ZOOLOGICAL SCIENCE 23: 963–968 (2006) © 2006 Zoological Society of Japan Characterization of Mouse Tissue Kallikrein 5 Sanath Rajapakse1,2, Katsueki Ogiwara1, Noriko Yamano3, Atsushi Kimura1, Kensaku Hirata3, Sumio Takahashi3 and Takayuki Takahashi1* 1Laboratory of Molecular and Cellular Interactions, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan 2Department of Molecular Biology and Biotechnology, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka 3Department of Biology, Faculty of Science, Okayama University, Okayama, Tsushima, Okayama 700-8530, Japan Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (Klk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and char- acterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein- 3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling. Key words: mouse, protease, kallikrein 5, recombinant enzyme, characterization female reproductive system. To establish a basis for further INTRODUCTION physiological studies of mK5, we prepared active recombi- Mouse tissue kallikreins (Klks) are members of a large, nant mK5 for biochemical characterization. The present data multigene family located as a gene cluster in cytogenic suggest that mK5 may play a role in various biological pro- region B2 on mouse chromosome number 7 (Diamandis et cesses. al., 2004; Olsson et al., 2004). Previous studies suggested MATERIALS AND METHODS that individual Klk gene products (mKs) are involved in many vital functions, such as kinin production, coagulation and Expression vector construction fibrinolysis, activation and/or inactivation of peptide hor- The expression vector for mK5 was prepared basically in the mones, regulation of growth factors, and extracellular matrix same way as for mK21 (Matsui and Takahashi, 2001). Briefly, a protein turnover (Margolius, 1998). It is now established that cDNA corresponding to amino acid residues 18–261 of the Klk5 gene, including the pro-enzyme region (DDBJ/EMBL/GenBank the mouse has 37 Klk genes, 26 of which can code for func- databases NM 008456), was inserted into the EcoRI and HindIII tional proteins (Diamandis et al., 2004; Olsson et al., 2004). sites of pET30a (Novagen, Madison, WI). A sense primer 5’-CCG- However, only about half of the 26 genes have been char- GAATTCGCACCTCCAGTCCAA-3’, which includes the EcoRI site, acterized to date by molecular biological and biochemical and an antisense primer 5’-CCCAAGCTTTCAGGCATTTTTAGC- approaches. Mouse tissue kallikrein 5 (Klk5) is one of the TAT-3’, which includes the HindIII site, were synthesized. PCR as-yet-uncharacterized Klk genes. At present, the nucleotide using KOD plus DNA polymerase (Toyobo, Osaka, Japan) was per- sequence is the only information available for this particular formed using mouse skeletal muscle cDNA as the template with the gene, and studies at the protein level are required to deter- above primers to create EcoRI sites at the 5’ ends and HindIII sites ° mine its biological role. at the 3’ ends. The PCR conditions were as follows: 3 min at 94 C, ° ° Our previous studies documented the expression of followed by 30 cycles of 30 sec at 94 C, 30 sec at 55 C, and 1 min at 68°C. The reaction product was sequentially digested with EcoRI Klk21 (Matsui and Takahashi, 2001), Klk24 (Matsui et al., and HindIII, then gel purified and ligated in frame between the EcoRI 2005a, b), and Klk27 (Matsui et al., 2000) in the testis and and HindIII sites of pET30a. The orientation and sequence of the their implications in the function of this male reproductive cDNA in the vector plasmid were confirmed by DNA sequencing. organ. In an attempt to examine by RT-PCR whether or not the Klk genes described above are expressed in other Production and purification of active mK5 mouse tissues, we happened to isolate a Klk5 fragment from The ligated vector (pET30a-mK5) was transformed to E. coli the uterus, suggesting that mK5 may play a role in the strain BL21. The cells were grown at 37°C, induced with isopropyl- 1-thio-β-D-galactoside, harvested by centrifugation, and lysed by * Corresponding author. Phone: +81-11-706-2748; freeze-thawing. The samples were washed twice with 0.5% Triton Fax : +81-11-706-4851; X-100, solubilized in 50 mM Tris-HCl (pH 7.8) containing 6 M urea E-mail: [email protected] and 0.5 M NaCl, and incubated for 12 h at room temperature in the doi:10.2108/zsj.23.963 same buffer containing 6 M urea and 0.5 M NaCl. The solubilized 964 S. Rajapakse et al. samples were fractionated on a Ni2+-chelate column (5-ml volume) (Novagen) previously equilibrated with 50 mM Tris-HCl (pH 7.8) containing 0.5 M NaCl and 6 M urea (Matsui and Takahashi, 2001). Elution was conducted with 50 mM histidine in the same buffer con- taining 0.5 M NaCl and 6 M urea. Eluted protein was extensively dialyzed against 50 mM Tris-HCl (pH 8.0). The protein thus pre- pared was a fusion protein containing at the N-terminus 51 extra amino acids, all of which originated from the plasmid sequence, in addition to the complete pro-mK5 sequence. This fusion protein was then incubated in 50 mM Tris-HCl (pH 8.0) with trypsin-Sepharose 4B, and the resulting active mK5 enzyme preparation was further purified by column chromatography on a Resource Q column (1-ml bed volume) (AKTA FPLC system) previously equilibrated with 50 mM Tris-HCl (pH 8.0). The materials retained were eluted with a lin- ear gradient of 0–0.5 M NaCl in the same buffer. One-milliliter frac- tions were collected and the enzyme activity of mK5 was assayed with a synthetic substrate, Pro-Phe-Arg-MCA, containing 4-methyl- coumaryl-7-amide (MCA) (Peptide Institute, Osaka, Japan). Fig. 1. Purity of purified recombinant mK5. Purified active mK5 Enzyme activity was subjected to SDS-PAGE analysis under reducing (lane 2) and Recombinant mK5 activity was determined according to the non-reducing (lane 3) conditions and visualized by Coomassie Bril- method of Barrett (1980) with a slight modification. Briefly, the reac- liant Blue R-250 staining. Lane 1, molecular mass standards (kDa). tion solution contained 0.1 M Tris-HCl buffer (pH 8.0), 0.1 mM MCA substrate, and the enzyme sample in a final volume of 0.5 ml. The reaction was initiated by the addition of the substrate and stopped by the addition of 2.5 ml of 30 mM sodium acetate buffer (pH 4.3) Table 1. Kinetic parameters of mK5 as measured on MCA-con- containing 100 mM monochloroacetic acid. The release of fluoro- taining substrates phore 7-amino-4-methylcoumarin (AMC) was measured by spec- Substrates V K k k /K trofluorometry at an excitation wavelength of 370 nm and an emis- max m cat cat m sion wavelength of 460 nm. μmol/min/mg mM min–1 mM–1·min–1 Pro-Phe-Arg-MCA 1.5 0.03 39 1300 Kinetic parameters Z-Phe-Arg-MCA 0.3 0.13 7.6 58 Kinetic parameters were determined for various MCA sub- Z-Leu-Arg-MCA 0.1 0.05 2.7 54 strates. Initial velocities, extrapolated from plots of product versus Pyr-Gly-Arg-MCA 0.1 0.03 1.6 53 time, were transformed into double-reciprocal plots. Maximum Z-Val-Val-Arg-MCA 0.2 0.11 4.7 43 velocities (Vmax), Km, and kcat values were obtained from the inter- Boc-Val-Leu-Lys-MCA ND cepts of these plots. The active mK5 concentration was determined Boc-Glu-Lys-Lys-MCA ND using the active-site titrant p- nitrophenyl-p’-guanidinobenzoate HCl Suc-Leu-Leu-Val-Tyr-MCA ND according to the method of Chase and Shaw (1967). Ac-Ile-Glu-Thr-Asp-MCA ND Suc-Ala-Ala-Pro-Phe-MCA ND Inhibition of mK5 by protease inhibitors Z, benzyloxycarbonyl; Pyr, pyroglutamyl; Boc, tert-butyloxycarbonyl; Active mK5 (0.1 μg) was pre-incubated with various concentra- Suc, succinyl; Ac, acetyl; ND, not determined because of no significant tions of protease inhibitors at 37°C for 15 min in 0.1 M Tris-HCl activity. buffer (pH 8.0). After addition of 0.1 mM Pro-Phe-Arg-MCA, the residual enzyme activity was measured. Table 2. Effects of protease inhibitors on mK5 activity Degradation of protein substrates by mK5 Gelatin zymography was performed according to the method Inhibitor Concentration Inhibition (%) previously described by Heussen and Dowdle (1980). Briefly, mK5 DFP 2.0 mM 87 was electrophoresed on 12% SDS-PAGE gels containing 1 mg/ml type A porcine skin gelatin (Sigma, St. Louis, MO) under non-reduc- Leupeptin 0.1 mM 70 ing conditions. After electrophoresis, gels were washed twice in Aprotinin 0.1 mg/ml 34 2.5% Triton X-100 for 30 min, and then incubated with shaking in Antipain 0.2 mM 13 0.1 M glycine-NaOH (pH 8.3) for 18 h at 37°C. The gels were SBTI 0.1 mg/ml 0 stained with 0.25% Coomassie Brilliant Blue to visualize zones of Benzamidine 1.0 mM 0 lysis. E-64 0.5 mM 12 Casein (Wako Pure Chemical Industries Ltd., Osaka, Japan) o-Phenanthroline 2.0 mM 10 was added to mK5 at a ratio of 10:1 (w/w) in 50 mM Tris-HCl buffer Pepstatin 0.1 mM 0 (pH 8.0), and the mixtures were incubated at 37°C.