Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry

Abstract

Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1-7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1-7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1-7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1-7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1-7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1-7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1-7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1-7) production pathways may have clinically important implications in patients with metabolic and renal disease.

Reduced thrombosis in Klkb1−/− mice is mediated by increased Mas receptor, prostacyclin, Sirt1, and KLF4 and decreased tissue factor

“…We did not observe increased plasma Ang-(1-7), the natural ligand for Mas, or increased activity for ACE2 or PRCP, the 2 main Ang-(1-7)-forming enzymes, in Klkb1 2/2 mice. 38 Our studies show that Mas elevation alone is sufficient to modify thrombosis risk. ; it is possible that it too may occur with Mas.…”

Angiotensin 1–7 mediates renoprotection against diabetic nephropathy by reducing oxidative stress, inflammation, and lipotoxicity

“…Our systemic ANG 1-7 treatment did increase renal ANG 1-7 levels. However, renal ANG II and ANG 1-7 levels in db/db kidneys did not change compared with wild-type kidneys despite variation in renal ACE2 levels and activity, suggesting alternative enzymatic pathways for processing angiotensin peptides in the kidneys, such as neprilysin and prolyl carboxypeptidase (18,50).…”

Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells

“…The present study indicates a high abundance of alternative ANG-(1-7)-forming enzymes, most likely prolyl carboxypeptidase or prolyl endopeptidase, in COS7 cell lysates but not cell media. Indeed, our laboratory recently identified compensatory ANG-(1-7) formation by prolyl carboxypeptidase in mouse kidneys (19). Involvement of prolyl carboxypeptidase or prolyl endopeptidase in renal ANG-(1-7) formation was also shown in human glomerular endothelial cells (47).…”

“…Enzymatic RAS activities were measured using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry as described with some modifications (12,18,19). For renin activity, COS7 cell lysates (ϳ10 -20 g protein) were incubated for 5 min at 37°C in 25 mM bicine buffer pH 7.6 containing Complete Lysis-M EDTA-Free protease inhibitors, 2.5 mM PMSF, and 1 g tetradecapeptide.…”