Prolyl oligopeptidase is involved in release of the antifibrotic peptide Ac-SDKP

Abstract

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous tetrapeptide hydrolyzed almost exclusively by angiotensin-converting enzyme (ACE). Chronic treatment with Ac-SDKP decreases cardiac and renal fibrosis and inflammatory cell infiltration in hypertensive rats. However, very little is known about endogenous synthesis of Ac-SDKP, except that thymosin-beta4 may be the most likely precursor. Two enzymes are potentially able to release Ac-SDKP from thymosin-beta4: prolyl oligopeptidase (POP) and endoproteinase asp-N. POP is widely present and active in several tissues and biological fluids, whereas endoproteinase asp-N appears to be lacking in mammals. Therefore, we hypothesized that POP is the main enzyme involved in synthesizing the antifibrotic peptide Ac-SDKP. We investigated in vitro and in vivo production of Ac-SDKP. Using kidney cortex homogenates, we observed that Ac-SDKP was generated in a time-dependent manner in the presence of exogenous thymosin-beta4, and this generation was significantly inhibited by several POP inhibitors (POPi), Z-prolyl-prolinal, Fmoc-prolyl-pyrrolidine-2-nitrile, and S17092. Long-term administration of S17092 in rats significantly decreased endogenous levels of Ac-SDKP in the plasma (from 1.76+/-0.2 to 1.01+/-0.1 nM), heart (from 2.31+/-0.21 to 0.83+/-0.09 pmol/mg protein), and kidneys (from 5.62+/-0.34 to 2.86+/-0.76 pmol/mg protein). As expected, ACE inhibitors significantly increased endogenous levels of Ac-SDKP in the plasma, heart, and kidney, whereas coadministration of POPi prevented this increase. We concluded that POP is the main enzyme responsible for synthesis of the antifibrotic peptide Ac-SDKP.

Figure 1

Time-dependent generation of Ac-SDKP in vitro in the presence of exogenous thymosin-β₄ and various POP inhibitors; 100-μL aliquots of the kidney homogenate were incubated at 37°C with: (1) thymosin-β₄ vehicle and POPi vehicle (time control); (2) thymosin-β₄ (2 μmol/L)+POPi vehicle; (3) thymosin-β₄+POPi (0.01 μmol/L); or (4) thymosin-β₄+POPi (0.1 μmol/L). A, Ac-SDKP generation in kidney homogenates containing exogenous thymosin-β₄ in the presence or absence of Z-prolyl-prolinal (Z-pro-pro) at 0.01 and 0.1 μmol/L; **P<0.001 vs time control; †P<0.01 and ‡P<0.001 vs thymosin-β₄. B, Ac-SDKP generation in kidney homogenates containing exogenous thymosin-β₄ in the presence or absence of Fmoc-prolyl-pyrrolidine-nitrile (Fmoc-Pro-PyrrCN) at 0.01 and 0.1 μmol/L; **P<0.001 vs time control; †P<0.05 and ‡P<0.001 vs thymosin-β₄. C, Ac-SDKP generation in kidney homogenates containing exogenous thymosin-β₄ in the presence or absence of S17092 at 0.01 and 0.1 μmol/L; **P<0.001 vs time control; †P<0.01 and ‡P<0.001 vs thymosin-β₄. Data are expressed as mean±SE.

Figure 2

Plasma and urinary levels of Ac-SDKP in rats chronically treated with POPi. Ac-SDKP concentrations in the plasma and urine of rats chronically treated with vehicle (peanut oil), S17092 (20 mg/kg per day), captopril (100 mg/kg per day), or captopril+S17092. **P<0.01 and *P<0.05. Data are expressed as mean±SE.

Figure 3

Endogenous levels of Ac-SDKP in rats treated with POPi long-term. Amount of Ac-SDKP per mg protein in heart and kidney cortex of rats chronically treated with vehicle (peanut oil), S17092 (20 mg/kg per day), captopril (100 mg/kg per day), or captopril+S17092. **P<0.01 and *P<0.05. Data are expressed as mean±SD.

Figure 4

Changes in mean arterial blood pressure of rats treated with POPi. Arterial blood pressure was monitored and changes in mean arterial pressure plotted during bolus injections of bradykinin (A) or angiotensin I (B) in rats treated long-term with vehicle (peanut oil), S17092 (40 mg/kg per day), or captopril (100 mg/kg per day). **P<0.01 and *P<0.05. Data are expressed as mean±SE.

Figure 5

Schematic diagram illustrating enzymatic generation and degradation of Ac-SDKP.