Hydrolysis of Biological Peptides by Human Angiotensin-converting Enzyme-related Carboxypeptidase
2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês
10.1074/jbc.m200581200
ISSN1083-351X
AutoresChad Vickers, Paul Hales, Virendar K. Kaushik, Lawrence R. Dick, James M. Gavin, Jin Tang, Kevin Godbout, Thomas F. Parsons, Elizabeth Baronas, F. Hsieh, Susan Acton, Michael A. Patane, Andrew J. Nichols, Peter J. Tummino,
Tópico(s)Renin-Angiotensin System Studies
ResumoHuman angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased ∼10-fold by Cl− and F− but is unaffected by Br−. ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar) (k cat/K m = 1.9 × 106m−1 s−1), apelin-13 (k cat/K m = 2.1 × 106m−1s−1), and dynorphin A 1–13 (k cat/K m = 3.1 × 106m−1 s−1). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar) as a substrate than with angiotensin I (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar, 9.Rovere C. Barbero P. Kitabgi P. J. Biol. Chem. 1996; 271: 11368-11375Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10.Bunning P. Riordan J.F. Biochemistry. 1983; 22: 110-116Crossref PubMed Scopus (99) Google Scholar). ACE2 also efficiently hydrolyzes des-Arg9-bradykinin (k cat/K m = 1.3 × 105m−1 s−1), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X (1–3 residues)-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid. Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased ∼10-fold by Cl− and F− but is unaffected by Br−. ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar) (k cat/K m = 1.9 × 106m−1 s−1), apelin-13 (k cat/K m = 2.1 × 106m−1s−1), and dynorphin A 1–13 (k cat/K m = 3.1 × 106m−1 s−1). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar) as a substrate than with angiotensin I (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar, 9.Rovere C. Barbero P. Kitabgi P. J. Biol. Chem. 1996; 271: 11368-11375Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10.Bunning P. Riordan J.F. Biochemistry. 1983; 22: 110-116Crossref PubMed Scopus (99) Google Scholar). ACE2 also efficiently hydrolyzes des-Arg9-bradykinin (k cat/K m = 1.3 × 105m−1 s−1), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X (1–3 residues)-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid. Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) 1The abbreviations used are: ACE2angiotensin-converting enzyme-related carboxypeptidaseACEangiotensin I-converting enzymeAng Iangiotensin I (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar, 9.Rovere C. Barbero P. Kitabgi P. J. Biol. Chem. 1996; 271: 11368-11375Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10.Bunning P. Riordan J.F. Biochemistry. 1983; 22: 110-116Crossref PubMed Scopus (99) Google Scholar)Ang IIangiotensin II (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar)Mca-APK(Dnp)((7-methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH)Mca-YVADAPK(Dnp)(7-methoxycoumarin-4-yl)acetyl-Tyr-Val-Ala-Asp-Ala-Pro-Lys(2,4-dinitrophenyl)-OHMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightHPLChigh pressure liquid chromatographyMES4-morpholineethanesulfonic acidCHES2- (cyclohexylamino)ethanesulfonic acidCAPS3-(cyclohexylamino) propanesulfonic acidbis-Tris propane1,3-bis[tris(hydroxymethyl)methylamino]propane is a close homolog of human endothelial angiotensin I-converting enzyme (ACE, EC 3.4.15.1), with 42% protein sequence identity between the catalytic domains (for sequence alignment, see Ref. 1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar). ACE, a component of the renin-angiotensin system, is a zinc metalloprotease that catalyzes cleavage of the C-terminal dipeptide from Ang I to produce the potent vasopressor octapeptide Ang II (2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar). ACE-inhibiting drugs have an antihypertensive effect and substantially lower the long-term risk of death, heart attack, stroke, coronary revascularization, heart failure, and complications related to diabetes mellitus (for review, see Ref.3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar). ACE also inactivates bradykinin by catalyzing the cleavage of the C-terminal dipeptide from the nonapeptide hormone (4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar), and ACE inhibitor-induced cough has been attributed to inhibition of bradykinin metabolism (5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar). angiotensin-converting enzyme-related carboxypeptidase angiotensin I-converting enzyme angiotensin I (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar, 9.Rovere C. Barbero P. Kitabgi P. J. Biol. Chem. 1996; 271: 11368-11375Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10.Bunning P. Riordan J.F. Biochemistry. 1983; 22: 110-116Crossref PubMed Scopus (99) Google Scholar) angiotensin II (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 2.Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (748) Google Scholar, 3.Francis G.S. N. Engl. J. Med. 2000; 342: 201-202Crossref PubMed Scopus (80) Google Scholar, 4.Blais C.J. Marceau F. Rouleau J.-L. Adam A. Peptides (Elmsford). 2000; 21: 1903-1940Crossref PubMed Scopus (121) Google Scholar, 5.Fox A.J. Lalloo U.G. Belvisi M.G. Bernareggi M. Chung K.F. Nat. Med. 1996; 2: 814-817Crossref PubMed Scopus (257) Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar, 7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8.Shapiro R. Holmquist B. Riordan J.F. Biochemistry. 1983; 22: 3850-3857Crossref PubMed Scopus (81) Google Scholar) ((7-methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH) (7-methoxycoumarin-4-yl)acetyl-Tyr-Val-Ala-Asp-Ala-Pro-Lys(2,4-dinitrophenyl)-OH matrix-assisted laser desorption ionization time-of-flight high pressure liquid chromatography 4-morpholineethanesulfonic acid 2- (cyclohexylamino)ethanesulfonic acid 3-(cyclohexylamino) propanesulfonic acid 1,3-bis[tris(hydroxymethyl)methylamino]propane Like ACE, ACE2 is expressed in endothelial cells, although its expression is restricted to fewer tissues, which include the heart, kidney, and testis (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar). ACE2 was identified as a zinc metalloprotease due to its canonical HEXXH sequence (amino acids 374–378) (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar), its inhibition by EDTA (6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar), and its sequence identity with the catalytic residues of ACE (7.Fernandez M. Liu X. Wouters M.A. Heyberger S. Husain A. J. Biol. Chem. 2001; 276: 4998-5004Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The ACE inhibitors captopril, lisinopril, and enalaprilat are not inhibitors of ACE2 (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar, 6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar). The physiological and pathophysiological role of ACE2 is not yet clearly understood. To better understand the physiological role of ACE2, a detailed biochemical analysis of ACE2 substrate preference was undertaken. We reported previously that secreted recombinant ACE2 expressed in Chinese hamster ovary cells catalyzes cleavage of the C-terminal residue of the biological peptides Ang I, des-Arg9-bradykinin, neurotensin 1–13, and kinetensin (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar). Similarly, Tipnis et al. (6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1745) Google Scholar) reported that unpurified ACE2 expressed in Chinese hamster ovary cells catalyzes the hydrolysis of the C-terminal residue of Ang I and Ang II. Herein is the first report of characterization of the catalytic activity of purified ACE2. A sensitive fluorogenic substrate was developed and used to assess the dependence of ACE2 hydrolytic activity on pH and on the presence of monovalent anions. Also, ACE2 substrates were identified from screening biological peptides, and the kinetic constants were determined for hydrolysis of those peptides. The identified peptides are candidate ACE2 physiological substrates. HPLC columns were purchased from the Waters Corp. (Milford, MA). Toyopearl columns were purchased from Tosoh Biosep (Montgomeryville, PA). The peptide Mca-APK(Dnp) was synthesized by Anaspec, Inc. (San Diego, CA). Biological peptides were purchased from Sigma-Aldrich Co., Bachem Bioscience (King of Prussia, PA), and American Peptide Co. (Sunnyvale, CA). Specifically, Ang I, Ang II, and dynorphin A 1–13 were purchased from Sigma-Aldrich Co. 7-Methoxycoumarin-4-yl)acetyl-YVADAPK(2,4-dinitrophenyl)-OH (M-2195), apelin-13, β-casomorphin, des-Arg9-bradykinin, Lys-des-Arg9-bradykinin, and neurotensin 1–8 were purchased from Bachem Bioscience. An expression vector was generated encoding a secreted form of human ACE2 (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar) (amino acids 1–740) in the pBac Pak9 vector (CLONTECH). Sf9 insect cells were infected at a multiplicity of infection of 0.1 with ACE2 baculovirus of titer 1.1 × 109 pfu/ml. A 10-liter fermentation run was carried out with SF9 cells grown to a density of 1.3 × 106 cells/ml in SF900II serum-free medium (Invitrogen), 18 mml-glutamine, and 1× antibiotic-antimycotic (from 100X stock; Invitrogen) at 27 °C. At 96 h after infection, cells were pelleted at 5000 × gcentrifugation, and the culture supernatant was collected, frozen, and stored at −80 °C. The thawed supernatant was filtered (0.2-μm filter) and loaded onto a Toyopearl QAE anion exchanger column, and the column was washed with buffer A (25 mm Tris-HCl, pH 8.0). A 0–50% gradient elution was then performed with increasing buffer B (1.0 mNaCl and 25 mm Tris-HCl, pH 8.0) using a total of 5 column volumes. The ACE2-containing fractions, as detected by Coomassie Blue-stained SDS-PAGE, were pooled, and (NH4)2SO4 was added to a final concentration of 1.0 m. The sample was then loaded onto a Toyopearl Phenyl column. After loading, the column was washed with buffer C (1.0 m(NH4)2SO4 and 25 mmTris-HCl, pH 8.0) using 5 column volumes and then gradient-eluted with buffer A (0–100%). The ACE2-containing fractions, as detected by Coomassie Blue-stained SDS-PAGE, were pooled and dialyzed against buffer A at 4 °C overnight. The dialyzed ACE2 protein sample was sequentially loaded onto MonoQ column (Amersham Biosciences) and gradient-eluted with buffer B. The ACE2-containing fractions from the MonoQ column, as detected by Coomassie Blue-stained SDS-PAGE, were concentrated with a Centricon (Millipore Corp., Bedford, MA) concentrator, with a molecular mass cutoff of 30 kDa. The concentrated sample was loaded onto a TSK G3000SWxl size exclusion column and eluted with buffer A. Mca-APK(Dnp) was dissolved in 100% Me2SO and quantitated by measuring absorbance at 350 nm using an extinction coefficient of 15,000m−1 cm−1. All reactions were performed in microtiter plates with a 100-μl total volume at ambient temperature. To each well, we added 75 μl of salt (NaCl, NaF, NaBr, or KCl), 5.0 μl of buffer (50 mm, final concentration), and 10 μl of Mca-APK(Dnp) (50 μm, final concentration), and the reaction was initiated by the addition of 10 μl of ACE2 (0.15 nm, final concentration). The buffers used in the pH dependence studies were sodium acetate, MES, bis-Tris propane, CHES, and CAPS, and the buffer used in the anion dependence studies was MES. The final Me2SO concentration in the assay was 0.7%. The assay was monitored continuously by measuring the increase in fluorescence (excitation = 320 nm, emission = 405 nm) upon substrate hydrolysis using a Polarstar Galaxy fluorescence plate reader (BMG Lab Technologies, Durham, NC). Initial velocities were determined from the rate of fluorescence increase over the 15–60-min time course corresponding to ≤10% product formed. The pH was found to have no significant effect on product fluorescence across the range of pH 5 to pH 10 in the assay buffers. Reactions were performed in microtiter plates at ambient temperature. To each well, we added 5 μl of 1 mm peptide (50 μm, final concentration) and 45 μl of buffer (50 mm MES, 300 mm NaCl, 10 μmZnCl2, and 0.01% Brij-35 pH 6.5), and reaction was initiated by the addition of 50 μl of 100 nm ACE2 (50 nm, final concentration) or buffer (control). Reactions were performed at room temperature for 2 h and quenched with 20 μl of 0.5 m EDTA. Samples were then analyzed by MALDI-TOF mass spectrometry for detection of hydrolysis and determination of products formed. Mass spectrometry was performed on a Voyager Elite biospectrometry MALDI-TOF spectrometer (PerSeptive Biosystems, Framingham, MA) as described previously (1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar). The peptides that were found to be hydrolyzed by ACE2 were re-assayed under the same conditions in the presence of a high concentration of a potent, specific inhibitor of ACE2 2E. Calderwood, N. Dales, A. Gould, B. Guan, T. Ocain, and M. Patane, unpublished results.to confirm specific cleavage by this protease. Rates of substrate hydrolysis were determined by reversed phase chromatography using a capillary HPLC system (Agilent, Palo Alto, CA). Reactions were performed in 100 μl in microtiter plates at ambient temperature. Reactions were initiated by the addition of 50 μl of ACE2 (0.025–0.70 nm, final concentration) to 50 μl of peptide in assay buffer (50 mmMES, 300 mm NaCl, 10 μm ZnCl2, and 0.01% Brij-35, pH 6.5). Reactions were performed at room temperature for 0, 15, 22.5, or 30 min and quenched by the addition of 10 μl of 0.5 m EDTA. Substrate concentrations ranged from 0.8–2000 μm, and hydrolysis was limited to ≤15% product formed (initial velocity conditions). Concentrations of the biological peptides were determined spectrophotometrically. Substrate and product peptides were resolved on a YMC ODS-A 1.0 × 50-mm 120A 5-μm column using a gradient of 10–45% B (A, water/0.1% trifluoroacetic acid (v/v); B, acetonitrile/0.1% trifluoroacetic acid (v/v)) and detected by absorbance at 215 nm. The peptide Mca-APK(Dnp) and its reaction product were resolved in the same manner using a gradient of 15–65% B and detected by absorbance at 350 nm. Injection volumes ranged from 0.5–20 μl. The extent of hydrolysis was determined from the areas of the substrate and product peaks (area of product peak/(area of product peak + area of substrate peak)) and converted to micromoles of product formed. Initial velocities (v) of substrate hydrolysis were calculated from the slopes of micromoles of product formed versus time. Initial velocities (v) were plotted versus substrate concentration and fit to the Michaelis-Menten equation (v =V max[S]/K m + [S]) using Grafit software (Erithacus Software Ltd., Surrey, United Kingdom). Turnover numbers (k cat) were calculated from the equation k cat = V max/[E], using a calculated ACE2 molecular mass of 85,314 Da and considering the enzyme sample to be essentially pure and fully active. Recombinant soluble human ACE2, encoding amino acids 1–740 of the 805-amino acid full-length enzyme and deleting the C-terminal transmembrane domain, was expressed in Chinese hamster ovary cells and isolated to ∼90% purity by SDS-PAGE (as described previously, Ref.1.Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: 1-9Crossref PubMed Google Scholar). This ACE2 sample was used to screen a number of commercially available intramolecularly quenched fluorescent peptides to identify a suitable fluorescent substrate for initial enzyme characterization. The caspase-1 substrate Mca-YVADAPK(Dnp) was found to be hydrolyzed by ACE2, as measured by a time-dependent increase in fluorescence (excitation = 320 nm, emission = 405 nm). Analysis of the reaction products by MALDI-TOF mass spectrometry indicated hydrolysis of the Pro-Lys(2,4-dinitrophenyl) peptide bond. A truncated peptide with more efficient intramolecular fluorescence quenching, Mca-APK(Dnp), was synthesized and assayed as an ACE2 substrate with the goal of improving the fluorescence signal of the assay. Complete hydrolysis of 40 μm Mca-APK(Dnp) resulted in a 300-fold fluorescence increase over background, whereas complete hydrolysis of the same concentration of Mca-YVADAPK(Dnp) resulted in a 21-fold increase over background. The Mca-APK(Dnp) substrate is hydrolyzed by ACE2 and was used for characterization of the enzyme activity. Recombinant soluble human ACE2 was expressed in Sf9 insect cells and isolated to >98% purity, based on SDS-PAGE (Fig. 1 C), by a four-step chromatography protocol. The purified protein sample was confirmed to be ACE2 by peptide mapping of trypsin-digested protein, analyzed by MALDI-TOF mass spectrometry (data not shown). The molecular mass of the purified ACE2 (89.6 kDa, as determined by MALDI-TOF mass spectrometry) is greater than that predicted from the peptide sequence (85.314 kDa). The higher molecular mass is likely to be due to glycosylation, as has been reported for ACE2 (6.Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text
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