Journal of Biochemistry Advance Access originally published online on July 29, 2008
Journal of Biochemistry 2008 144(4):499-506; doi:10.1093/jb/mvn093
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© 2008 The Japanese Biochemical Society.
Species Differences Between Human and Rat in the Substrate Specificity of Cathepsin K
,¶Novartis Institutes for BioMedical Research, Tsukuba, Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
¶To whom correspondence should be addressed. Tel: +81-78-991-1919, Fax: +81-78-991-1942, E-mail: gohda.keigo{at}sysmex.co.jp
||Correspondence may also be addressed: Tel: +81-29-865-2290, Fax: +81-298-65-2308, E-mail: naoki.teno{at}novartis.com
Received May 23, 2008; Accepted July 11, 2008
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Abstract |
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Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.
Key Words: cathepsin K, hydroxyproline, S2–P2 interaction, species difference, substrate specificity
Abbreviations: Abz, o-aminobenzoic acid; Cbz, carbobenzyloxy; Hyp, hydroxyproline; MCA, 4-methylcoumaryl-7-amide; Phe(NO2), p-nitro-l-phenylalanine
*Present address: Central Research Laboratories, Sysmex Co., 4-4-4, Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
These two authors contributed equally to this work.
College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1, Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
Computer-Aided Molecular Modeling Research Center Kansai (CAMM Kansai), 1-3-12, Honjyo-cho, Higashinada-ku, Kobe 658-0012, Japan.