An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE.HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE--between Arg1203 and Ser1204--was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another.The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase)

A novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP

BACKGROUND: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S 1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis

A Novel Angiotensin I-Converting Enzyme Mutation (S333W) Impairs N-Domain Enzymatic Cleavage of the Anti-Fibrotic Peptide, AcSDKP

Background: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis.Results: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE.Conclusions and Significance: A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.</p

Contact residues involved in hydrogen bonding at the interface of native and mutated ACE based on PISA analysis.

<p>The residue column gives the description of the chain, residue name, residue number and atom involved in hydrogen bonding. The distance column represents the calculated distance between the two interacting atoms. Bonding with Y465 is marked by bold. It was observed that transition from the native to the single followed by the double mutation results in systematic loss of H-bonding at the interface.</p

MALDI TOF/TOF spectrum of mutant (Y465D) ACE after tryptic digest.

<p>An in-gel tryptic digest was performed on mutant (Y465D) ACE, the cysteines were protected using iodoacetamide, and the total digest subjected to MALDI TOF/TOF using the matrix α-cyano-4-hydroxycinmanic acid. Masses corresponding to predicted ACE peptides are labeled. The masses corresponding to peptide containing Y465D and the C-terminal cleavage peptide are indicated in bold.</p

A putative model of an ACE dimer attached to cell membrane.

<p>The yellow and red “dumbbells” correspond to the EM model of porcine ACE. ACE monomers <b>A</b> (red) and <b>B</b> (yellow) are oriented such that they form a “back-to-back” complex. The N domain is rendered grey, C domain – beige.</p

Dimer interactions observed at the interface of N domain.

<p><b>A.</b> X-ray model showing all the residues at the interface. The color scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025952#pone-0025952-g008" target="_blank">Fig. 8</a>. Hydrogen bonds and non-covalent interactions between Y465 (red) and other residues are shown by dashed lines. <b>B.</b> A schematic representation of the protein-protein interactions at the dimer interface identified by PDBSum [<a href="http://www.ebi.ac.uk/pdbsum/" target="_blank">http://www.ebi.ac.uk/pdbsum/</a>]. The residues and their interactions are colored using the following notation: hydrogen bonds –blue+dashed lines, non-bonded contacts - dashed lines, positive residues - blue, negative residues - red, neutral residues - green, proline residues - orange, aromatic residues – magenta. Y465 is involved in a hydrogen bond with D462 and hydrophobic non-bonded interaction with F461.</p

MALDI MS/MS analysis of sACE-Y465D after tryptic digest.

<p>Masses (<i>m/z</i>) corresponding to ACE peptides, the peptide containing Y465D (<i>underlined</i>) and the C-terminal peptide are indicated in <i>bold</i>.</p

Western blot analysis of normal and mutant ACEs.

<p><b>A.</b> The lysates and culture medium of HEK cells expressing WT and mutant (Y465D) ACE (normalized by equal ACE activity loading −5 mU/ml) were subjected to SDS-PAGE (7.5% gel) in reducing conditions for Western blotting with mAbs 3C5 and 5C8 recognizing different sequential epitopes on the C domain of ACE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025952#pone.0025952-Balyasnikova4" target="_blank">[45]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025952#pone.0025952-Balyasnikova6" target="_blank">[47]</a>. Proteins transferred on PVDF-Plus membrane were revealed with 2 µg/ml of indicated mAb. Molecular weight markers are shown by arrows on the left of panel A, which is a representative experiment. <b>B.</b> Revelation of WT and mutant ACE presented in panel A (with mAb 3C5) and with mAb 5C8 (not shown) was quantified by densitometry of the bands. Data presented as a ratio of density with mAb 5C8 to that with mAb 3C5 by the image analysis (densitometry) using ImageJ software (NIH). Data are expressed as mean ± SD of 3 independent experiments. <b>C.</b> Western blot analysis was performed on total cell lysate and concentrated medium from CHO cells transfected with WT and ACE-Y465D. Samples were separated by 6% SDS-PAGE in the presence (<i>right</i>) or absence (<i>left</i>) of 2-mercaptoethanol. Immunoblotting was performed and ACE was detected with the C domain-specific mAb 1D8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025952#pone.0025952-Balyasnikova6" target="_blank">[47]</a>. <b>D.</b> Densitometric analysis of cell lysates separated by Western blot, calculated as the percentage dimer of monomer (D/M). Data is the mean ± SD of samples prepared in duplicate.</p

Binding of mAbs to mutant ACE on the surface of CHO cells (Cell ELISA).

<p>Cell ELISA assay was performed on CHO cells expressing WT and mutant (Y465D) ACE using the panel of 16 mAbs directed to different epitopes on the N- and C domains of human ACE. <b>A.</b> Binding of mAbs to WT ACE determined via secondary Ab - anti-mouse IgG conjugated to peroxidase - optical density at 405 nm. Non-specific binding of the secondary mAbs to control non-immune mouse IgG was subtracted. <b>B.</b> Data are expressed as a percentage of mAbs binding to surface of CHO cells expressing mutant ACE to that expressing WT ACE and normalized for surface ACE expression. Data presented as a mean of 3–4 independent determinations in duplicate. mAbs showing significant changes in binding compared to WT are colored (red- increase >20%, yellow-decrease >20%). * p<0.05 indicates ratio shown is significantly different from 1.</p