ACE phenotyping in human heart

Abstract

AimsAngiotensin-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.Methods and resultsWe 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.ConclusionsSignificant 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.

Conformational fingerprint of blood and tissue ACEs: Personalized approach

“…As ACE glycosylation could be both cell- and tissue-specific due to different post-translational modification of ACE globule in different cells, the local topography of ACE surface produced by different cells could be also unique. We demonstrated previously that the pattern of ACE binding by a set of mAbs to 16 epitopes of human ACE–"conformational fingerprint of ACE"–is indeed cell- and tissue-specific [5, 9–10] and, moreover, confirmed that tissue-specific glycosylation of ACE is an important structural requirement for this specific “conformational fingerprint” [10–12].…”

“…In order to save valuable mAbs, we used not more than 3 μg/ml of pure mAbs (or 1/20 dilution of hybridoma cell culture medium) which was found to be sufficient for coating (Fig 1C and 1D). Usually the amount of ACE immunoreactive protein was estimated using the strongest mAb to ACE, clone 9B9 [16, 20], while a pattern of ACE activity precipitation by a panel of mAbs to different epitopes on ACE—conformational fingerprint of ACE—was used for the estimation of local conformational differences in ACE surface topography due to disease [5, 7, 21] or due to tissue origin of ACE [5, 9, 10].…”

“…Seminal fluid was pooled from ejaculates of more than 30 individuals. Heart and lung homogenates were prepared from tissues of individual donors as described [9] using 1:9 (weight: volume of PBS) ratio. Previously we found that ACE activity in tissue homogenates (or cell lysates) prepared with 0.25% Triton X-100 do not tolerate long storage as frozen samples (unpublished).…”

“…ACEs were purified from corresponding sources using anion-exchange chromatography on DEAE-Toyopearl 650M and lisinopril affinity chromatography as in [13], then further washed and concentrated on Vivaspin filtration membranes (GE Healthcare, Sartorius Corp., Bohemia, NY) with 100 kDa limit as in [9]. Recombinant soluble ACE was obtained from culture fluid of CHO cells transfected with ACE lacking transmembrane anchor [14], kindly provided by F. Alhenc-Gelas (then INSERM Unit 352, Paris, France).…”

“…Recent ACE studies with mAbs recognizing different conformational epitopes on the surface of the catalytically active N domain (eight mAbs) and the C domain (eight mAbs) of human ACE molecule revealed that the pattern of mAb binding to ACE is potentially a very sensitive marker of the local conformation of ACE globule [5]. The changes of this pattern could be definitely attributed to the changes of the topography of the epitopes for the distinct mAbs due to denaturation of ACE globule, chemical modification, mutations, or the binding of inhibitors, as well as protein and low molecular weight (LMW) effectors [5–9]. It is noteworthy that ACE contains 17 potential N-glycosylation sites and the epitopes of all mAbs contain n at least one site.…”