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J. Biol. Chem., Vol. 282, Issue 4, 2405-2422, January 26, 2007
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1
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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1-antitrypsin, 2-antiplasmin, antithrombin III, and 1-antichymotrypsin with second order rate constants (k+2/Ki) 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 |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A (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 AmiconTM 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 HiTrapTM 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 SuperscriptTM 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 GenBankTM 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 pcDNA3.1TMD/V5-His-TOPO® (Invitrogen) using the pcDNA3.1TM 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 GlutaMaxTMI (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, SimplyBlueTM SafeStain (Invitrogen), and/or by silver staining with the Silver XpressTM 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 SimplyBlueTM 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 Victor2TM 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 (kcat/Km) 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 Victor2TM 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 SimplyBlueTM 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 Victor2TM 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 Victor2TM 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 SimplyBlueTM SafeStain or with the Silver XpressTM 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 Victor2TM 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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).
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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.
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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).
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Substrate Specificity of KLK14
The substrate specificity of KLK14 was assessed by determining its relative hydrolytic activity and corresponding kinetic parameters (i.e. Km and kcat) 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 kcat/Km 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.
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The P3 preference of KLK14 was assessed by examining kcat/Km 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 (KI, 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/KI) 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.
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64, 90, and 77 kDa formed between KLK14 and PAI-1, AP, and AT, respectively (Fig. 7, A–C). However, two complexes of 83 and 67 kDa formed between ATIII and KLK14 (Fig. 7D). The lower molecular weight complex may represent an interaction between ATIII and autodegradation fragment I of KLK14, which may retain partial serine protease activity, as discussed above. In addition to the inhibitory pathway, KLK14 also reacts with these serpins via the substrate pathway, at varying extents, as evidenced by the presence of lower molecular weight bands pertaining to cleaved serpin fragments (i.e. 38 kDa for PAI-1 (Fig. 7A); 50 and 10 kDa for AP (Fig. 7B); 50 kDa for AT (Fig. 7C; 53 and 4 kDa for ATIII (Fig. 7D); and 50 kDa for ACT (Fig. 7E)). Based on our kinetic analysis, ACT is an inefficient inhibitor of KLK14 compared with the other serpins studied. A likely explanation may be that KLK14 reacts with ACT primarily via the substrate pathway, as reflected in Fig. 5E by the presence of a major 50-kDa band, below that of intact ACT, which corresponds to cleaved ACT. Because of the minor contribution of the inhibitory pathway, the complex between KLK14 and ACT was not visualized under the given experimental conditions. Notably, other KLKs also react with serpins via the substrate pathway, for instance the interaction between KLK3/PSA and ACT (35) and of KLK5 with AP (27). 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.
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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 (AS4.5), 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.
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); 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.
) 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.
), 0.4 (
). B, AT final concentration (µM):0(
). C, ATIII final concentration (µM):0(
), 0.5 (•), 1.0 (
