Purification and characterization of angiotensin converting enzyme 2 (ACE2) from murine model of mesangial cell in culture

Abstract

Angiotensin converting enzyme 2 (ACE2) is a component of the renin-angiotensin system (RAS) which converts Ang II, a potent vasoconstrictor peptide into Ang 1-7, a vasodilator peptide which may act as a negative feedback hormone to the actions of Ang II. The discovery of this enzyme added a new level of complexity to this system. The mesangial cells (MC) have multiple functions in glomerular physiology and pathophysiology and are able to express all components of the RAS. Despite of being localized in these cells, ACE2 has not yet been purified or characterized. In this study ACE2 from mice immortalized MC (IMC) was purified by ion-exchange chromatography. The purified enzyme was identified as a single band around 60-70 kDa on SDS-polyacrylamide gel and by Western blotting using a specific antibody. The optima pH and chloride concentrations were 7.5 and 200 mM, respectively. The N-terminal sequence was homologous with many species ACE2 N-terminal sequences as described in the literature. ACE2 purified from IMC was able to hydrolyze Ang II into Ang 1-7 and the K(m) value for Ang II was determined to be 2.87 ± 0.76 μM. In conclusion, we purified and localized, for the first time, ACE2 in MC, which was able to generate Ang 1-7 from Ang II. Ang 1-7 production associated to Ang II degradation by ACE2 may exert a protective effect in the renal hemodynamic.

Fig. 1

Ion-exchange chromatography using Resource-Q column of concentrated cell lysate from IMC. ACE2 was eluted in one peak (P1) with enzymatic activity using Abz-SPY(NO2) as substrate. Fractions (0.5 mL) were collected at a flow rate of 0.5 m/min with a linear gradient (0–100% NaCl 1 M). () OD 280 nm (–●–) ACE2 activity (-----) % NaCl.

Fig. 2

(A) pH profiles of ACE. The optimal pH ACE activity was assayed using Abz-SPY(NO2) as substrate in buffers with pH ranging from 4.0 at 9.0. (B) Effect of NaCl on the activity ACE2. The influence of Cl⁻ on ACE2 activity was determined using Abz-SPY(NO2) as substrate in buffer with NaCl concentrations ranging from 50 to 1500 mM.

Fig. 3

(A) SDS-PAGE (7.5%) of reduced purified ACE2. Lane A: molecular mass marker; lane B: purified material. Protein was stained with the Bio-Rad Silver reagent (Bio-Rad, USA). (B) Western blotting of purified ACE2. Proteins were separated by SDS/PAGE (7.5% gel), transferred to a nitrocellulose membrane and revealed with anti-ACE2 antibody. Lane A: molecular mass marker; lanes B and C: purified material.

Fig. 4

Identification of peptide bond in ANG II hydrolyzed by purified ACE2. The conditions for reverse-phase HPLC are given in Section 2. (A) Zero time of incubation of ACE2 with ANG II. (B) After 24 h of incubation of ACE2 with ANG II resulting in ANG 1–7 generation.

Fig. 5

Lineweaver–Burk plot for ACE2 activitu using ANG II as substrate. ANG II concentrations were expressed in μM. Velocity was expressed as μM/min.

Fig. 6

Confocal fluorescence microscopy of the ACE2 localization in mice IMC. (A) Immortalized mesangial cells (DIC image). (B) Nuclei labeling with DAPI. (C) Localization of ACE2 in immortalized mesangial cells using anti-ACE2 polyclonal antibody. Original magnification 40×. Bar: 10 μm.