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J. Biol. Chem., Vol. 282, Issue 4, 2405-2422, January 26, 2007

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Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue Kallikrein 14^*

Carla A. Borgoño, Iacovos P. Michael, Julie L. V. Shaw, Liu-Ying Luo, Manik C. Ghosh, Antoninus Soosaipillai, Linda Grass, Dionyssios Katsaros, and Eleftherios P. Diamandis¹

From the Department of Pathology and Laboratory Medicine, Mount Sinai Hospital and the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada and the Department of Gynecology, Gynecologic Oncology Unit, University of Turin, Turin 10126, Italy

Received for publication, August 31, 2006 , and in revised form, November 14, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins ₁-antitrypsin, ₂-antiplasmin, antithrombin III, and ₁-antichymotrypsin with second order rate constants (k₊₂/K_i) of 49.8, 23.8, 1.48, and 0.224 µM^–1 min^–1, respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I–IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Proteases include a group of enzymes that catalyze peptide bond hydrolysis. Serine proteases (SP),² characterized by the presence of a nucleophilic serine residue at the active site, account for 30% of all proteases within the human degradome (1). Based on structural homology, SP are categorized into 12 clans, the most highly populated being clan PA(S), and are further classified into families that share high sequence similarity (2).

Human tissue kallikreins (KLK, EC 3.4.21) form a subgroup of 15 secreted (chymo)tryptic-like SP within the S1A family of clan PA(S) and are encoded by the largest contiguous cluster of protease genes in the entire genome, located on chromosome 19q13.4 (1, 3, 4). Because KLKs are widely expressed, KLK activity is implicated in an assorted array of normal physiological processes (e.g. blood pressure regulation, skin homeostasis, and semen liquefaction) and in the pathobiology of several diseases (e.g. cancer, neurodegenerative disorders, and dermatoses) (3–5). Because of the frequent dysregulation of KLK expression in malignancy, KLKs have been intensely studied in terms of their clinical applicability as cancer biomarkers. In addition to human kallikrein 3 (KLK3, also known as prostate-specific antigen (PSA)), the premier biomarker in clinical medicine for prostate cancer (6), most other KLKs have emerged as promising markers of diagnosis, prognosis, prediction of therapeutic response, and monitoring for several cancer types, particularly ovarian carcinoma (5).

Along with seven other KLK genes, human tissue kallikrein gene 14 (KLK14) was independently identified in our laboratory by positional candidate cloning in 2001 (7, 8). Subsequent studies demonstrated that the KLK14 gene is under steroid-hormone regulation (9, 10) and is most highly expressed in the glandular epithelia of the breast (10, 11) and prostate (8, 10) and in the epidermis (i.e. stratum granulosum) and appendages (i.e. hair follicular epithelium and eccrine sweat glands) of the skin (10, 12, 13). Consequent to its secretion, KLK14 forms a constituent of seminal plasma (10) and sweat (14) and is present in its catalytically active form in the stratum corneum of the skin (13–15). Furthermore, aberrant KLK14 expression has been detected in patients with breast (7, 10, 11, 16), ovarian (7, 9, 10), prostate (7, 17), and testicular (7) cancers and peeling skin syndrome (18), at the tissue and/or serum level. Correlative clinical data have also linked elevated KLK14 mRNA expression with aggressive forms of breast (11, 16) and prostate cancer (17) and with the prognosis of breast (16) and ovarian (9) cancer patients. Thus, KLK14 represents a potential biomarker and therapeutic target for several pathologic conditions.

As with all S1A family SP, KLK14 is synthesized as an inactive precursor or zymogen (herein denoted pro-KLK14), containing a 6-amino acid N-terminal pro-peptide that maintains its latency (7, 8). Proteolytic removal of the pro-peptide at Lys²⁴–Ile²⁵ is required to generate active KLK14, a process that may be performed by KLK5 (19). Because of the presence of Asp¹⁹⁸ in its S1 binding pocket (7, 8), KLK14 is predicted to exert trypsin-like substrate specificity with a preference for basic P1 residues (Schechter and Berger nomenclature (20) is used to describe the interaction between protease subsites (Sn-S1;S1'–Sn') and corresponding substrate residues (Pn-P1;P1'–Pn'), where P1–P1' denotes the scissile bond). However, we (21) and others (19), through the use of phage-display technology and chromogenic substrates, respectively, have recently demonstrated that KLK14 can accommodate both basic (Arg and Lys) and hydrophobic (Tyr) P1 residues at the S1 subsite with a preference for Arg. Hence, KLK14 manifests dual, trypsin-like and chymotrypsin-like, substrate specificity.

To date, putative biological substrates and (patho)physiological roles for KLK14 have been inferred from its expression pattern, substrate specificity, and the known function of co-localized KLKs. Phage-displayed pentapeptide motifs preferred by KLK14 were identified within several intact proteins, including the extracellular matrix (ECM) molecules laminin, collagen type IV, and matrilin-4 (21). The latter may support a role for KLK14 in cancer progression via ECM digestion, particularly in breast, ovarian, and prostate tumors that bear elevated KLK14 levels. Because of its presence and prominent activity in the stratum corneum of the skin (13–15), KLK14 is also implicated in epidermal desquamation (i.e. shedding) via degradation of intercellular (corneo)desmosomal adhesion molecules that link adjacent corneocytes (19). More recently, our group has reported that KLK14 may also mediate pleiotropic effects by signaling through proteinase-activated receptors 1, 2, and 4 (22). KLK14 may exercise its functions alone or in a proteolytic cascade pathway, as KLK5 may activate pro-KLK14 and vice versa (19).

Collectively, studies on KLK14 reveal that this novel SP may play important (patho)physiological roles at the main sites of its expression and bears clinical utility. To gain further insights into the biological significance of KLK14 action, this study examines KLK14 expression, specificity, activity against candidate substrates, and several modes of regulation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning, Expression, and Purification of Recombinant KLK14
Mature KLK14—Mature recombinant KLK14 was produced in the Easyselect^TM Pichia pastoris expression system (Invitrogen), as described previously (21). Briefly, PCR-amplified KLK14 cDNA encoding the mature enzyme form of KLK14 (amino acids 25–251 based on GenBank^TM accession number AAK48524 [GenBank] ) was cloned into P. pastoris expression vector pPICZA (Invitrogen) at EcoRI and XbaI restriction enzyme sites between the 5' promoter and the 3' terminator of the AOX1 gene, in-frame with the yeast -mating factor (for secretion), using standard techniques. The PmeI-linearized pPICZA-KLK14 construct was transformed into chemically competent P. pastoris yeast strains X-33, GS115, and KM71. A stable X-33 transformant was selected, and recombinant KLK14 expression was induced with 1% methanol/day for 6 days at 30 °C in a shaking incubator (250 rpm).

Mature recombinant KLK14 was purified to homogeneity from culture supernatant by a two-step procedure consisting of cation-exchange and affinity chromatography. Typically, 1 liter of culture supernatant was clarified by centrifugation and concentrated 20-fold by positive pressure ultrafiltration in an Amicon^TM stirring chamber (Millipore Corporation, Bedford, MA) with a 10-kDa cutoff nitrocellulose membrane (Millipore). The concentrated supernatant was diluted 1:4 in 10 mM MES (pH 5.3) and fractionated on a pre-equilibrated 5-ml cation-exchange HiTrap^TM SP HP column (GE Healthcare), with the automatedÁKTA FPLC system (GE Healthcare). Adsorbed KLK14 was eluted with a multistep salt gradient using 1 M KCl in 10 mM MES (pH 5.3) at a flow rate of 3 ml/min as follows: (a) continuous linear gradient of 0–0.3 M KCl for 17 min, (b) maintenance at 0.3 M KCl for 20 min, (c) followed by a continuous linear gradient from 0.3–1 M KCl for 50 min. Elution fractions (3 ml) containing KLK14 were identified (as described below), pooled, and concentrated using Biomax-10 Ultrafree®-15 centrifugal filter device (Millipore Corp., Bedford, MA). The concentrated fractions were diluted 1:4 in 100 mM Tris-HCl (pH 7.8) binding buffer and incubated with 1 ml of soybean trypsin inhibitor-agarose beads (Sigma) overnight at 4 °C. The beads were then packed into an Econo-Pac open column (Bio-Rad) and washed three times with binding buffer. KLK14 was eluted with 0.1 M glycine buffer (pH 3.0).

Pro-KLK14—First-strand cDNA synthesis was performed by reverse transcriptase using the Superscript^TM preamplification system (Invitrogen) with 2 µg of total human cerebellum RNA (Clontech) as a template. The cDNA encoding prepro-KLK14 (amino acids 1–251 based on GenBank^TM accession number AAK48524 [GenBank] ) was PCR-amplified in a 50-µl reaction mixture containing 1 µl of cerebellum cDNA as a template, 100 ng of primers (forward, 5' CACC ATG TTC CTC CTG CTG ACA GCA CTT; reverse, 5' AGA CCA TCA TTT GTC CCG CAT CGT TTC CT, containing CACC sequence required for TOPO® cloning (underlined) and the native KLK14 stop codon (italics)), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 µM deoxynucleoside triphosphates (dNTPs), and 0.75 µl (2.6 units) of Expand Long Template PCR polymerase mix (Roche Diagnostics) on an Eppendorf master cycler (Eppendorf, Westbury, NY). PCR conditions were 94 °C for 2 min, followed by 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s for 40 cycles, and a final extension at 68 °C for 7 min. The PCR product was cloned into the mammalian expression vector pcDNA3.1^TMD/V5-His-TOPO® (Invitrogen) using the pcDNA3.1^TM Directional TOPO® expression kit (Invitrogen), according to the manufacturer's protocol. The KLK14 sequence within the construct, denoted pcDNA3.1-KLK14, was confirmed with an automated DNA sequencer using vector-specific primers in both directions.

Human embryonic kidney (HEK)-293 cells (American Type Culture Collection (ATCC), Manassas, VA) were stably transfected with 3 µg of PmeI-linearized pcDNA3.1-KLK14 by lipofection using PolyFect transfection reagent (Qiagen, Valencia, CA), according to the manufacturer's instructions. Transfected HEK-293 cells were incubated in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS), in a humidified atmosphere containing 5% CO₂. After 48 h, cells were re-plated into 35-mm dishes, and the selection agent Geneticin (G418, 300 µg/ml final concentration; Invitrogen) was added. Resistant clones were chosen over 14 days under continuous G418 selection and cultured separately in 24 wells followed by 6-well plates. One clone, denoted P1–4, was selected for large scale expression of pro-KLK14 and grown in DMEM with 10% FBS until 80% confluency. At this stage, the media were replaced with serum-free CD Chinese hamster ovary media supplemented with GlutaMax^TMI (8 mM final concentration; Invitrogen). Conditioned media was collected by centrifugation after 10 days of incubation.

Recombinant pro-KLK14 was purified to homogeneity from the conditioned serum-free media of HEK-293 cells stably transfected with pcDNA3.1-KLK14 sequentially by cation-exchange and reversed-phase chromatography. Prior to purification, 1 liter of media was prepared as described above for yeast culture supernatant. Concentrated CM was diluted 1:4 in 10 mM MES (pH 5.3) and applied to a previously equilibrated 5-ml SP HP-Sepharose column (GE Healthcare), previously equilibrated with 10 mM MES (pH 5.3), on theÁKTA fast performance liquid chromatography system at a flow rate of 1 ml/min. Pro-KLK14 was eluted with a multistep salt gradient of 1 M KCl in 10 mM MES (pH 5.3) at a flow rate of 3 ml/min as follows: (a) continuous linear gradient of 0–0.25 M KCl for 17 min, (b) maintenance at 0.25 M KCl for 33 min, (c) linear gradient of 0.25–0.35 M KCl for 8.3 min, (d) maintenance at 0.35 M KCl for 33 min, and (e) followed by a linear gradient of 0.35-0.8 M KCl for 33 min. Elution fractions (3 ml) were analyzed by our newly developed enzyme-linked immunosorbent assay (ELISA; described herein) and by detection methods mentioned below. Fractions containing pro-KLK14 were then pooled, concentrated, and supplemented with a final concentration of 1% trifluoroacetic acid and loaded in 0.1% trifluoroacetic acid in H₂O onto a 1-ml Vydac C4 reversed-phase column (The Separations Group, Inc., Hesperia, CA) connected to an Agilent series 1100 HPLC system equipped with a diode array detector (Agilent Technologies, Inc, Palo Alto, CA). Elution was performed with a multistep acetonitrile (ACN) gradient at a flow rate of 0.8 ml/min consisting of 5-min linear gradients of 20–25, 25–30, 30–32, 32–35, 35–40, and 40–100% ACN intervened by 15-min steps at 25, 30, 32, and 35% ACN. ACN was removed from each fraction by evaporation with nitrogen gas at a pressure of 15 p.s.i.

For both recombinant KLK14 proteins, purity was assessed on silver-stained SDS-polyacrylamide gels (described below). Concentration was determined by the BCA method (Pierce), and protein identity was confirmed by tandem mass spectrometry, as described previously (23). Proteins were aliquoted and stored at –80 °C in 0.1 M sodium acetate buffer (pH 5.0).

Detection of Recombinant KLK14
To monitor recombinant KLK14 production, purification, and/or activity, samples were analyzed by SDS-PAGE, Western blot, and zymography.

SDS-PAGE—SDS-PAGE was performed using the NuPAGE BisTris electrophoresis system and precast 4–12% gradient polyacrylamide gels at 200 V for 30 min (Invitrogen). Proteins were visualized with a Coomassie G-250 staining solution, SimplyBlue^TM SafeStain (Invitrogen), and/or by silver staining with the Silver Xpress^TM kit (Invitrogen), according to the manufacturer's instructions.

Western Blots—For immunodetection of KLK14, proteins resolved by SDS-PAGE were subsequently transferred onto a Hybond-C Extra nitrocellulose membrane (GE Healthcare) at 30 V for 1 h. The membrane was blocked with Tris-buffered saline/Tween (0.1 mol/liter Tris-HCl buffer (pH 7.5) containing 0.15 mol/liter NaCl and 0.1% Tween 20) supplemented with 5% nonfat dry milk overnight at 4 °C and probed with a KLK14 polyclonal rabbit antibody (produced in-house; diluted 1:2000 in Tris-buffered saline/Tween) for 1 h at room temperature. The membrane was washed three times for 15 min with Tris-buffered saline/Tween and treated with alkaline phosphatase-conjugated goat anti-rabbit antibody (1:10,000 in Tris-buffered saline/Tween; Jackson ImmunoResearch) for 1 h at room temperature. Finally, the membranes were washed again as above, and fluorescence was detected on x-ray film using chemiluminescent substrate (Diagnostic Products Corp., Los Angeles).

Zymography—The proteolytic activity of recombinant KLK14 proteins was visualized by gelatin zymography (Invitrogen). Recombinant KLK14 was diluted 1:1 in Tris-glycine SDS sample buffer and electrophoresed on precast Novex® 10% gelatin zymogram gels (Invitrogen) at 125 V for 2.5 h at 4 °C. The gels were subsequently incubated in zymogram renaturing buffer (Invitrogen) for two 30-min intervals at room temperature, followed by incubation in zymogram developing buffer (Invitrogen) for 3 h at 37°C. Gels were stained with SimplyBlue^TM SafeStain and destained until the white lytic bands corresponding to areas of protease activity were visible.

Generation of Antibodies against KLK14
New Zealand White female rabbits and BALB/c female mice were repeatedly immunized with purified recombinant mature KLK14 (100 µg) for polyclonal and monoclonal antibody development, respectively. KLK14 was diluted 1:1 in complete Freund's adjuvant for the first injection and in incomplete Freund's adjuvant for subsequent injections. Injections were repeated three times for mice and six times for rabbits at 3-week intervals. The polyclonal sera were tested every 2 weeks by an immunofluorometric method described in detail elsewhere (10), until the highest antibody titers against KLK14 were detected.

Monoclonal antibodies against KLK14 were produced by standard hybridoma technology. The splenocytes from preselected immunized mice were fused with the Sp2/0 myeloma cells using polyethylene glycol 1500. The resultant hybridoma cells were cultured in 96-well plates in DMEM (Invitrogen) containing 20% FBS, 200 mM glutamine, 1% OPI (oxaloacetic acid, pyruvic acid, insulin), and 2% HAT (hypoxanthine, aminopterin, thymidine; Sigma) for selection at 37 °C, 5% CO₂ for 10–14 days. Hybridoma supernatants were collected and screened by an immunofluorometric technique described previously (24). Positive clones were then expanded sequentially in 24- and 6-well plates in complete media (reducing the FBS to 15% and changing the HAT to HT). Antibody isotyping was subsequently performed using a mouse immunoglobulin subtype identification kit (Sigma) to isolate clones producing anti-KLK14 monoclonal antibodies of the IgG class. One positive hybridoma clone, denoted 2E9, was expanded in flasks in serum-free CD Chinese hamster ovary media (Invitrogen) containing 200 mM glutamine, for large scale antibody production. Anti-KLK14 monoclonal antibody 2E9 was purified from the culture supernatant using the Affi-Gel Protein-A MAPS II kit (Bio-Rad), according to manufacturer's instructions, and used in subsequent immunoassay development.

Development of an ELISA for KLK14
A "sandwich-type" ELISA, with a mouse monoclonal/rabbit polyclonal configuration, was developed as follows. The KLK14-specific monoclonal antibody (clone 2E9; developed inhouse) was first immobilized on a 96-well white polystyrene plate (500 ng/well) by incubating in coating buffer (50 mmol/liter Tris, 0.05% sodium azide (pH 7.8)) overnight at room temperature. The plate was then washed three times with washing buffer (50 mmol/liter Tris, 150 mmol/liter NaCl, 0.05% Tween 20 (pH 7.8)). KLK14 standards or samples were then pipetted into each well (100 µl/well, diluted 1:1 in assay buffer (50 mmol/liter Tris, 6% bovine serum albumin, 10% goat IgG, 2% mouse IgG, 1% bovine IgG, 0.5 mol/liter KCl, 0.05% sodium azide (pH 7.8)), incubated for 2 h with shaking, and then washed six times, as above. Subsequently, 100 µl of rabbit anti-KLK14 polyclonal sera (developed in-house) diluted 1000-fold in assay buffer was added and incubated for 1 h. After incubation, the plate was washed as above, and alkaline phosphatase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), diluted 3000-fold in assay buffer, was applied and plates were incubated for 45 min. After washing as above, the alkaline phosphatase substrate diflunisal phosphate (100 µl of a 1 mM solution) in substrate buffer (0.1 mol/liter Tris (pH 9.1), 0.1 mol/liter NaCl, and 1 mmol/liter MgCl₂) was added to each well and incubated for 10 min followed by addition of developing solution (100 µl, containing 1 mol/liter Tris base, 0.4 mol/liter NaOH, 2 mmol/liter TbCl₃, and 3 mmol/liter EDTA) for 1 min. The resultant fluorescence was measured by time-resolved fluorometry on the Cyberfluor 615 Immunoanalyzer (MDS Nordion, Kanata, Ontario, Canada). Calibration and data reduction were performed automatically, as described elsewhere (25).

The optimal assay configuration and conditions described above were obtained by testing several monoclonal antibody and polyclonal sera dilutions and diluents, as well as different incubation times for each step of the procedure. Assay sensitivity (detection limit), specificity (including cross-reactivity to other KLK family members), linearity, recovery, and within-run and between-run imprecisions were determined as described previously (10).

Tissue Extracts, Biological Fluids, and Cancer Cell Lines
The concentration of KLK14 in normal and cancerous tissue extracts, biological fluids, and conditioned media of various cancer cell lines was determined with the newly developed KLK14-specific ELISA.

Tissue Extracts—A panel of 40 normal tissue extracts were prepared as described elsewhere (10), including adrenal gland, axillary lymph node, bone, breast, colon, endometrium, esophagus, fallopian tube, kidney, liver, lung, mesenteric lymph node, skeletal muscle, ovary, pancreas, prostate, seminal vesicle, skin, small intestine, spleen, stomach, testis, thyroid, trachea, ureter, uterus, salivary gland, pharyngeal tonsil, placenta, cervix, cerebellum, frontal lobe cortex, hippocampus, medulla, midbrain, occipital cortex, pituitary, pons, temporal lobe cortex, and spinal cord. Cancerous ovarian tissues, containing >80% tumor cells, were obtained from patients with primary epithelial ovarian cancer who underwent surgery and treatment for ovarian cancer at the Department of Gynecology of the University of Turin, Turin, Italy, between July of 1991 and April of 1999. Ovarian tumor cytosolic extracts were prepared as described previously (26). Total protein content of the extracts was determined using the bicinchoninic acid method, with bovine serum albumin as a standard (Pierce).

Biological Fluids—Cerebrospinal fluid, breast milk, amniotic fluid, seminal plasma, ascites fluid from ovarian cancer patients, breast cancer cytosolic extracts, and sera from males and females without known disease and of prostate cancer patients were residual samples submitted previously for routine biochemical testing and stored at –80 °C. Investigations were carried out in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 1983, and have been approved by the Institutional Review Board of Mount Sinai Hospital, Toronto, Canada, and the Institute of Obstetrics and Gynecology, Turin, Italy.

Cell Lines—The astrocytoma cell line, SW-1088 (HTB12); breast cancer cell lines MDA-MB-468 (HTB132), BT-474 (HTB-20), T-47D (HTB-133), MCF7 (HTB-22), and ZR-75, MFM223, MDA-MB-231 (HTB-26), MDA-MB-453 (HTB-131), MDA-MB-361 (HTB27), BT-20 (HTB19); the colorectal adenocarcinoma cell line, COLO 320HSR (CCL-220.1); the ovarian cancer cell lines Caov-3 (HTB-75), MDAH 2774 (CRL-10303), TOV-21G (CRL-11730), TOV112D (CRL-11731), OVCAR-3 (HTB-161), OV-90 (CRL-11732), ES2 (CRL-1978); the cervical cancer cell lines HeLa (CCL-2), C-4 I (CRL-1594), ME-180 (HTB-33), HT-3 (HTB-32); neuroblastoma cell lines SK-N-DZ (CRL-2149) and IMR-32 (CCL-127) and neuroepithelioma cell line Sk-N-MC (HTB-10); the pancreatic cancer cell line, MIA PaCa-2 (CRL-1420); and the prostate cancer cell line LNCaP (CRL-1740) were purchased from ATCC. The BG-1 ovarian cancer cell line was kindly provided by Dr. Henri Rochefort (Montpellier University, Montpellier, France), and the PC-3 cell line, stably transfected with androgen receptor (PC-3 (AR)₆); was provided by Dr. Theodore Brown (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada). Cells were cultured to near confluency in RPMI medium (Invitrogen) supplemented with glutamine (200 nM) and FBS (100 ml/liter) in plastic flasks.

Protease Assays and Kinetic Constant Determinations
7-Amino-4-methylcoumarin (AMC) was purchased from Sigma. The following synthetic AMC substrates were obtained from Bachem Bioscience (King of Prussia, PA): Boc-Phe-Ser-Arg-AMC (FSR-AMC), Boc-Val-Pro-Arg-AMC (VPR-AMC), H-Pro-Phe-Arg-AMC (PFR-AMC), benzyloxycarbonyl-Gly-Gly-Arg-AMC (GGR-AMC), Boc-Leu-Gly-Arg-AMC (LGR-AMC), Boc-Leu-Lys-Arg-AMC (LKR-AMC), Boc-Leu-Arg-Arg-AMC (LRR-AMC), Boc-Gln-Arg-Arg-AMC (QRR-AMC), Boc-Gln-Ala-Arg-AMC (QAR-AMC), Boc-Gln-Gly-Arg-AMC (QGR-AMC), Tos-Gly-Pro-Arg-AMC (GPR-AMC), Tos-Gly-Pro-Lys-AMC (GPK-AMC), Boc-Glu-Lys-Lys-AMC (EKK-AMC), Boc-Val-Leu-Lys-AMC (VLK-AMC), and Suc-Ala-Ala-Pro-Phe-AMC (AAPF-AMC). Suc-Leu-Leu-Val-Tyr-AMC (LLVY-AMC) was purchased from the Peptide Institute Inc. (Osaka, Japan). All substrates were diluted in dimethyl sulfoxide (Me₂SO) to a final concentration of 80 mM and stored at –20 °C.

Individual AMC-peptide substrates (final concentrations ranging from 0.004 to 2 mM) were incubated with recombinant mature KLK14 (final concentration of 12 nM) at a final volume of 100 µl under optimal buffer conditions within 96-well white polystyrene microtiter plates. Reaction mixtures contained less than 5% (v/v) Me₂SO. Initial rates of KLK14-mediated peptide hydrolysis was monitored by measuring free AMC fluorescence on the Wallac 1420 Victor^2TM fluorometer (PerkinElmer Life Sciences) with excitation and emission filters set at 380 and 480 nm, respectively, at 1-min intervals for 20 min at 37 °C. KLK14-free reactions, for each peptide concentration, were used as negative controls, and background counts obtained were subtracted from each value. A standard curve was constructed using known concentrations of AMC in order to calculate the rate of free AMC release. The slope of the resultant AMC standard curve was 19.184 AMC fluorescence counts/nM free AMC. The steady-state (Michaelis-Menten) kinetic constants (k_cat/K_m) were then calculated by nonlinear regression analysis using Enzyme Kinetics Module 1.1 (Sigma Plot). All experiments were performed in triplicate and repeated at least twice.

Pro-KLK14 Activation Studies
The possible activation of pro-KLK14 by recombinant mature KLK5 (produced in-house in P. pastoris as described previously (27)) was monitored via complex formation between KLK14 and the serine protease inhibitor (serpin) ₁-antitrypsin (AT), an inhibitor of KLK14, but not of KLK5 (27). Pro-KLK14 was preincubated with KLK5 in a final volume of 10 µl at different molar ratios and incubation times. AT was then added at a 2-fold molar excess to a final volume of 15 µl. The reaction was incubated for 30 min at room temperature. As controls, AT was incubated alone and with recombinant mature KLK14 and pro-KLK14 and with mature KLK5. Reaction mixtures were resolved by SDS-PAGE under reducing conditions, and resultant gels were silver-stained.

Inhibition Assays, Serpins—Serine protease inhibitors (serpins), AT (SERPINA1), ₁-antichymotrypsin (ACT; SERPINA3), antithrombin III (ATIII; SERPINC1), ₂-antiplasmin (AP; SERPINF2), and plasminogen activator inhibitor-1 (PAI-1; SERPINE1) were purchased from Calbiochem. Inhibitors displayed >95% purity on Coomassie Blue-stained polyacrylamide gels, were diluted in water to a final concentration of 1 mg/ml, and were stored at –20 °C. To calculate second-rate constants, the inhibition of KLK14 by serpins was performed under pseudo-first order conditions, as described previously (28–30). KLK14 was preincubated with each serpin for various lengths of time (0–5 min for AT, AP, and PAI-1; 0–10 min for ATIII, and 0–60 min for ACT) and at different KLK14:serpin molar ratios at room temperature (for AT, AP, PAI-1 and ATIII) or 37 °C (for ACT) with gentle agitation. The reaction was quenched by diluting 10 µl in 190 µl of optimal buffer containing 0.2 mM QAR-AMC. AMC fluorescence was measured on the Wallac 1420 Victor^2TM fluorometer, as described above. All experiments were performed in triplicate.

To monitor KLK14-serpin complex formation, KLK14 (0.5 µg; 1 µM final concentration) was incubated with each serpin at molar ratios of 1:1, 1:2, 1:5, and 1:10 at room temperature for 30 min (for AT, AP, PAI-1 and ATIII) or at 37 °C for 1 h (for ACT). Reaction mixtures were resolved by SDS-PAGE under reducing conditions; gels were stained with SimplyBlue^TM SafeStain.

Effect of Ions on KLK14 Activity
KLK14 was incubated with each cation and citrate (solutions prepared from salts of ZnCl₂, ZnSO₄, Zn(CH₃COO)₂, CaCl₂, MgCl₂, NaCl, KCl, sodium citrate, at final concentrations of 0, 12, 60, 120, 1200, and 12,000 nM) in optimal assay buffer at a final volume of 100 µl for 10 min at 37 °C with gentle agitation. At this point, QAR-AMC (at a final concentration of 0.5 mM) was applied to each reaction mixture, and AMC fluorescence was measured on the Wallac 1420 Victor^2TM fluorometer, as described above. Residual KLK14 activity against QAR-AMC after incubation with individual cations/citrate was calculated.

Reversal of Zn²⁺ Inhibition of KLK14 by EDTA
KLK14 (12 nM final concentration) was incubated alone or with the following: (a) Zn²⁺ (1.2 µM final concentration ZnCl₂), (b) EDTA (1 mM final concentration), (c) Zn²⁺ and EDTA, and (d) Zn²⁺ followed by the addition of EDTA after 20 min, at a final volume of 100 µl in optimal assay buffer in microtiter plate wells for 10 min at 37 °C with gentle agitation. After incubation, QAR-AMC (final concentration of 0.5 mM) was applied to each well. AMC fluorescence was measured on the Wallac 1420 Victor^2TM fluorometer, as described above. KLK14-free reactions were used as negative controls, and background fluorescence was subtracted from each value. All experiments were performed in triplicate.

In Vitro Proteolysis Experiments
The following substrates were incubated with KLK14: mouse sarcoma collagen type I, chicken sternal cartilage collagen type II, calf skin collagen type III, human placenta collagen type IV, human foreskin fibroblast fibronectin, human plasma vitronectin, laminin, and low molecular weight kininogen were purchased from Sigma. Plasminogen from human plasma and recombinant human insulin-like growth factor-binding proteins 2 and 3 (IGFBP-2 and IGPBP-3) were obtained from R&D Systems Inc. (Minneapolis, MN).

Collagen I, II, and III (2 µg); collagen IV, laminin, fibronectin, kininogen (5 µg); fibrinogen, plasminogen (10 µg); and vitronectin, IGFBP-2, and IGFBP-3 (300 ng) were incubated with specific amounts of KLK14 (6 ng to 1 µg) in optimal assay buffer over time at room temperature or 37 °C with gentle agitation. KLK14-free reactions were included as negative controls. Reactions were terminated by freezing in liquid nitrogen and resolved by reducing SDS-PAGE, as described above. Gels were stained with SimplyBlue^TM SafeStain or with the Silver Xpress^TM kit (Invitrogen).

Furthermore, KLK14 (0.4 and 0.8 µg) was incubated with fluorescent conjugates of collagen types I and IV (2.5 µg) and fibrinogen (3.75 µg) (Molecular Probes, Eugene, OR) in optimal assay buffer (final volume 200 µl) at room temperature. Fluorescence was measured every 10 min for 2 h on the Wallac 1420 Victor^2TM fluorometer set at 492 nm for excitation and 535 nm for emission. Enzyme-free reactions were used as negative controls, and background fluorescence was subtracted from each value. All experiments were performed in triplicate.

N-terminal Sequencing
N-terminal sequence analysis was performed on KLK14-generated fragments derived from recombinant mature KLK14, fibronectin, collagen IV, plasminogen, kininogen, fibrinogen, and vitronectin. KLK14 was incubated alone (3 µg) or with each intact protein (2.5–5 µg) for different time points as specified by the proteolysis experiments, separated by reducing SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (GE Healthcare), previously immersed in 100% methanol at 30 V for 1 h. After the transfer, the membrane was removed and rinsed with de-ionized water, stained with Coomassie Blue R-250 (0.1% solution in 40% methanol) for 5 min, and de-stained for 5 min in 50% methanol. The membrane was then thoroughly washed with de-ionized water and air-dried. Fragments were excised and subjected to automated N-terminal Edman degradation (31) consisting of five cycles of Edman chemistry on a 492 Procise cLC sequencer (Applied Biosystems, Foster City, CA), followed by analysis of resultant phenylthiohydantoin-derivative residues on an HPLC column.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Recombinant Mature and Pro-KLK14
Recombinant mature and pro-KLK14 proteins were produced in their native forms, without fusion tags, in yeast (P. pastoris) and mammalian (HEK-293) expression systems, respectively. Secreted expression was achieved by cloning KLK14 cDNA in-frame with the yeast -factor for mature KLK14 and with the native KLK14 signal sequence in the case of pro-KLK14. As monitored by SDS-PAGE, Western blot analysis and/or ELISA, mature KLK14 was detected in the yeast culture supernatant after 1 day of methanol induction, with the highest levels produced after 6 days; maximal pro-KLK14 levels were attained after a 10-day incubation period in serum-free media (data not shown). After their respective two-step chromatographic purification procedures, recombinant mature and pro-KLK14 were obtained with >95% purity, as verified by silver-stained polyacrylamide gels (Fig. 1A). The yield of purified mature and pro-KLK14 from 1-liter culture supernatants was in the range of 1.5–3 mg and 25–50 µg, respectively, as determined by ELISA and total protein assays.

Prepro-KLK14 is a 251-amino acid polypeptide consisting of a signal sequence (Met¹–Ser¹⁸), followed by a 6-amino acid pro-peptide (Gln¹⁹–Lys²⁴) and a 227-amino acid trypsin-like serine protease domain (Ile²⁵–Lys²⁵¹) with a predicted zymogen activation site at Lys²⁴–Ile²⁵ (7, 8). The resultant masses of both recombinant proteins were consistent with those inferred from the primary KLK14 sequence, indicating a lack of glycosylation or other post-translational modifications. Purified mature KLK14 appeared as an enzymatically active intact band of 25 kDa along with 8 less abundant lower molecular weight bands, identified as products of autodegradation (Fig. 1, lane 1, and Fig. 4). Purified pro-KLK14 was visualized as a single, enzymatically inactive band of 27 kDa, as expected (Fig. 1, lane 2).

View larger version (48K): [in this window] [in a new window]   FIGURE 1. Purified recombinant mature and pro-KLK14. A–C, lane 1, mature KLK14; lane 2, pro-KLK14. A, silver-stained reducing SDS-PAGE of KLK14 (300 ng/lane). B, immunodetection of KLK14 using a primary rabbit polyclonal antibody (1:2000) generated against mature KLK14. C, gelatin zymography of KLK14. Intact mature KLK14 is represented by a single band of 25 kDa (); lower molecular weight bands likely correspond to autodegradation products and are indicated with arrows (). Pro-KLK14 is visualized as a single band of 27 kDa (). Zymographic analysis reveals that only mature KLK14 is active, as expected. M, molecular mass standards in kDa.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Concentration of KLK14 in adult human tissue extracts. The amount of KLK14 in tissue extracts is corrected for the total protein content and is expressed as nanograms of KLK14 per g of total protein.

Distribution of KLK14 in Tissues, Fluids, and Cell Lines
A novel KLK14-specific ELISA was established to quantify KLK14 levels in normal and cancerous tissue extracts, biological fluids, and cell lines. The optimized KLK14-ELISA, consisting of monoclonal antibody 2E9 (capture antibody) and polyclonal rabbit antiserum (detection antibody) generated against recombinant mature KLK14, is sensitive (detection limit of 0.05 µg/liter), specific for KLK14 (<0.1% cross-reactivity with other KLKs), recognizes both mature and pro-KLK14 recombinant proteins, and is linear from 0.05 to 10 µg/liter with between-run and within-run coefficients of variation of <10%.

Among the 40 normal adult human tissues screened for KLK14 content, 18 contained detectable levels of KLK14 (Fig. 2). The highest KLK14 levels were found in skin (245 ng/g total protein), followed by breast (224 ng/g total protein) and prostatic (131 ng/g total protein) tissue extracts, in general accord with previous studies (7;8;10;11;13–15;17). Comparatively lower levels were observed in axillary lymph nodes (62.8 ng/g total protein) and medulla (59.2 ng/g total protein) extracts with minor amounts (<50 ng/g total protein) in the remaining tissues.

The concentration of KLK14 in biological fluids from healthy individuals was quantified (Table 1). In agreement with our prior study (10), the highest levels of KLK14 were measured in seminal plasma (15.8 ± 16.0 µg/liter), followed by amniotic fluid (8.1 ± 6.0 µg/liter) and saliva (4.4 ± 2.1 µg/liter). Follicular fluid and breast milk contained lower concentrations (1.2 µg/liter). KLK14 was not detected in the cerebrospinal fluid samples examined. Compared with normal, KLK14 levels were elevated in the serum of patients with prostate cancer (Fig. 3), a likely consequence of KLK14 overexpression and secretion by prostate tumor tissues (17). We also detected KLK14 in ascites fluids from ovarian cancer patients (0.34 ± 0.7 µg/liter) as well as in cytosolic extracts of ovarian (1.77 ± 5.13 µg/liter) and breast (0.26 ± 1.17 µg/liter) tumors.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Elevation of KLK14 in serum of prostate cancer patients. Distribution of KLK14 levels (µg/liter) in the sera of healthy males (normal) and patients with prostate cancer. The p value was determined by the Mann-Whitney U test. Horizontal lines represent the median values.

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Autodegradation of KLK14. A, Coomassie Blue-stained SDS-polyacrylamide gel of fragmented mature recombinant KLK14 (5 µg). Intact KLK14 corresponds to the 25-kDa band. Lower molecular weight autodegradation fragments of KLK14 are labeled I–VII. The region that each peptide fragment spans within the KLK14 protein sequence is in parentheses. Note that protein bands III and VII are comprised of 2 and 3 individual peptides, respectively. M, molecular mass standards in kDa. B, location of the N-terminal sequence of each KLK14 degradation fragment within the primary KLK14 protein sequence. Prepro-KLK14 is formed of a signal peptide (italics), followed by a pro-peptide (underlined) and the mature sequence containing the serine protease domain (boldface). Catalytic triad (His67, Asp111, and Ser204) residues are indicated with an asterisk. The N-terminal sequence of KLK14 degradation fragments is boxed, with the corresponding label above. Cleavage sites are indicated by an arrow; the P1 amino acid is numbered. A and B, KLK14 sequences are numbered from the N terminus of prepro-KLK14 based on GenBankTM accession number AAK48524. C, location of autolytic cleavage sites within the theoretical tertiary structure of mature KLK14, as predicted by homology modeling. The ribbon plot of mature KLK14 is shown in the traditional serine protease standard orientation (i.e. looking into the active site cleft) (86). Secondary structure elements are displayed as arrows (-strands) and ribbons (-helices). The position of the autolysis loop is indicated. N- and C-terminal residues are shown in black; the side chains of the catalytic triad residues are shown in green; S1 side chain is colored orange. KLK14 cleavage sites, i.e. P1 and P1' residues, are shown in red and blue, respectively, and P1-R residues are labeled. D, inactivation of KLK14 by autodegradation. KLK14 was incubated alone at 4 °C (•), 25 °C (), and 37 °C () over time (0–48 h). E, SDS-PAGE analysis of KLK14 autodegradation over time at 4, 25, and 37 °C. Time points are indicated above each lane. M, molecular mass standards in kDa.

Activation of Pro-KLK14
As with other S1A family SP, pro-KLK14 requires the removal of the inhibitory N-terminal pro-peptide sequence at Lys²⁴–Ile²⁵ for conversion into its enzymatically active form. Autoactivation is not expected to occur because P1-Lys residues are unfavored by KLK14 (21) (see below). Indeed, purified recombinant pro-KLK14 remained as an enzymatically inactive, intact 27-kDa band on SDS-PAGE and zymographic analyses after prolonged incubation (data not shown), which is supported by previous findings (19).

View larger version (60K): [in this window] [in a new window]   FIGURE 5. Lack of pro-KLK14 activation by KLK5. Pro-KLK14 (0.5 µM) was incubated with KLK5 (0.1 µM) for 0, 10, and 30 min and 1 and 3 h at 37°C, after which AT, at a KLK14-AT molar ratio of 1:2, was added for 30 min at room temperature (lanes 5–9, respectively). Activation of pro-KLK14 was monitored by formation of the AT-KLK14 complex. As controls, AT was incubated alone (lane 1) and with mature KLK14 (lane 2), pro-KLK14 (lane 3), and mature KLK5 (lane 4). Note that an AT-KLK14 complex was not observed after incubation of pro-KLK14 with KLK5, indicating a lack of activation.

Substrate Specificity of KLK14
The substrate specificity of KLK14 was assessed by determining its relative hydrolytic activity and corresponding kinetic parameters (i.e. K_m and k_cat) against a panel of 16 synthetic substrates containing an AMC fluorogenic leaving group (Table 2). Among these AMC-peptides, 14 contain a basic P1 residue (Arg, Lys) and 2 contain bulky, hydrophobic amino acids at P1 (Tyr, Phe) and thereby represent substrates for trypsin-like and chymotrypsin-like serine proteases, respectively. In accord with our previous study (21), KLK14 displayed trypsin-like specificity with a greater catalytic efficiency for Arg versus Lys at the P1 position, as substrates with the highest k_cat/K_m values contain P1-Arg. The P1-Arg preference of KLK14 was further demonstrated by the location of KLK14 cleavage sites within intact proteins (Fig. 4; Table 5), all of which occurred C-terminal of Arg residues. However, in contrast to prior findings (19, 21), we could not ascribe chymotryptic-like specificity to KLK14 under the experimental conditions chosen, i.e. no hydrolytic activity against either AAPF-AMC or LLVY-AMC was detected. Possible explanations for this finding include the following: 1) the inherent incompatibility of the AMC-substrates used in our study with KLK14 (i.e. lack of P' residues and the presence of unfavored residues at P2 and P3), 2) possible steric effects attributed to the interaction between AMC and the P1 amino acid (e.g. cleavage of a chymotrypsin-like chromogenic substrate, Ala-Pro-Tyr-p-nitroanilide, by KLK14 was reported in a previous study (19)), and 3) the final enzyme concentration used.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Kinetics of inactivation of KLK14 by 2-antiplasmin (A), 1-antitrypsin (B), antithrombin III (C), and 1-antichymotrypsin (D). KLK14 (final concentration, 0.1 µM) was incubated with increasing concentrations of each serpin for various time intervals after which residual activity against QAR-AMC (0.2 mM final concentration) was measured. A, AP final concentration (µM):0(), 0.1 (•), 0.2 (), 0.4 (). B, AT final concentration (µM):0(), 0.1 (•), 0.2 (), 0.4 (), 0.6 (). C, ATIII final concentration (µM):0(), 0.4 (), 0.6 (), 0.8 (), 1.0 (•). D, ACT final concentration (µM): 0 (), 0.5 (•), 1.0 (), 1.5 (), 2.0 (), 2.5 (). The insets show a double-reciprocal plot of the pseudo-first order rate constant (k') and the inhibitor concentration. The line drawn is a least squares fit of the experimental points. The equation of the line for AP is Y = 42.02X + 0.32 (r = 0.981); AT, Y = 20.07X + 0.12 (r = 0.980); ATIII, Y = 673.7X + 3.57 (r = 0.998); ACT, Y = 5.54X + 2.0 (r = 0.996). r is the Pearson correlation coefficient.

The P3 preference of KLK14 was assessed by examining k_cat/K_m values in substrate subgroups bearing the same P1 and P2 amino acids as follows: 1) VPR-AMC and GPR-AMC; 2) LGR-AMC, QGR-AMC, and GGR-AMC; and 3) QRR-AMC and LRR-AMC. Yet, unlike its relatively stringent P1 and modest P2 preferences, KLK14 possesses a lower specificity at P3. Specifically, the S3 subsite can accommodate a variety of amino acids, including basic residues and those with large and small aliphatic chains (21).

Regulation of KLK14 Activity
Serpins—Several extracellular inhibitory members of the serpin superfamily, namely ₁-antitrypsin (AT), ₁-antichymotrypsin (ACT), antithrombin III (ATIII), ₂-antiplasmin (AP), and plasminogen activator inhibitor-1 (PAI-1), were assayed for their inhibitory capacity against KLK14. Such inhibitory serpins behave as irreversible suicide substrate inhibitors and are cleaved by their protease targets at a scissile bond (P1-P1') within their exposed reactive site loops (RSL), after which the interaction may proceed via the "inhibitory" or "substrate" pathway, resulting in complex formation or an inactive serpin and an active protease, respectively (34). The kinetics of inactivation of KLK14 activity in the presence of increasing serpin concentrations are displayed graphically in Fig. 6. The corresponding kinetic constants (K_I, k₊₂) are listed in Table 3 and were calculated from the double-reciprocal plots of the pseudo-first order rate constant k' versus inhibitor concentrations (Fig. 6, insets). Based on the apparent second order rate constants (k₊₂/K_I) determined, the rank order for KLK14 inhibition by serpins from highest to lowest efficiency was AT > AP ATIII >> ACT (Table 3). AT was 2 times more effective than AP at KLK14 inactivation and 34- and 220-fold more efficient than ATIII and ACT, respectively. For instance, a 50% decrease in KLK14 activity was induced by AT (0.1 µM) in 1.2 min, by AP (0.1 µM) in 2.3 min, by ATIII (0.6 µM) in 5.7 min, and by ACT (1.0 µM) in 26 min. PAI-1 also inactivated KLK14; however, the reaction proceeded at a much higher rate than with other serpins; hence rate constant determination was not possible. For example, incubation of PAI-1 with KLK14 at a 1:1 molar ratio for 10 s at room temperature resulted in 100% inhibition of KLK14 activity. Thus, we speculate that PAI-1 is a more efficient inhibitor of KLK14 than AT.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. SDS-PAGE analysis of the interaction between KLK14 and the serpins plasminogen activator inhibitor-1 (A), 2-antiplasmin (B), 1-antitrypsin (C), antithrombin III (D), and 1-antichymotrypsin (E). KLK14 was incubated at 1:1 (lane 3), 1:2 (lane 4), 1:5 (lane 5), and 1:10 (lane 6) molar ratios with different serpins at either 37 °C or at room temperature for time points specified under "Experimental Procedures." KLK14 and serpins incubated alone are shown in lanes 1 and 2, respectively. The mixtures were subsequently resolved by SDS-PAGE under reducing conditions, and the gels were stained with Coomassie Blue. Intact serpins are indicated with arrows. Complexes (; labeled C) were formed between KLK14 and all serpins, with the exception of 1-antichymotrypsin. Interactions between KLK14 and all serpins also generated lower molecular weight fragments (), indicative of substrate pathway contributions. M, molecular mass standards in kDa.

Ions—The relative hydrolytic activity of KLK14 against QAR-AMC in the presence of the anion citrate and the cations Zn²⁺, Ca²⁺, Mg²⁺, Na⁺, and K⁺ is shown in Table 4. The presence of citrate seemed to have an activating effect on KLK14 activity, particularly at higher citrate concentrations (12–120 µM). Citrate has analogous effects on KLK3 (36), KLK5 (33), and KLK6 (37) activity.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Zinc inhibition of KLK14 activity (A) and its reversal by EDTA (B). A, KLK14 (12 nM) was incubated with increasing concentrations (12, 60, 120, 1200, 12,000 and 120,000 nM) of ZnCl2, ZnSO4, and Zn(CH3COO)2 for 30 min after which residual KLK14 activity was measured against QAR-AMC. Zinc ions (Zn2+) efficiently attenuate the activity of KLK14 with an average IC50 value of 1.46 nM. B, hydrolysis of QAR-AMC by KLK14 (12 nM) in the presence of: optimal buffer only (•), 1 mM EDTA (), 1.2 mM ZnCl2 (), and 1.2 mM ZnCl2 prior to and after the addition of 1 mM EDTA (). The arrow denotes the addition of 1 mM EDTA.

Extracellular Matrix Components
Incubation of KLK14 with fibronectin resulted in the generation of several fragments, ranging in size from 32 to 180 kDa (Fig. 9A). Based on the N-terminal sequence of fragments F1–F6, three major KLK14 cleavage sites were identified at Arg²⁹⁰–Ala²⁹¹, Arg⁹⁰³–Ser⁹⁰⁴, and Arg²⁰⁷⁰–His²⁰⁷¹, all of which lie between fibronectin-type domains, i.e. areas most susceptible to proteolysis (43) (Table 5). In terms of ligand-binding regions, the first site, Arg²⁹⁰–Ala²⁹¹, lies between the first fibrin/heparin-binding region and the collagen/gelatin-binding region; the second site, Arg⁹⁰³–Ser⁹⁰⁴, is flanked by the collagen/gelatin-binding and cell-binding regions, whereas the third site occurs within "connecting strand 3," which links the second heparin-binding and fibrin-binding regions (43).

Both and chains of laminin were completely digested by KLK14 after a 4-h incubation period (Fig. 9B). In our previous study, we identified and confirmed one KLK14 cleavage site at Arg²⁴²¹–Asp²⁴²² within the laminin 5 chain (21).

KLK14 was able to effectively process collagens I–IV in their denatured forms at 37 °C (Fig. 9, C–F), but not in their native conformations at 25 °C, as shown for collagen IV (Fig. 9F). KLK14 cleavage sites were identified within the collagen IV 2 chain at Arg¹⁰⁷⁷–Ala¹⁰⁷⁸ and Arg¹¹⁰⁹–Gly¹¹¹⁰. An additional putative cleavage site has also been discovered by our group in the less abundant 3 chain isoform at Arg⁸³³–Gly⁸³⁴ (21).

Moreover, fluorescent conjugates of collagen I, collagen IV, and fibrinogen were also incubated with KLK14, and a progressive increase in fluorescence emission resulting from substrate degradation was observed (data not shown).

Plasma Proteins
KLK14 induced rapid and extensive degradation of the plasma proteins kininogen, plasminogen, fibrinogen, and vitronectin (Fig. 10). Proteolysis of low molecular weight kininogen (LMWK) by KLK14 over time yielded a number of kininogen fragments (Fig. 10A). One KLK14 cleavage site was revealed at Arg¹¹⁴–Ser¹¹⁵ within the first of three cystatin-like domains that include LMWK (44), which harbor inhibitory activity against cysteine proteases. Based on our data, it was not possible to determine whether KLK14 can release lysyl-bradykinin (kallidin) from LMWK, a major consequence of KLK1 action (45).

KLK14 could completely digest fibrinogen chains A and B after a 15-min incubation period. The chain, however, displayed higher resistance to KLK14-mediated proteolysis (Fig. 10B). The N-terminal sequence of several major degradation fragments, denoted G1–G4, indicated that KLK14 cleaved the A chain at Arg³⁸–Val³⁹ and Arg⁵¹⁰–His⁵¹¹ and the B chain at Arg⁴⁴–Gly⁴⁵ (Table 5). Cleavage at the latter site by thrombin leads to the release of fibrinopeptide B, thereby unmasking a polymerization site that drives fibrin assembly (46). The same cleavage site within the B chain was identified for KLK5 (27). Moreover, KLK4 (47) and KLK6 (48) have also been shown to digest fibrinogen, in vitro.

KLK14 digests plasminogen into three fragments of 30 (fragment P2), 50 (fragment P1), and 70 kDa, which result from cleavage at Lys⁹⁶–Lys⁹⁷ and Arg⁵⁴⁹–Lys⁵⁵⁰ (Fig. 10C; Table 5). Fragment P1 (Lys⁹⁷–Arg⁵⁴⁹) corresponds to angiostatin 4.5 (AS_4.5), an isoform of angiostatin consisting of the first four and 85% of the fifth kringle domain, which acts as a potent inhibitor of angiogenesis (49). Fragment P2 (Lys⁵⁵⁰–Asn⁸¹⁰) represents microplasmin, which consists of the remainder of the fifth kringle domain followed by the serine protease domain (50). Similarly, additional KLKs, including KLK3 (51), KLK5 (27), KLK6 (52), and KLK13 (53), can also generate angiostatin-like fragments from plasminogen in vitro.

View larger version (77K): [in this window] [in a new window]   FIGURE 9. KLK14-mediated degradation of the extracellular matrix proteins fibronectin (A), laminin (B), collagen I (C), collagen II (D), collagen III (E), and collagen IV (F). KLK14 was incubated with 5 µgof fibronectin, laminin, and collagen IV and 2 µg of collagens I–III. A–E, lane 1 represents each matrix protein incubated alone for 2 (in A) or 4 h (in B–E). A, lanes 2–7 represent 0-, 5-, 15-, and 30-min and 1- and 2-h incubation periods, respectively. B–E, lanes 2–7 correspond to 0-, 15-, and 30-min and 1-, 2-, and 4-h time points, respectively. F, increasing amounts of KLK14, shown in micrograms above each lane, were incubated with collagen IV for 8 h at either 25 or 37 °C. The mixtures were resolved by SDS-PAGE under reducing conditions, and the gels were stained with Coomassie Blue (A, B, and F) or silver (C, D, and E). Major KLK14-generated fragments are indicated by open arrowheads; arrows, and corresponding labels (F1–6 and C1–2) denote fragments subjected to N-terminal sequence analysis (results are listed in Table 5). M, molecular mass standards in kDa.

IGFBPs
KLK14 was able to completely digest IGFBP-2 and IGFBP-3 within 4-h and 15-min incubation periods, respectively (Fig. 11). Major fragments of 28, 20, and 14 kDa were generated from IGFBP-2 (Fig. 11A), and products of 28 and 14 kDa were obtained from IGFBP-3 (Fig. 11B). Although KLK14 cleavage sites within either IGFBP were not determined, the degradation products obtained are comparable with those generated by KLK2 (55), KLK3 (55, 56), and KLK5 (33). Given this, it may be inferred that KLK14, akin to other KLKs and many other proteases, may target IGFBPs within their middle linker region (57), which is flanked by areas that mediate binding to insulin-like growth factors I and II (IGF-1 and IGF-II) and may thereby decrease the affinity of IGFBPs for IGFs (58).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
KLK14 is a relatively uncharacterized extracellular serine protease of the human tissue kallikrein family. Thus, its physiological targets, inhibitors, and role(s) are not well understood. The present data reveal a broad picture of KLK14 action, providing additional clues into this enzyme's selectivity for protein substrates, several regulatory mechanisms that control its activity, and potential biological and pathological consequences of KLK14 function.

View larger version (77K): [in this window] [in a new window]   FIGURE 10. KLK14-mediated degradation of the plasma proteins kininogen (A), fibrinogen (B), plasminogen (C), and vitronectin (D). KLK14 was incubated with 5 µg of kininogen, 10 µg of fibrinogen and plasminogen, and 300 ng of vitronectin at 37 °C. A–C, lanes 1–6 correspond to 0-, 15-, and 30-min and 1-, 2-, and 4-h time points, respectively. D, lanes 1–6 represent 0-, 5-, 15-, and 30-min and 1- and 2-h incubation periods, respectively. A–D, lanes 7 and 8, plasma protein and KLK14, respectively, incubated alone for 4 (A–C) or 2 h (D). Reaction mixtures were resolved by reducing SDS-PAGE, and gels were stained with Coomassie Blue (A–C) or Silver (D). Major KLK14-generated fragments are indicated by open arrowheads; fragments subjected to N-terminal sequence analysis are indicated by arrows and labels on the right (results are listed in Table 5). M, molecular mass standards in kDa.

View larger version (37K): [in this window] [in a new window]   FIGURE 11. Degradation of IGFBP2 and -3 by KLK14. A and B, lane 1 shows each IGFBP incubated alone for 4 h; lanes 2–7 represent KLK14 (30 ng) incubated with IGFBPs (300 ng) for 0, 0.25, 0.5, 1, 2, and 4 h, respectively, at 37 °C. Intact IGFBPs are indicated by solid arrowheads; major KLK14-generated fragments are indicated by open arrowheads. M, molecular mass standards in kDa.

Numerous reports indicate that KLK14 is dysregulated in malignancy (7, 9–11, 16, 17). In this study, we demonstrate that KLK14 is expressed in cancerous breast and ovarian tissue extracts and cell lines. We have shown previously that KLK14 is elevated in the serum of breast and ovarian cancer patients (10). This study is the first to report a significant elevation of KLK14 serum levels in prostate cancer patients compared with healthy males, which correlates with and may be attributable to the overexpression of KLK14 transcripts observed in cancerous prostatic tissues versus normal counterparts (17). Thus, serum KLK14 might function as a complementary biomarker to PSA/KLK3 for prostatic diseases. KLK14-expressing breast cancer cell lines, MCF7 and MDA-MB-468, are derived from metastatic sites and likely represent more aggressive forms of this disease; the ovarian cancer cells that produce KLK14 are of various grades and histotypes, including serous (OV-90), endometrioid (TOV112D; MDAH-2774), and clear cell (TOV-21G). In addition to breast and ovarian carcinoma, the clinical applicability of KLK14 expression in prostate cancer warrants further investigation. Taken together, the normal and/or diseased skin, breast, prostate, and ovary and their associated secretions likely represent the key tissues and fluids of KLK14 action.

Defining the specificity of an SP, i.e. its discrimination among substrates, allows for the identification of potential biological targets, a further understanding of its (patho)physiological role, and can direct the design of specific inhibitors. Although the S1 subsite is recognized as the primary determinant of substrate specificity in SP, the contribution of additional interactions between S4-S4' and P4-P4' can also be critical (59). To elucidate the primary and extend substrate specificity of KLK14, we have examined its optimal occupancy across S4-S4' subsites based on the P4-P4' residues within selected phage-displayed pentapeptides (21), fluorogenic AMC-peptides, and macromolecular substrates and inhibitors that efficiently interact with KLK14. We and others (19, 21) have shown previously that KLK14 manifests dual trypsin and chymotrypsin-like enzymatic specificity, with a rather strict preference for Arg and Tyr over Lys at the P1 position. However, as demonstrated in this study and by other independent investigators (19), the chymotrypsin-like activity of KLK14 cannot be readily assayed with commercially available chymotrypsin-like fluorogenic or chromogenic peptides using the same enzyme concentration as with trypsin-like peptides. Hence, it is likely that this activity requires prime side residues for optimal interactions as we have shown by phage display substrate (21) and by the ability of KLK14 to specifically cleave the ectodomain of desmoglein-1 at Tyr⁵²⁸–Ser⁵²⁹ in vitro.³ With respect to its extended specificity, we uncovered relatively modest amino acid preferences for KLK14 at S4-S2 and S1'-S4'. Although a variety of P4, P3, P2, P1', P2' and P3' residues are accommodated by KLK14, 60% of all side chains on average contain either aliphatic (Leu, Val, and Gly) or hydroxyl groups (Thr and Ser). The most predominant residues at P4, P3, P2, P1',P2', and P3' present in 20% of all substrates are Thr, Gly, Leu, Ser, Ser, and Thr, respectively. A similar preference for P1'-Ser has also been documented for other KLKs, including KLK1 and KLK2 (60). With the exception of P2'-Tyr/Phe and P3'-Arg/Lys found in 9 and 11% of substrates, respectively, KLK14 generally does not favor hydrophobic or charged residues in its extended subsites. Notably, KLK14 exhibits selectively for Pro at P4', as this residue was present in 30% of all substrates, indicating that P4' may be an important contributor to KLK14 specificity.

Because the catalysis of peptide bonds by proteases is irreversible, several regulatory mechanisms have evolved to prevent unnecessary and potentially damaging protein degradation. In this study, we examined four regulatory mechanisms that control KLK14 activity as follows: 1) zymogen activation, 2) internal cleavage, 3) endogenous inhibitors, and 4) ions.

We examined pro-KLK14 activation by KLK5 and could not demonstrate the efficient conversion of pro-KLK14 into its active form via KLK5 action, even after a 3-h incubation at a 5:1 molar ratio. However, under similar conditions, Brattsand et al. (19) reported 25% activation of pro-KLK14 by KLK5, which increased to 80% after 24 h. Catalytic efficiency is often the main criterion used in the selection of an activating enzyme. Thus, if KLK5 could activate pro-KLK14 in vivo, the conversion reaction would occur at an exceedingly slow rate, which may not be biologically relevant. In actuality, KLK5 may be the physiological activator of pro-KLK2 and pro-KLK3 in prostatic tissues and fluids, a process that occurs within 10 min in vitro (33). The stringent P1-Arg specificity of KLK5 (27) may explain its differential activation capacity toward pro-KLK14 and pro-KLK2/KLK3, which require limited proteolysis after P1-Lys and P1-Arg residues, respectively, for maturation. Given the latter, candidate activators that co-localize with pro-KLK14 in vivo and possess a compatible specificity for P1-Lys may include KLK4 (47, 61), KLK8 (41), and membrane-type serine protease 1 (62).

Autodegradation leading to inactivation may represent another means by which the activity of KLK14 is regulated. Intermolecular KLK14-mediated processing events may begin with the proteolysis of the most solvent-accessible P1-Arg residues first, resulting in destabilization of KLK14 tertiary structure, which subsequently leads to its complete degradation. Similar cases of inactivation by internal cleavage have been made for a number of SP and KLK family members, including KLK2 (autolytic and/or KLK5-mediated cleavage at Arg¹²⁵–Leu¹²⁶ and Arg¹⁶⁸–Ser¹⁶⁹ (33, 63)), KLK3 (KLK5-mediated processing at Lys¹⁶⁹–Lys¹⁷⁰ and Lys²⁰⁶–Ser²⁰⁷ (33, 64)), KLK6 (autolysis at Arg⁸⁰–Glu⁸¹ (48, 65)), KLK11 (plasmin-mediated cleavage at Arg¹⁵⁶–Leu¹⁵⁷ (66)), and KLK13 (autolytic processing at Arg¹¹⁴–Ser¹¹⁵ (53)). Most internal cleavage sites occur at analogous locations within exposed surface loops, for instance the scissile bonds Arg¹⁶⁸–Ser¹⁶⁹, Lys¹⁶⁹–Lys¹⁷⁰, Arg¹⁵⁶–Leu¹⁵⁷, and Arg¹⁵⁷–Tyr¹⁵⁸ of KLK2, KLK3, KLK11, and KLK14, respectively, all reside in the autolysis loop. Furthermore, clipped forms of KLK5 (67) and KLK7 (68) also exist in vitro and/or in vivo. In addition to abolishing catalytic activity, internal cleavage at specific sites may also alter SP specificity, as reported for thrombin (69).

Akin to many other KLKs (4), we found that KLK14 activity is inhibited by several serpins, including PAI-1, AT, AP, ATIII, and ACT, ranked in order of highest to lowest inhibitory efficiency. The P1-Arg preference of KLK14 and the presence of P1-Arg (PAI-1, AP, ATIII), P1-Met (AT), and P1-Leu (ACT) in the RSL of the serpins studied (34) may explain their relative inhibitory potencies against KLK14. As serpins play pivotal roles in the control of KLK activity in serum, amniotic fluid, breast milk, seminal plasma, and the prostate (4), the serpins studied may also represent in vivo inhibitors of KLK14 proteolytic function in similar physiological settings, with the exception of ACT, which displays exceedingly slow inhibitory kinetics against KLK14.

We have also demonstrated that KLK14 is positively and negatively regulated by citrate and zinc, respectively. Although these ions are secreted by a variety of cell types, a likely in vivo scenario for ion-mediated modulation of KLK14 activity may be within the prostate and seminal plasma, the latter of which contains 19–34 mM citrate and 1–4 mM Zn²⁺ (70), well above or within the range used in this study, respectively. Citrate also enhances the activity of other KLK family members (33, 36, 37) and has been shown to induce a conformational change in KLK3 leading to a more active configuration, likely via a thermodynamic solvent effect rather than a direct interaction (36). Zinc ions also exert an inhibitory effect on other KLKs (33, 38–41), and previous studies have defined the allosteric zinc-binding site, which is comprised of three His residues, within KLK3 (71) and KLK2 (38). However, only one corresponding His residue is present in KLK14, suggesting that zinc may bind and regulate KLK14 in a different manner than KLK2 and KLK3. As zinc inhibition is reversible by EDTA in vitro, and possibly by semenogelins within the seminal plasma in vivo (33), this mechanism is tightly regulated and highly dependent on the composition of the extracellular milieu. Furthermore, cations (e.g. zinc) have also been shown to alter the substrate specificity of a number of SP (e.g. prostasin) (72, 73); this effect on KLK14 may be worth examining in the future.

Based on its dysregulation in several malignancies and its in vitro substrate repertoire, KLK14 activity may be implicated in several aspects of tumor progression, including tumor growth, invasion, and angiogenesis. As with other KLKs (5) and proteases (74), the consequence of KLK14 action may be either stimulatory or inhibitory, depending on the cancer type and tumor microenvironment. Digestion of IGFBP-2 and IGFBP-3 by KLK14 may not only abolish the intrinsic tumor-suppressive functions of IGFBPs but may also cause the release of IGF-I and IGF-II, mitogenic peptides that stimulate the growth of normal and malignant cells (58). By cleaving vitronectin directly within its integrin-binding site (Arg⁶⁴–Gly⁶⁵), KLK14 may decrease integrin-mediated as well as urokinase receptor-mediated cell adhesion (75) and thereby enable tumor cell detachment. Via degradation of ECM components (e.g. collagen I–III and fibronectin) and proteins of vascular basement membranes (e.g. collagen IV and laminin), KLK14 may facilitate tumor cell invasion and metastasis. Yet, because KLK14 could only process collagens in their denatured and not native conformations in vitro, KLK14 likely acts sequential to collagenases (i.e. a subfamily of MMPs), which digest and thereby denature native collagens in vivo (76). Taken together, these tumor-promoting effects of KLK14 may explain why its levels are often associated with parameters of poor prognosis in breast and prostate cancers.

KLK14 may also exert tumor-suppressive functions, by unmasking cryptic cleavage sites within ECM and plasma proteins leading to liberation of fragments with angiostatic functions. For instance, KLK14 is able to generate AS_4.5 from plasminogen via cleavage at Lys⁹⁶–Lys⁹⁷ and Arg⁵⁴⁹–Lys⁵⁵⁰, and may release a fibronectin fragment containing anastellin by cleavage at Arg²⁹⁰–Ala²⁹¹ and Arg⁹⁰³–Ser⁹⁰⁴. Both AS_4.5 and anastellin possess anti-angiogenic and/or anti-metastatic functions (49, 77). It remains to be investigated whether KLK14 can also cleave at cryptic sites within collagen IV and laminin (78), thereby releasing additional anti-angiogenic fragments. These indirect tumor-inhibitory activities of KLK14 may form the basis of its correlation with a favorable prognosis in ovarian cancer patients (9).

In addition to tumor progression, KLK14 may also potentiate the development of arthritic disease. This may occur directly, via degradation of collagenous (e.g. collagen II) and noncollagenous (e.g. matrilin-4 (21, 79)) structural molecules that form cartilage, and/or indirectly, by generating 29- and 45-kDa fibronectin fragments, via cleavage at Arg²⁹⁰–Ala²⁹¹ and Arg⁹⁰³–Ser⁹⁰⁴, that destroy cartilage by inducing the expression of MMPs and catabolic cytokines (80, 81) and by also mediating chronic pro-inflammatory responses via proteinase-activated receptor 2 (22), which is implicated in the pathology of arthritis (82). Recently we have immunohistochemically localized KLK14, and several other KLKs, within chondrocytes (83), which further supports our hypothesis that KLK14, and possibly other KLKs, may contribute to the progression of arthritic diseases.

Accumulating evidence suggests that cross-talk exists among members of the KLK family and with other SP and MMP (5, 19). Data presented here suggest that KLK14 may interact with other serine proteases by indirectly regulating their activities. KLK14 cleaves vitronectin at Arg⁶⁴–Gly⁶⁵ and releases the somatomedin B domain, the primary high affinity binding site for active PAI-1 (54). The interaction between vitronectin and PAI-1 is biologically important, as vitronectin not only stabilizes and increases the half-life of PAI-1 but also localizes its activity in plasma and tissues during several (patho)physiological processes, including fibrinolysis and tumor progression (54). Although the action of KLK14 releases the intact somatomedin B domain, which has been reported to stabilize PAI-1 (84), PAI-1 activity would no longer be localized, leading to the dysregulation of urokinase-mediated pericellular proteolysis.

Given the clinical data that link KLK14 expression to disease and the possible involvement of KLK14 in tumor progression and other (patho)physiological processes, the inhibition of KLK14 activity may represent a promising therapeutic strategy. To this end, we have already constructed highly specific recombinant serpins for KLK14 by replacing the RSL of AT and ACT with KLK14-selected pentapeptides from our phage-displayed library screen (85), which may be useful in future functional studies on KLK14 and in assessing its utility as a therapeutic target.

In summary, this study provides a number of functional inferences into the action of the novel serine protease, KLK14. Our data may help to direct future research needed to resolve the role(s) of KLK14 in normal and pathological states.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario M5G 1X5, Canada. Tel.: 416-586-8443; Fax: 416-586-8628; E-mail: ediamandis{at}mtsinai.on.ca.

² The abbreviations used are: SP, serine proteases; ACT, ₁-antichymotrypsin; AP, ₂-antiplasmin; AS_4.5, angiostatin4.5; ATIII, antithrombin III; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ECM, extracellular matrix; IGFBP, insulin-like growth factor-binding protein; HPLC, high performance liquid chromatography; KLK, kallikrein; LMWK, low molecular weight kininogen; MMP, matrix-metalloprotease; PAI-1, plasminogen activator inhibitor; RSL, reactive site loop; serpin, serine protease inhibitor; MES, 4-morpholineethanesulfonic acid; PSA, prostate-specific antigen; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AMC, 7-amino-4-methylcoumarin; ACN, acetonitrile; IGF, insulin-like growth factor; Tos, tosyl; Boc, t-butoxycarbonyl; Suc, succinyl; AT, ₁-antitrypsin.

³ C. A. Borgoño, I. P. Michael, N. Komatsu, A. Jayakumar, R. Kapadia, G. L. Clayman, G. Sotiropoulou, and E. P. Diamandis, submitted for publication.

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