Identification of dipeptidyl peptidase 3 as the Angiotensin-(1–7) degrading peptidase in human HK-2 renal epithelial cells

• Published in: Peptides (New York, N.Y. : 1980) Vol. 83; pp. 29 - 37
• Main Authors: Cruz-Diaz, Nildris, Wilson, Bryan A., Pirro, Nancy T., Brosnihan, K. Bridget, Marshall, Allyson C., Chappell, Mark C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2016
• Subjects: Angiotensin I - genetics; Angiotensin I - metabolism; Angiotensin II - genetics; Angiotensin II - metabolism; Angiotensin-(1–7); Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - genetics; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - metabolism; DPP 3; Epithelial Cells - metabolism; HK-2 cells; Humans; Hydrolysis; Kidney - drug effects; Kidney - metabolism; Oligopeptides - administration & dosage; Peptide Fragments - genetics; Peptide Fragments - metabolism; Reactive Oxygen Species - metabolism; Renin angiotensin system; Renin angiotensin system; Angiotensin-(1–7); DPP 3; HK-2 cells
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2016.06.005

•DPP 3 is identified as the Ang-(1–7) degrading peptidase activity in the HK-2 cells.•DPP3 cleaves Ang-(1–7) to Ang-(3–7) which is more rapidly hydrolyzed to Ang-(5–7).•Kinetic analysis revealed a greater kcat/Km for Ang-(3–7) versus Ang-(1–7).•Treatment with a DPP 3 inhibitor tended to augment the intracellular levels of Ang-(1–7). Angiotensin-(1–7) (Ang-(1–7)) is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic and pro-oxidant effects of the Ang II-AT1 receptor axis. We previously identified a peptidase activity from sheep brain, proximal tubules and human HK-2 proximal tubule cells that metabolized Ang-(1–7); thus, the present study isolated and identified the Ang-(1–7) peptidase. Utilizing ion exchange and hydrophobic interaction chromatography, a single 80kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80kDa band was excised, the tryptic digest peptides analyzed by LC–MS and a protein was identified as the enzyme dipeptidyl peptidase 3 (DPP 3, EC: 3.4.14.4). A human DPP 3 antibody identified a single 80kDa band in the purified enzyme preparation identical to recombinant human DPP 3. Both the purified Ang-(1–7) peptidase and DPP 3 exhibited an identical hydrolysis profile of Ang-(1–7) and both activities were abolished by the metallopeptidase inhibitor JMV-390. DPP 3 sequentially hydrolyzed Ang-(1–7) to Ang-(3–7) and rapidly converted Ang-(3–7) to Ang-(5–7). Kinetic analysis revealed that Ang-(3–7) was hydrolyzed at a greater rate than Ang-(1–7) [17.9 vs. 5.5 nmol/min/μg protein], and the Km for Ang-(3–7) was lower than Ang-(1–7) [3 vs. 12μM]. Finally, chronic treatment of the HK-2 cells with 20nM JMV-390 reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1–7). We conclude that DPP 3 may influence the cellular expression of Ang-(1–7) and potentially reflect a therapeutic target to augment the actions of the peptide.