Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular Title matrix proteins Author(s) Rajapakse, Sanath; Ogiwara, Katsueki; Takano, Naoharu; Moriyama, Akihiko; Takahashi, Takayuki Febs Letters, 579(30), 6879-6884 Citation https://doi.org/10.1016/j.febslet.2005.11.039 Issue Date 2005-12-01 Doc URL http://hdl.handle.net/2115/985 Type article (author version) File Information FL579-30.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular matrix proteins Sanath Rajapaksea, Katsueki Ogiwaraa, Naoharu Takanoa, Akihiko Moriyamab, Takayuki Takahashia,* aDivision of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan bDivision of Biomolecular Science, Institute of Natural Sciences, Nagoya City University, Nagoya 467-8501, Japan *Correspondence: Takayuki Takahashi, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Tel: 81-11-706-2748 Fax: 81-11-706-4851 E-mail: [email protected] 1 Abstract Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg275-Ile276. This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells. Keywords: human kallikrein 8; enzymatic characterization; extracellular matrix proteins; tissue-type plasminogen activator 2 1. Introduction The tissue kallikreins are a subgroup of trypsin- and chymotrypsin-like serine proteases that are characterized by their homology to true tissue kallikrein, which is encoded by KLK1/Klk1 gene in many species. These proteases share a high degree of sequence and structural similarity [1-3]. In recent studies, at least 15 genes encoding serine proteases have been identified in the human glandular kallikrein locus on chromosome 19q13.3-q13.4 [4-6]. Detailed mapping of the human kallikrein gene locus revealed that KLK4-14 are located telomeric to KLK2, which encodes human glandular kallikrein 2 (hK2), while KLK15 is located between KLK1, which encodes classical tissue kallikrein, and KLK3, which encodes prostate-specific antigen (PSA) [7]. Twelve of these fifteen KLK genes—i.e., all but KLK1-3—were discovered in recent years. Existing evidence suggests that all of the new KLK genes encode functional proteins. However, our knowledge of the KLK gene products is very limited at present. The chemical identities and molecular properties of human kallikrein 6 (hK6) [8-10], and, more recently, hK13 [11] and hK5 [12] have been determined. KLK8 (also known as neuropsin/ovasin) was cloned from a human skin cDNA library as a homologue of mouse neuropsin [13]. In the mouse, KLK8 mRNA is localized at high concentration in the skin and brain, and the gene product has been shown to play an important role in normal hippocampal function [14]. KLK8 mRNA was also detected in the hippocampus of normal human brains. Interestingly, the KLK8 transcript level in the hippocampus of patients with Alzheimer’s disease was found to be elevated 11.5-fold compared to that in controls, suggesting that KLK8 may play a role in 3 neurodegeneration [15]. Furthermore, human KLK8 mRNA and its protein (hK8) have been shown to be more highly expressed in ovarian cancer tissues than in normal ovarian tissue [16, 17]. These findings led to the proposal that hK8 protein may serve as a new ovarian cancer marker [17, 18]. However, despite the increasing biological and clinical interest in hK8, there have been no reports describing the biochemical and enzymatic properties of this protein. In the present study, active recombinant hK8 was produced and characterized. The present data indicate that hK8 is a trypsin-like enzyme that degrades some extracellular matrix (ECM) proteins, such as collagen type IV and fibronectin. The enzyme is also capable of converting single-chain tissue-type plasminogen activator (tPA) to its two-chain protein. These results suggest that hK8 plays a role in the turnover of ECM proteins. 2. Methods 2.1. hK8 expression vector construction The expression vectors for hK8 were constructed by inserting its cDNA (DDBJ/EMBL/GenBank databases AB009849) including the mature enzyme region (amino acids 33-260) with an enteropeptidase cleavage site at the 5’ end into the KpnI and EcoRI sites of pET30a. To amplify the insert, two oligonucleotide primers were synthesized: the sense primer 5’-CGGGGTACCGACGACGACGACAAGGTGCTGGGGGGTCAT-3’, which contained the KpnI site, the enteropeptidase susceptibility site DDDDK, and the 5 33 37 NH2-terminal residues (Val to His ) of mature hK8, and the antisense primer 5’-CCGGAATTCTCAGCCCTTGCTGCC-3’, which included the EcoRI site and the 4 4 COOH-terminal residues (Gly257 to Gly260) of the mature enzyme. PCR was performed with KOD plus DNA polymerase (Toyobo, Osaka, Japan) using human adult ovary total RNA (Stratagene, La Jolla, CA) as a template. The PCR conditions were 3 min at 94°C for heating, followed by 30 cycles of 30 sec at 94°C for denaturing, 30 sec at 60°C for annealing and 1 min at 68°C for extension. The reaction product was sequentially digested with KpnI and EcoRI, gel purified and ligated in frame between the KpnI and EcoRI sites of pET30a. The orientation and sequence of the cDNA in the vector (pET30a-hK8) were confirmed by DNA sequencing. 2.2. Preparation of active recombinant hK 8 The ligated vector (pET30a-hK8) was transformed to E.coli strain BL21. The cells were grown at 37°C, induced with isopropyl-1-thio-β-D-galactoside. Cells were harvested by centrifugation and lysed by freeze-thawing. The materials were washed twice with 0.5% Triton X-100 and solubilized by being dissolved and then incubated in 50 mM Tris-HCl (pH 7.8) containing 6 M urea and 0.5 M NaCl for 12 h at room temperature. The solubilized materials were directly applied to a Ni2+-chelate column (5 ml volume) (Novagen, Madison, WI) previously equilibrated with 50 mM Tris-HCl (pH 7.8) containing 0.5 M NaCl and 6 M urea. The column was washed extensively with the above buffer containing 20 mM imidazole (100 ml), and then eluted with 50 mM histidine in the same buffer containing 0.5 M NaCl and 6 M urea. Eluted proteins were dialyzed against 50 mM Tris-HCl (pH 8.0) and stored at -20°C. The enzymatically inactive fusion protein thus produced was activated with recombinant enteropeptidase from the medaka fish (Oryzias latipes), a small freshwater 5 teleost. Medaka enteropeptidase was immobilized on CNBr-activated Sepharose 4B (Sigma, St. Louis, MO) prior to use according to the manufacturer’s protocol. hK8 was incubated with the immobilized enteropeptidase for 16 h at room temperature. After removing the immobilized protease by filtration, the activity of hK8 was assayed by a 4-methylcoumaryl-7-amide (MCA)-containing synthetic substrate, Pro-Phe-Arg-MCA (Peptide Institute, Osaka, Japan). The molecular cloning, expression, and characterization of medaka enteropeptidase will be reported elsewhere. 2.3. Enzyme activity assay Unless otherwise stated, hK8 activity was determined at 37°C with Pro-Phe-Arg-MCA according to the method of Barrett [19], with the slight modifications reported in our previous study [20]. The release of fluorophore 7-amino-4-methyl coumarin (AMC) was measured by spectrofluorometry using an excitation wavelength of 370nm and an emission wavelength of 460nm. Active hK8 was preincubated with various protease inhibitors at 37°C in 0.1 M Tris-HCl buffer (pH 8.0). After incubation for 15 min, enzyme activity was measured using Pro-Phe-Arg-MCA as substrate. 2.4. Kinetic parameters Kinetic parameters were determined for various MCA substrates. Initial velocities, extrapolated from the plot of product versus time, were transformed into double-reciprocal plots [21]. Maximum velocities (Vmax), Km, and kcat values were obtained from the intercepts of these plots. 6 The active rhK8 concentration was determined using the active site titrant p- nitrophenyl-p’-guanidinobenzoate HCl [22]. 2.5. Digestion of protein substrates Forty micrograms of human plasma fibronectin (Chemicon, Temecula, CA), porcine type 1 gelatin (Daiichi Fine Chemicals, Toyama, Japan), mouse laminin (Biomedical Technologies Inc., Stoughton, MA), human fibrinogen (Merk Biosciences, Tokyo, Japan), human high-molecular-weight kininogen (Calbiochem), and casein (Sigma) were incubated at 37°C in 200 μl of 50 mM Tris-HCl buffer (pH 8.0) with 4 μg of hK8. Aliquots of 20 μl mixtures were taken at various time points (0 to 18 h), and mixed with 5 μl of SDS-PAGE sample buffer. The mixtures were boiled and aliquots of 20 μl samples were subjected to SDS-PAGE under reducing conditions. After electrophoresis, gels were stained with 0.25% Coomassie Brilliant Blue. Twenty-five micrograms of FITC-conjugated bovine collagen type I and type IV (Yagai Corporation, Yamagata, Japan) were incubated with hK8 (0.25 μg) at 33°C for 16 h in 100 μl of 50 mM Tris-HCl buffer (pH 8.0).