Identification of dipeptidyl peptidase 3 as the Angiotensin-(1–7) degrading peptidase in human HK-2 renal epithelial cells |
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| Institution: | 1. Department of Surgery, Hypertension & Vascular Research, Cardiovascular Sciences Center, Wake Forest University School of Medicine, Winston-Salem, NC, United States;2. TKL Research Inc., Fairlawn, NJ, United States;1. Department of Chemistry, University of Puerto Rico, PO Box 23346, San Juan 00931-3346, Puerto Rico;2. Department of Biological and Pharmaceutical Chemistry, University of London, School of Pharmacy, London WC1N 1AX, UK;3. Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon 1649-028, Portugal;4. Laboratório de Química Medicinal e Computacional, Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP 13563-120, Brazil;5. Chemical Biology Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362 Esch-sur-Alzette, Luxembourg;6. Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, CH-4002 Basel, Switzerland;7. University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland;8. School of Chemistry, National University of Ireland, Galway, Ireland;1. Multimedia Systems Department, Gdansk University of Technology, Faculty of Electronics, Telecommunications and Informatics, Narutowicza 11/12, 80-233 Gdansk, Poland;2. Audio Acoustics Laboratory, Gdansk University of Technology, Faculty of Electronics, Telecommunications and Informatics, Narutowicza 11/12, 80-233 Gdansk, Poland;1. Service d’urologie et transplantation rénale, CHRU de Besançon, 3, boulevard A.-Fleming, 25030 Besançon, France;2. Université de Franche-Comté, 25000 Besançon, France;3. Service d’urologie, CHI de Haute-Saône, 25000 Vesoul, France;4. Service de chirurgie plastique, CHRU de Besançon, 25000 Besançon, France;5. Service d’oncologie médicale, CHRU de Besançon, 25000 Besançon, France;6. Inserm UMR 1098, 25000 Besançon, France |
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| Abstract: | 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 80 kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80 kDa 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 80 kDa 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. 12 μM]. Finally, chronic treatment of the HK-2 cells with 20 nM 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. |
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| Keywords: | Renin angiotensin system Angiotensin-(1–7) HK-2 cells DPP 3 |
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