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Journal of Biochemistry Advance Access originally published online on October 17, 2007
Journal of Biochemistry 2007 142(6):671-680; doi:10.1093/jb/mvm182
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© 2007 The Japanese Biochemical Society.

Cloning, Characterization and Site-Directed Mutagenesis of Canine Renin

Yuri Bukhtiyarov*, Marianne Zecher{dagger}, Reshma Panemangalore, Zhongren Wu, Joseph G. Bruno, Jing Yuan, Zhenrong Xu, Lawrence W. Dillard, Gerard M. McGeehan, Richard K. Harrison{dagger} and Boyd B. Scott

Department of Discovery Biology, Vitae Pharmaceuticals Inc., Fort Washington, PA 19034, USA

*To whom correspondence should be addressed. Department of Discovery Biology, Vitae Pharmaceuticals Inc., Fort Washington, PA 19034, USA. Tel: +215-461-2051, Fax: +215-461-2006, E-mail: ybukhtiyarov{at}vitaerx.com

Received June 20, 2007; Accepted August 27, 2007


  Abstract

Inhibition of renin has been shown to be successful in managing hypertension and maintaining cardiac health. Canine models have played a key role in preclinical assessment of renin inhibitors. Here we report the cloning of canine prorenin gene. The amino acid sequence of mature canine renin was ~70% identical to that of human renin. The full-length prorenin was expressed in HEK 293 cells, purified and converted to its active form by trypsin-mediated cleavage of the 43 residue propeptide. The mature enzyme was characterized by steady-state kinetics using a peptide corresponding to the canine angiotensinogen sequence, Ac–Asp–Arg–Val–Tyr–Ile–His–Pro–Phe–His–Leu–Leu–Val–Tyr–Ser–OH (cleavage between Leu10–Leu11). The reaction followed Michaelis–Menten kinetics with a KM of 120 µM and a second-order rate constant (kcat/KM) of 1.7 x 105 M–1s–1. The enzyme was inhibited by various human renin inhibitors, but at reduced potency compared to the human renin. The basis of the species specificity was investigated by mutagenesis. Based on primary sequence and structural alignments, three mutants were prepared (G149S-S150T, V286L, G149S-S150T-V286L). Each mutant yielded catalytically active enzymes with lower specific activities than native canine renin. V286L had the greatest effect on substrate specificity, while G149S, S150T mutations produced enzymes with inhibitor profiles similar to human renin.

Key Words: hypertension, proteases, recombinant-canine-renin, renin–angiotensin–aldosterone system, renin-inhibition, species-specificity

Abbreviations: AI, angiotensin I; AII, angiotensin II; BES, N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; RACE, Rapid amplification of cDNA ends; RAAS, renin–angiotensin–aldosterone system; TFA, trifluoroacetic acid

{dagger}Present addresses: Marianne Zecher, 2104 Dunhill Drive, Wilmington, DE 19810, USA

Richard K. Harrison, Wyeth Research, 500 Arcola Road N3338, Collegeville, PA 19426, USA


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