Conformational fingerprint of blood and tissue ACEs: Personalized approach.

BackgroundThe pattern of binding of monoclonal antibodies (mAbs) to 18 epitopes on human angiotensin I-converting enzyme (ACE)-"conformational fingerprint of ACE"-is a sensitive marker of subtle conformational changes of ACE due to mutations, different glycosylation in various cells, the presence of ACE inhibitors and specific effectors, etc.Methodology/principal findingsWe described in detail the methodology of the conformational fingerprinting of human blood and tissue ACEs that allows detecting differences in surface topography of ACE from different tissues, as well detecting inter-individual differences. Besides, we compared the sensitivity of the detection of ACE inhibitors in the patient's plasma using conformational fingerprinting of ACE (with only 2 mAbs to ACE, 1G12 and 9B9) and already accepted kinetic assay and demonstrated that the mAbs-based assay is an order of magnitude more sensitive. This approach is also very effective in detection of known (like bilirubin and lysozyme) and still unknown ACE effectors/inhibitors which nature and set could vary in different tissues or different patients.Conclusions/significancePhenotyping of ACE (and conformational fingerprinting of ACE as a part of this novel approach for characterization of ACE) in individuals really became informative and clinically relevant. Appreciation (and counting on) of inter-individual differences in ACE conformation and accompanying effectors make the application of this approach for future personalized medicine with ACE inhibitors more accurate. This (or similar) methodology can be applied to any enzyme/protein for which there is a number of mAbs to its different epitopes

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs

ACE phenotyping in human heart

<div><p>Aims</p><p>Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, is expressed as a type-1 membrane glycoprotein on the surface of different cells, including endothelial cells of the heart. We hypothesized that the local conformation and, therefore, the properties of heart ACE could differ from lung ACE due to different microenvironment in these organs.</p><p>Methods and results</p><p>We performed ACE phenotyping (ACE levels, conformation and kinetic characteristics) in the human heart and compared it with that in the lung. ACE activity in heart tissues was 10–15 lower than that in lung. Various ACE effectors, LMW endogenous ACE inhibitors and HMW ACE-binding partners, were shown to be present in both heart and lung tissues. “Conformational fingerprint” of heart ACE (i.e., the pattern of 17 mAbs binding to different epitopes on the ACE surface) significantly differed from that of lung ACE, which reflects differences in the local conformations of these ACEs, likely controlled by different ACE glycosylation in these organs. Substrate specificity and pH-optima of the heart and lung ACEs also differed. Moreover, even within heart the apparent ACE activities, the local ACE conformations, and the content of ACE inhibitors differ in atria and ventricles.</p><p>Conclusions</p><p>Significant differences in the local conformations and kinetic properties of heart and lung ACEs demonstrate tissue specificity of ACE and provide a structural base for the development of mAbs able to distinguish heart and lung ACEs as a potential blood test for predicting atrial fibrillation risk.</p></div

Conformational characteristics of different ACEs.

<p>Conformational fingerprinting of the heart and lung ACEs was performed with a set of 17 mAbs to the two-domain ACE. Immunoprecipitated ACE activity from purified ACEs solutions (<b>A</b>), tissue homogenates from 10 donors (<b>B</b>), or ACEs after perfusion into rat blood circulation (<b>C</b>) are presented as % (“binding ratio”) for heart ACE from that of lung ACE. Ratios increased more than 20% are highlighted in orange, more than 50% in dark orange, and more than 200% in red, while decreased more than 20% are highlighted in yellow and more than 50% in deep blue. Data are mean ± SD of at least 3 experiments (each in duplicates), p<0.01.</p

Effect of dilution on the apparent ACE activity in the heart and lung homogenates.

<p>ACE activity was measured in the heart and lung homogenates from 10 donors at different dilutions using two substrates, ZPHL and HHL (as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181976#pone.0181976.g001" target="_blank">Fig 1</a>). Data are expressed as % from the ACE activity in undiluted homogenates (<b>A,B</b>), as well as % of ZPHL/HHL ratio from that for undiluted homogenates (<b>C</b>,<b>D</b>). Each value is a mean of several (2–3) experiments in duplicates, p<0.01.</p

Dependence of ACE activity on pH.

<p>The assays of the activity of the purified heart and lung ACEs toward 0.5 mM Z-Phe-His-Leu (<b>A</b>) and 1.3 mM Hip-His-Leu (<b>B</b>) were performed in 25 mM acetate-MES-Tris-borate buffer, containing 0.15 M NaCl and 1 μM ZnCl₂. Each value is a mean of several (2–3) experiments in duplicates.</p

Differences between ACE protein level and ACE activity in the heart chambers.

<p>ACE activity was measured in the undiluted homogenates of human heart chambers and at 1/10 dilution as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181976#pone.0181976.g001" target="_blank">Fig 1</a>. ACE protein level was quantified after precipitation of ACE by a set of mAbs and washing out putative endogenous ACE inhibitors/effectors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181976#pone.0181976.ref026" target="_blank">26</a>]. Data are expressed as a percentage from the corresponding data for left ventricle homogenate. Each value is a mean of several (3) experiments in duplicates. Bars highlighted in orange represent the samples with values higher than 20% of mean ± SD for control samples (left ventricle homogenate), * p<0.05, ** p<0.01.</p

Effect of dilution on ACE activity in the homogenates of heart chambers.

<p>ACE activity and ZPHL/HHL ratio were measured in the homogenates of human heart chambers at different dilutions using two substrates (as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181976#pone.0181976.g001" target="_blank">Fig 1</a>). Data are expressed as absolute values (<b>A</b>-<b>C</b>) and as % from homogenates at maximal dilution—1/30 (<b>D</b>-<b>F</b>). Each value is a mean of several (2–3) experiments on separate homogenates in duplicates.</p

Kinetic parameters of the hydrolysis of different substrates by ACEs isolated from human heart and lung tissues.

<p>Kinetic parameters of the hydrolysis of different substrates by ACEs isolated from human heart and lung tissues.</p

Conformational fingerprinting of ACE in different heart chambers.

<p>Conformational fingerprinting of ACE in the homogenates (1:9) of different heart chambers was performed with a set of 17 mAbs to ACE as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181976#pone.0181976.g003" target="_blank">Fig 3</a>. Immunoprecipitated ACE activity from these homogenates undiluted and further diluted 1/10 is presented as % (“binding ratio”) from the immunoprecipitated ACE activity from left ventricle homogenate (undiluted and diluted, correspondingly). Ratios increased more than 20% are highlighted in orange. Data are mean ± SD of at least 3 experiments (each in duplicates), p<0.01.</p