2 and other peptidases, including ACE, may playkey signaling roles in heart and kidney. Although the functionsof Ang1-5 and Ang1-9 are unknown, Ang1-7 is known tofunction as a vasodilator (for review, see Reference 3). Thus,ACE2 could function to increase local vasodilation through itsability to produce a precursor to Ang1-7. In addition, ACE2could also alter vasomotor tone by decreasing the availability ofAng I, a well-established substrate for ACE and precursor of thevasoconstrictor Ang II (Figure 10). Thus, ACE2 activity in the

Figure 8. Ang1-9 is a substrate for ACE. Ang1-9 was incubatedalone (A, time50) or with ACE (B, time55 seconds at 20°C; C,time530 minutes at 37°C), and products were analyzed bymass spectrometry.

Figure 9. Conversion of Ang1-9 to products by rat neonatalcardiac myocyte culture. [Sar1]Ang1-9 was added to serum-freecultures of primary rat neonatal cardiac myocytes for 0 hours (A)and 48 hours in the absence (B) and presence (C) of lisinopril.The resulting products in the conditioned medium were ana-lyzed by mass spectrometry.

TABLE 2. Peptides Tested for Cleavage by ACE2

C-Terminal 4-Amino Acids

Cleaved*

Angiotensin I P-F-H*L

des-Arg bradykinin F-S-P*F

Neurotensin P-Y-I*L

Kinetensin P-Y-F*L

Not Cleaved

Angiotensin (1-9) H-P-F-H

Met enkephalin G-G-F-M

Leu enkephalin G-G-F-L

Vasopressin C-P-R-G

Oxytocin C-P-L-G

Atrial natriuretic peptide S-F-R-Y

Adrenocorticotropic hormone P-L-E-F

Bradykinin S-P-F-R

Eledoisin I-G-L-M

Neuromedin C G-H-L-M

Litorin G-H-F-M

Ranatensin G-H-F-M

Bombesin G-H-L-M

Luteinizing hormone–releasing hormone L-R-P-G

Neuropeptide Y R-Q-R-Y

Mast cell–degranulating peptide HR1 K-K-V-L

*Site of cleavage.




heart could induce local vasodilation, thereby maintaining myo-cardial perfusion under conditions of generalized systemic va-soconstriction such as acute blood loss. Local ACE2 activity inthe endothelium and tubular epithelium of the kidney maymodulate renal blood flow distribution and salt and waterhandling and thereby regulate blood pressure.

It remains to be determined whether Ang I is indeed aphysiological substrate for ACE2 in vivo and whether othersubstrates for ACE2 exist. Our in vitro studies demonstrate that,in addition to Ang I, ACE2 is capable of cleaving at least threeother vasoactive peptides, des-Arg bradykinin, neurotensin, andkinetensin. Des-Arg bradykinin is involved locally in vesseldilation through binding to the B1 receptor that is expressedunder conditions of tissue damage or inflammation.19 This isconsistent with a role for ACE2 in local regulation of vasomotortone, through both Ang I and des-Arg bradykinin cleavage. Incontrast, ACE cleaves the vasodilator bradykinin, which actssystemically through the B2 receptor. Neurotensin has diverseeffects in the cardiovascular, nervous, and digestive systems (forreview, see Reference 20), whereas kinetensin, a related peptidederived in vitro, stimulates mast cell degranulation and vascularpermeability.21 Degradation of these peptides by ACE2 mayserve to modulate these activities.

The elucidation of ACE2 adds a potential new dimension tothe complexity of the RAS. Enzymes other than ACE, eg, heartchymase, convert Ang I to Ang II (for review, see References 2and 22), and Ang II can act through at least three distinctreceptors (for review, see References 23 and 24). In addition,multiple other angiotensin-related peptides including Ang1-9and Ang1-7 are present and may have unique effects. Targetingof the cardiac RAS at several of these points is a mainstay ofdrug treatment for hypertension and heart failure. ACE2, ex-pressed specifically in heart and kidney and capable of cleaving

Ang I and other key vasoactive peptides, provides additionalpossibilities for the development of novel therapeutics.

AcknowledgmentsThis work was supported by funding from Eli Lilly & Company,Indianapolis, Ind. We gratefully acknowledge the skilled technicalassistance of Stephen Katz, Yong Yao Xu, Roslyn Feeley, Ron Meyer,and Lin Zhang. We appreciate the gift of a left ventricular heart failuretissue sample from George Sandusky and Nancy Bowling of Eli Lilly &Company, Indianapolis, Ind. The advice and support of Andrew Nicholsand Geoff Ginsburg are also greatly appreciated.

References1. Ehlers MRW, Riordan JF. Angiotensin-converting enzyme. Zinc- and

inhibitor-binding stoichiometries of the somatic and testis isozymes.Bio-chemistry. 1991;30:7118–7126.

2. Hubert C, Houot AM, Corvol P, Soubrier F. Structure of the angiotensinI-converting enzyme gene: two alternate promoters correspond to evolu-tionary steps of a duplicated gene.J Biol Chem. 1991;266:15377–15383.

3. Stock P, Liefeldt L, Paul M, Ganten D. Local renin-angiotensin systems incardiovascular tissues: localization and functional role.Cardiology. 1995;86:2–8.

4. Urata H, Nishimura H, Ganten D, Chymase-dependent angiotensin IIforming system in humans.Am J Hypertens. 1996;9:277–284.

5. Ferrario CM, Chappell MC, Dean RH, Iyer SN. Novel angiotensin peptidesregulate blood pressure, endothelial function, and natriuresis.J Am SocNephrol. 1998;9:1716–1722.

6. Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments ona microcomputer.Comput Appl Biosci. 1989;5:151–153.

7. Long CS, Henrich CJ, Simpson PC. A growth factor for cardiac myocytes isproduced by cardiac nonmyocytes.Cell Regul. 1991;2:1081–1095.

8. Kozak M. Interpreting cDNA sequences: some insights from studies ontranslation.Mamm Genome. 1996;7:563–574.

9. Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear G,Corvol P. Two putative active centers in human angiotensin I-convertingenzyme revealed by molecular cloning.Proc Natl Acad Sci U S A. 1988;85:9386–9390.

10. Ehlers MR, Schwager SL, Chubb AJ, Scholle RR, Brandt WF, Riordan JF.Proteolytic release of membrane proteins: studies on a membrane-protein-solubilizing activity in CHO cells.Immunopharmacology. 1997;36:271–278.

11. Sadhukhan R, Sen GC, Ramchandran R, Sen I. The distal ectodomain ofangiotensin-converting enzyme regulates its cleavage-secretion from the cellsurface.Proc Natl Acad Sci U S A. 1998;95:138–143.

12. Skeggs L Jr. Historical overview of the renin-angiotensin system. In: DoyleAE, Bearn AG, eds.Hypertension and the Angiotensin System: TherapeuticApproaches. New York, NY: Raven Press; 1984:31–45.

13. Johnson H, Kourtis S, Waters J, Drummer OH. Radioimmunoassay forimmunoreactive [des-Leu10]-angiotensin I.Peptides. 1989;10:489–492.

14. Drummer OH, Kourtis S, Johnson H. Effect of chronic enalapril treatment onenzymes responsible for the catabolism of angiotensin I and formation ofangiotensin II.Biochem Pharmacol. 1990;39:513–518.

15. Dostal DE, Rothblum KN, Conrad KM, Cooper GR, Baker KM. Detection ofangiotensin I and II in cultured rat cardiac myocytes and fibroblasts.Am JPhysiol. 1992;263:C851–C863.

16. Snyder RA, Wintroub BU. Inhibition of angiotensin-converting enzyme bydes-Leu10-angiotensin I: a potential mechanism of endogenous angiotensin-converting enzyme regulation.Biochim Biophys Acta. 1986;871:1–5.

17. Changaris DG, Miller JJ, Levy RS. Angiotensin II generated by a humanrenal carboxypeptidase.Biochem Biophys Res Commun. 1986;138:573–579.

18. Miller JJ, Changaris DG, Levy RS. Conversion of angiotensin I to angioten-sin II by cathepsin A isoenzymes of porcine kidney.Biochem Biophys ResCommun. 1988;154:1122–1129.

19. Marceau F. Kinin B1 receptors: a review.Immunopharmacology. 1995;30:1–26.

20. Vincent JP, Mazella J, Kitabgi P. Neurotensin and neurotensin receptors.Trends Pharmacol Sci. 1999;20:302–309.

21. Sydbom A, Ware J, Mogard MH. Stimulation of histamine release by thepeptide kinetensin.Agents Actions. 1989;27:68–71.

22. Danser AH, Saris JJ, Schuijt MP, van Kats JP. Is there a local renin-angio-tensin system in the heart?Cardiovasc Res. 1999;44:252–265.

23. Lorell BH. Role of angiotensin AT1, and AT2 receptors in cardiac hypertro-phy and disease.Am J Cardiol. 1999;83:48H–52H.

24. Riordan JF. Angiotensin II: biosynthesis, molecular recognition, and signaltransduction.Cell Mol Neurobiol. 1995;15:637–651.

Figure 10. Schematic of Ang I and bradykinin peptide process-ing by ACE and ACE2. A, Proposed pathways of Ang I cleavageto products via ACE and ACE2. B, Proposed pathways of bra-dykinin and des-Arg bradykinin degradation to products by ACEand ACE2.




and Susan ActonStagliano, Michael Donovan, Betty Woolf, Keith Robison, Raju Jeyaseelan, Roger E. Breitbart Mary Donoghue, Frank Hsieh, Elizabeth Baronas, Kevin Godbout, Michael Gosselin, Nancy

Angiotensin I to Angiotensin 1-9Related Carboxypeptidase (ACE2) Converts−A Novel Angiotensin-Converting Enzyme

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2000 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.87.5.e12000;87:e1-e9Circ Res.

http://circres.ahajournals.org/content/87/5/e1World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circres.ahajournals.org//subscriptions/

is online at: Circulation Research Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer about this process is available in the

located, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is

can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:


http://circres.ahajournals.org/content/87/5/e1

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circres.ahajournals.org//subscriptions/


a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to...

Documents