Inhibitory antibodies to human angiotensin-converting enzyme

Fine epitope mapping and mechanism of action

Olga E. Skirgello, Irina V. Balyasnikova, Petr V. Binevski, Zhu Li Sun, Igor I. Baskin, Vladimir A. Palyulin, Andrei B. Nesterovitch, Ronald F. Albrecht, Olga A. Kost, Sergei M. Danilov*

*Corresponding author for this work

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the SD-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E-mAb and E·S·mAb complexes, respectively; however, only complex E·S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

Original languageEnglish (US)
Pages (from-to)4831-4847
Number of pages17
JournalBiochemistry
Volume45
Issue number15
DOIs
StatePublished - Apr 18 2006

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Epitope Mapping
Peptidyl-Dipeptidase A
Epitopes
Antibodies
Monoclonal Antibodies
Enzymes
Amino Acids
Bearings (structural)
Substrates
Lisinopril
Mutagenesis
Mutation
Kinetics
CHO Cells
Hinges
Catalysis
Conformations
Assays
Catalytic Domain
Enzyme-Linked Immunosorbent Assay

ASJC Scopus subject areas

  • Biochemistry

Cite this

Skirgello, O. E., Balyasnikova, I. V., Binevski, P. V., Sun, Z. L., Baskin, I. I., Palyulin, V. A., ... Danilov, S. M. (2006). Inhibitory antibodies to human angiotensin-converting enzyme: Fine epitope mapping and mechanism of action. Biochemistry, 45(15), 4831-4847. https://doi.org/10.1021/bi052591h
Skirgello, Olga E. ; Balyasnikova, Irina V. ; Binevski, Petr V. ; Sun, Zhu Li ; Baskin, Igor I. ; Palyulin, Vladimir A. ; Nesterovitch, Andrei B. ; Albrecht, Ronald F. ; Kost, Olga A. ; Danilov, Sergei M. / Inhibitory antibodies to human angiotensin-converting enzyme : Fine epitope mapping and mechanism of action. In: Biochemistry. 2006 ; Vol. 45, No. 15. pp. 4831-4847.
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abstract = "Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the SD-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E-mAb and E·S·mAb complexes, respectively; however, only complex E·S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the {"}closed{"} conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.",
author = "Skirgello, {Olga E.} and Balyasnikova, {Irina V.} and Binevski, {Petr V.} and Sun, {Zhu Li} and Baskin, {Igor I.} and Palyulin, {Vladimir A.} and Nesterovitch, {Andrei B.} and Albrecht, {Ronald F.} and Kost, {Olga A.} and Danilov, {Sergei M.}",
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Skirgello, OE, Balyasnikova, IV, Binevski, PV, Sun, ZL, Baskin, II, Palyulin, VA, Nesterovitch, AB, Albrecht, RF, Kost, OA & Danilov, SM 2006, 'Inhibitory antibodies to human angiotensin-converting enzyme: Fine epitope mapping and mechanism of action', Biochemistry, vol. 45, no. 15, pp. 4831-4847. https://doi.org/10.1021/bi052591h

Inhibitory antibodies to human angiotensin-converting enzyme : Fine epitope mapping and mechanism of action. / Skirgello, Olga E.; Balyasnikova, Irina V.; Binevski, Petr V.; Sun, Zhu Li; Baskin, Igor I.; Palyulin, Vladimir A.; Nesterovitch, Andrei B.; Albrecht, Ronald F.; Kost, Olga A.; Danilov, Sergei M.

In: Biochemistry, Vol. 45, No. 15, 18.04.2006, p. 4831-4847.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Inhibitory antibodies to human angiotensin-converting enzyme

T2 - Fine epitope mapping and mechanism of action

AU - Skirgello, Olga E.

AU - Balyasnikova, Irina V.

AU - Binevski, Petr V.

AU - Sun, Zhu Li

AU - Baskin, Igor I.

AU - Palyulin, Vladimir A.

AU - Nesterovitch, Andrei B.

AU - Albrecht, Ronald F.

AU - Kost, Olga A.

AU - Danilov, Sergei M.

PY - 2006/4/18

Y1 - 2006/4/18

N2 - Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the SD-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E-mAb and E·S·mAb complexes, respectively; however, only complex E·S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

AB - Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the SD-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E-mAb and E·S·mAb complexes, respectively; however, only complex E·S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

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