Recombinant Expression and Characterization of Human and Murine ACE2: Species-Specific Activation of the Alternative Renin-Angiotensin-System - PubMed (original) (raw)

Recombinant Expression and Characterization of Human and Murine ACE2: Species-Specific Activation of the Alternative Renin-Angiotensin-System

Marko Poglitsch et al. Int J Hypertens. 2012.

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase of the renin-angiotensin-system (RAS) which is known to cleave several substrates among vasoactive peptides. Its preferred substrate is Angiotensin II, which is tightly involved in the regulation of important physiological functions including fluid homeostasis and blood pressure. Ang 1-7, the main enzymatic product of ACE2, became increasingly important in the literature in recent years, as it was reported to counteract hypertensive and fibrotic actions of Angiotensin II via the MAS receptor. The functional connection of ACE2, Ang 1-7, and the MAS receptor is also referred to as the alternative axis of the RAS. In the present paper, we describe the recombinant expression and purification of human and murine ACE2 (rhACE2 and rmACE2). Furthermore, we determined the conversion rates of rhACE2 and rmACE2 for different natural peptide substrates in plasma samples and discovered species-specific differences in substrate specificities, probably leading to functional differences in the alternative axis of the RAS. In particular, conversion rates of Ang 1-10 to Ang 1-9 were found to be substantially different when applying rhACE2 or rmACE2 in vitro. In contrast to rhACE2, rm ACE2 is substantially less potent in transformation of Ang 1-10 to Ang 1-9.

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Figures

Figure 1

Figure 1

Batch quality of recombinant human and murine ACE2. (a) Equal amounts (5 _μ_g) rmACE2 and rhACE2 were analyzed by size-exclusion chromatography. The elution was continuously monitored by in-line measurement of the absorbance at 280 nm. The eluted peaks for purified rmACE2 (blue) and rhACE2 (red) are shown in the chromatogram and given in absorption units (mAU). (b) Equal amounts (7 _μ_g) of rmACE2 (m) and rhACE2 (h) were subjected to SDS-PAGE analysis followed by in-gel protein staining with Coomassie Brilliant Blue. Selected marker bands are depicted in the graph to allow the estimation of molecular weights. (c) Equal amounts (2 _μ_g) of rmACE2 (m) and rhACE2 (h) were analyzed in native PAGE followed by in-gel protein staining with Coomassie Brillant Blue as described in Section 2.

Figure 2

Figure 2

In vitro turnover rates of rhACE2 and rmACE2. The turnover rates for natural ACE2 substrates were determined by in vitro incubation with excess amounts of substrate followed by reversed-phase HPLC analysis. Therefore, 14 nM rmACE2 or rhACE2 were coincubated with 65 _μ_M Ang 1–8 (a) in MES buffer and aliquots were taken at indicated time points. The resulting time courses are shown in the graphs. The turnover rates for Ang 1–10 were determined in MES buffer containing 1.5 _μ_M rmACE2 or rhACE2 and 65 _μ_M Ang 1–10 (b) as described in Section 2. Each time point was analyzed in true triplicates and standard deviations are given in the graphs together with linear regressions of the measured values.

Figure 3

Figure 3

Interference of human and murine ACE2 with the endogenous RAS in human plasma. Anticoagulated plasma samples were incubated at 37°C in the presence of 100 pg/mL recombinant human renin (a, b). rhACE2 or rmACE2 were added as indicated at a final plasma concentration of 5 _μ_g/mL. RAS-Fingerprints were measured by LC-MS/MS as indicated in Section 2. Samples were incubated in the absence (a) or presence (b) of 10 _μ_M of the ACE inhibitor Lisinopril. In this figure, the diameter of the spheres reflects the concentration of the respective peptide metabolite, which is also given in pg/mL next to each individual sphere. 0 pg/mL indicates concentrations below quantification limits, which are defined by a signal-to-noise ratio below 10. Furthermore, the amino acid sequence of each angiotensin metabolite is schematically given in brackets beside the corresponding sphere. The sequence annotation is based on the decapeptide Angiotensin I (1–10) which is N- or C-terminally cleaved by the indicated proteases. Proteases are illustrated by arrows connecting their substrate and product. AP: aminopeptidases; NEP: neutral endopeptidase; DAP: di-aminopeptidase; ACE: angiotensin-converting enzyme. (c) Indicated concentrations of recombinant human renin were added to Lisinopril-treated plasma samples. Control samples (black bars), rmACE2- (grey bars), and rhACE2- (white bars) treated samples were subjected to RAS-Fingerprinting, and resulting concentrations for Ang 1–10 (left) and Ang 1–9 (right) are given in pg/mL.

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