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.

Enzymatic processing of angiotensin peptides by human glomerular endothelial cells

“…Furthermore, expression of ACE2 was reported in human aortic endothelial cells in culture (20). Therefore, GEnCs could be an additional source of glomerular ACE2, along with podocytes (58, 63) and mesangial cells (2).…”

“…After a 1-h starvation period, cells were maintained in serum-free medium and spiked with 1 M ANG I [ANG- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) the addition of the ANG substrate. Conditioned media were collected at various time points and stored at Ϫ20°C until assayed.…”

“…ANG II [ANG- (1)(2)(3)(4)(5)(6)(7)(8)] is an octapeptide that is generated from the cleavage of its decapeptide precursor ANG I by a carboxypeptidase, primarily ANG-converting enzyme (ACE), or by other peptidases, such as chymase or cathepsin G. However, ANG I can be converted into other fragments by the action of various carboxy-, endo-, or aminopeptidases. An outline of the major RAS enzymes and corresponding cleaving sites are shown in Table 1.…”

Is the kidney a target of SARS-CoV-2?

“…In the kidney, ACE2 is present in podocytes, mesangial cells, parietal epithelium of Bowman's capsule, proximal cell brush border, and collecting ducts (3,32,40,84). Several models of nephropathy have shown that ACE2 is implicated in reducing glomerular and tubular damage as well as fibrosis (43,48,63,84).…”

Alternative pathways for angiotensin II production as an important determinant of kidney damage in endotoxemia

“…Nevertheless, the angiotensinconverting enzyme 2 (ACE2) was identified as being homologous to ACE but responsible for cleaving ANG II into ANG (1-7), which presents anti-proliferative and vasodilator effects. Thus, the balance between ANG II and ANG (1-7) levels is important for controlling RAS activity (2,9,14,15,23,28,33).…”