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

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 α1-antitrypsin, α2-antiplasmin, antithrombin III, and α1-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.

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)(4)(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 prostatespecific 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)(14)(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 Lys 24 -Ile 25 is required to generate active KLK14, a process that may be performed by KLK5 (19). Because of the presence of Asp 198 in its S1 binding pocket (7,8), KLK14 is predicted to exert trypsinlike 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;S1Ј-SnЈ) and corresponding substrate residues (Pn-P1;P1Ј-PnЈ), where P1-P1Ј 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 colocalized 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)(14)(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.

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) was cloned into P. pastoris expression vector pPICZ␣A (Invitrogen) at EcoRI and XbaI restriction enzyme sites between the 5Ј promoter and the 3Ј terminator of the AOX1 gene, in-frame with the yeast ␣-mating factor (for secretion), using standard techniques. The PmeI-linearized pPICZ␣A-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 automated Á KTA 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 g 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) was PCR-amplified in a 50-l reaction mixture containing 1 l of cerebellum cDNA as a template, 100 ng of primers (forward, 5Ј CACC ATG TTC CTC CTG CTG ACA GCA CTT; reverse, 5Ј 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 MgCl 2 , 200 M deoxynucleoside triphosphates (dNTPs), and 0.75 l (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 TM D/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 g 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% CO 2 . After 48 h, cells were re-plated into 35-mm dishes, and the selection agent Geneticin (G418, 300 g/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 TM I (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 the Á KTA fast performance liquid chromatography system at a flow rate of 1 ml/min.  (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 H 2 O 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 Ϫ80°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, Sim-plyBlue 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 Trisbuffered saline/Tween and treated with alkaline phosphataseconjugated 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 Simply-Blue 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 g) 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% CO 2 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.
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 Ͼ80% 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 Ϫ80°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 l under optimal buffer conditions within 96-well white polystyrene microtiter plates. Reaction mixtures contained less than 5% (v/v) Me 2 SO. 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. KLK14free 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 l at different molar ratios and incubation times. AT was then added at a 2-fold molar excess to a final volume of 15 l. 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.

Effect of Ions on KLK14 Activity
KLK14 was incubated with each cation and citrate (solutions prepared from salts of ZnCl 2 , ZnSO 4 , Zn(CH 3 COO) 2 , CaCl 2 , MgCl 2 , 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 l 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 Zn 2؉ Inhibition of KLK14 by EDTA
KLK14 (12 nM final concentration) was incubated alone or with the following: (a) Zn 2ϩ (1.2 M final concentration ZnCl 2 ), (b) EDTA (1 mM final concentration), (c) Zn 2ϩ and EDTA, and (d) Zn 2ϩ followed by the addition of EDTA after 20 min, at a final volume of 100 l 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 g); collagen IV, laminin, fibronectin, kininogen (5 g); fibrinogen, plasminogen (10 g); and vitronec-tin, IGFBP-2, and IGFBP-3 (300 ng) were incubated with specific amounts of KLK14 (6 ng to 1 g) 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 Simply-Blue TM SafeStain or with the Silver Xpress TM kit (Invitrogen).
Furthermore, KLK14 (0.4 and 0.8 g) was incubated with fluorescent conjugates of collagen types I and IV (2.5 g) and fibrinogen (3.75 g) (Molecular Probes, Eugene, OR) in optimal assay buffer (final volume 200 l) 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 KLK14generated fragments derived from recombinant mature KLK14, fibronectin, collagen IV, plasminogen, kininogen, fibrinogen, and vitronectin. KLK14 was incubated alone (3 g) or with each intact protein (2.5-5 g) 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.

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 twostep chromatographic purification procedures, recombinant mature and pro-KLK14 were obtained with Ͼ95% 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 g, respectively, as determined by ELISA and total protein assays.
Prepro-KLK14 is a 251-amino acid polypeptide consisting of a signal sequence (Met 1 -Ser 18 ), followed by a 6-amino acid pro-peptide (Gln 19 -Lys 24 ) and a 227-amino acid trypsin-like serine protease domain (Ile 25 -Lys 251 ) with a predicted zymogen activation site at Lys 24 -Ile 25 (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 ϳ25 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 ϳ27 kDa, as expected (Fig. 1, lane 2).

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 g/liter), specific for KLK14 (Ͻ0.1% cross-reactivity with other KLKs), recognizes both mature and pro-KLK14 recombinant proteins, and is linear from 0.05 to 10 g/liter with between-run and within-run coefficients of variation of Ͻ10%.
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 (Ͻ50 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 Ϯ 16.0 g/liter), followed by amniotic fluid (8.1 Ϯ 6.0 g/liter) and saliva (4.4 Ϯ 2.1 g/liter). Follicular fluid and breast milk contained lower concentrations (ϳ1.2 g/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 Ϯ 0.7 g/liter) as well as in cytosolic extracts of ovarian (1.77 Ϯ 5.13 g/liter) and breast (0.26 Ϯ 1.17 g/liter) tumors.

Autodegradation of KLK14 in Vitro
Purified recombinant mature KLK14 consisted of a mixture of peptides visualized as multiple bands under reducing SDS-PAGE (Fig. 4A). Western blot (Fig. 1B) and N-terminal sequence analysis confirmed the identity of the low molecular weight (Ͻ25 kDa) protein bands as internal fragments of mature KLK14 likely arising from auto-proteolytic cleavage (Fig. 4). All KLK14 cleavage sites occurred after P1-Arg residues (Fig. 4B), and the majority reside in exposed, solvent-accessible surface loops, which are most susceptible to proteolytic cleavage (Fig. 4C). Among KLK14 autodegradation fragments, only fragment I retains all residues of the catalytic triad (His 67 , Asp 111 , and Ser 204 ; see Fig. 4B) and may exhibit residual serine protease activity. However, prolonged incubation (24 h) of mature KLK14 at 37°C resulted in complete hydrolysis of the   intact 25-kDa band and a corresponding decrease in enzymatic activity (Fig. 4, D and E). Auto-inactivation of KLK14 by autolytic cleavage may represent a potential regulatory mechanism.

Optimal pH and Buffer Composition for KLK14 Activity
Two buffer systems, 100 mM Na 2 HPO 4 (pH 7-10) and 100 mM Tris-HCl (pH 6 -9), were chosen to determine the optimal pH for KLK14 activity. Although KLK14 was most active at a pH of 8.0 in both systems, KLK14 activity was 1.4-fold higher in phosphate versus Tris buffer. Furthermore, KLK14 displayed enhanced activity (13% increase) in the presence of 0.01% Tween 20 compared with 0.2% bovine serum albumin in which activity decreased 30% (Tween 20 and bovine serum albumin prevent adsorption of KLK14). Hence, the buffer composition utilized in subsequent studies was 100 mM Na 2 HPO 4 , 0.01% Tween 20 (pH 8.0).

Activation of Pro-KLK14
As with other S1A family SP, pro-KLK14 requires the removal of the inhibitory N-terminal pro-peptide sequence at Lys 24 -Ile 25 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 inac-tive, intact ϳ27-kDa band on SDS-PAGE and zymographic analyses after prolonged incubation (data not shown), which is supported by previous findings (19).
To test the potential activation of pro-KLK14 by KLK5, which is often co-expressed with KLK14 in vivo (32), we incubated pro-KLK14 with KLK5 and monitored the reaction via complex formation with AT. However, we were unable to provide evidence for the activation of pro-KLK14 by KLK5. Neither dose-dependent (data not shown) nor time-dependent activation of pro-KLK14 by KLK5 was observed in our study, as confirmed by the lack of KLK14-AT complex formation after incubation of pro-KLK14 with KLK5 (Fig. 5, lanes 5-9). As expected, mature KLK14 readily formed a complex with AT, whereas pro-KLK14 and KLK5 did not (Fig. 5, lanes 2-4). Our results are further substantiated by the fact that KLK5 is not able to cleave a heptapeptide encompassing the activation site of KLK14 (33) and that KLK5 generally favors Arg relative to Lys at P1 (27), i.e. the best P1-Arg containing substrate (VPR-AMC) was hydrolyzed at a 133-fold higher catalytic efficiency by KLK5 than the best P1-Lys containing substrate (VLK-AMC) (27). A prior study by Brattsand et al. (19) reported that KLK5 is an efficient activator of pro-KLK14. However, pro-KLK14 activation occurred after 24 h of incubation with KLK5, suggesting an inefficient conversion reaction, which may not be physiologically relevant. Furthermore, the reaction was monitored with a chromogenic substrate (Pro-Phe-Arg-p-nitroanilide) equally hydrolyzed by both KLK5 and KLK14. In our study, we utilized the serpin AT, an inhibitor that targets KLK14 but not KLK5 (27), to monitor pro-KLK14 activation over 3 h. Taken together, these results call into question the ability of KLK5 to efficiently activate pro-KLK14.

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 PЈ 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-pnitroanilide, by KLK14 was reported in a previous study (19)), and 3) the final enzyme concentration used. The P2 specificity of KLK14 was examined by comparing the k cat /K m values among substrate subgroups with invariable P1 and P3 residues as follows: 1) QAR-AMC, QGR-AMC, and QRR-AMC; 2) LKR-AMC, LRR-AMC, and LGR-AMC; and 3) GPR-AMC and GRR-AMC. As shown in Table 2, KLK14 exerts minimal, if any, catalytic activity toward substrates containing basic residues (i.e. Arg and Lys) at P2 and instead prefers relatively small, aliphatic amino acids (i.e. Gly and Ala) at this position. These results concur with those of our phage displayed library screening, because the vast majority of KLK14-selected pentapeptides contained P2 amino acids with aliphatic side chains (i.e. Gly, Ala, Val, and Leu), whereas none possessed basic P2 residues (21).
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-P1Ј) 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 ϩ2 ) are listed in Table 3 and were calculated from the double-reciprocal plots of the pseudo-first order

Enzymatic Action of KLK14
rate constant kЈ versus inhibitor concentrations (Fig. 6,  insets). Based on the apparent second order rate constants (k ϩ2 /K I ) determined, the rank order for KLK14 inhibition by serpins from highest to lowest efficiency was AT Ͼ AP ATIII Ͼ Ͼ ACT (Table 3). AT was ϳ2 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 ; however, the reaction proceeded at a much higher rate than with other serpins; hence rate constant determina-tion 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. Because the hallmark of the serpin inhibitory mechanism is the formation of a covalent complex between the serpin and its cognate protease (34), we visualized stable complexes between KLK14 and PAI-1, AP, AT, and ATIII but not ACT by SDS-PAGE under reducing conditions (Fig. 7). Single complexes of ϳ64, 90, and 77 kDa formed between KLK14 and PAI-1, AP, and AT, respectively (Fig. 7, A-C). However, two complexes of ϳ83 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. ϳ38 kDa for PAI-1 (Fig. 7A); ϳ50 and 10 kDa for AP (Fig.  7B); ϳ50 kDa for AT ( Fig. 7C; ϳ53 and 4 kDa for ATIII (Fig.  7D); and ϳ50 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 ϳ50-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 Zn 2ϩ , Ca 2ϩ , Mg 2ϩ , Na ϩ , and K ϩ is shown in Table 4. The presence of citrate seemed to have an activating effect on KLK14 activity, particularly at higher citrate concentrations (12-   Enzymatic Action of KLK14 JANUARY 26, 2007 • VOLUME 282 • NUMBER 4 respectively ( Fig. 8A; Table 4). Zinc inhibition was completely reversed by EDTA (Fig. 8B). In a similar fashion, Zn 2ϩ also regulates the activity of KLK2 (38), KLK3 (39), KLK5 (33), KLK7 (40), and KLK8 (41). Mg 2ϩ was also able to attenuate KLK14 activity, but to a much lesser extent, in contrast to its stimulatory effect on KLK8 activity (41). The remaining cations Ca 2ϩ , Na ϩ , and K ϩ had no significant effect on KLK14 activity up to concentrations of 1.2 mM, in contrast to the positive and negative influence of Na ϩ on the activity of KLK3 (36,42) and KLK6 (37), respectively.

Digestion of Potential Substrates by KLK14
ECM (fibronectin, laminin, and collagens I-IV) components, plasma proteins (fibrinogen, kininogen, plasminogen, and vitronectin), and insulin-like growth factor-binding proteins were highly susceptible to degradation by KLK14 in vitro . In all cases, degradation was not observed in the absence of KLK14. In the presence of KLK14, however, all proteins were readily digested at varying efficiencies in a time-dependent manner, resulting in the generation of numerous proteolytic fragments (Figs. 9 -11). The N-terminal sequence of several KLK14-generated fragments was identified, allowing for the determination of KLK14 cleavage sites within the intact molecules (Table 5).

Extracellular Matrix Components
Incubation of KLK14 with fibronectin resulted in the generation of several fragments, ranging in size from ϳ32 to 180 kDa (Fig. 9A). Based on the N-terminal sequence of fragments F1-F6, three major KLK14 cleavage sites were identified at Arg 290 -Ala 291 , Arg 903 -Ser 904 , and Arg 2070 -His 2071 , 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, Arg 290 -Ala 291 , lies between the first fibrin/heparin-binding region and the collagen/gelatin-binding region; the second site, Arg 903 -Ser 904 , 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 Arg 2421 -Asp 2422 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 Arg 1077 -Ala 1078 and Arg 1109 -Gly 1110 . An additional putative cleavage site has also been discovered by our group in the less abundant ␣3 chain isoform at Arg 833 -Gly 834 (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 Arg 114 -Ser 115 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 Arg 38 -Val 39 and Arg 510 -His 511 and the B␤ chain at Arg 44 -Gly 45 ( 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.
Incubation of KLK14 with vitronectin resulted in the generation of several distinct fragments of ϳ60 and 20 kDa (Fig. 10D). N-terminal sequencing of fragment V2 revealed that KLK14 cleaves at peptide bond Arg 64 -Gly 65 , which is located directly within the cell attachment sequence (i.e. the integrin-binding site Arg 64 -Gly 65 -Asp 66 ) and adjacent to the C-terminal boundary of the somatomedin B domain (i.e. a binding site for plasminogen activator inhibitor-1 and of the urokinase receptor) (54). The N terminus of V3 indicated that a second KLK14 cleavage site exists at Arg 197 -Asp 198 , which lies within the middle of the molecule between its collagen and plasminogen-binding sites (54). KLK5 also processes vitronectin in vitro, but at cleavage sites distinct from those of KLK14 (27).

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 ϳ28, 20, and 14 kDa were generated from IGFBP-2 (Fig. 11A), and products of ϳ28 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
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.  Table 5). M, molecular mass standards in kDa.
Multiple studies have investigated the expression of KLK14 at both the mRNA (7-9, 12, 16, 17) and protein level (10,11,14,15) and concur that epithelial tissues of the normal and/or diseased breast, ovary, prostate, and skin are among the most abundant in KLK14. Our group was the first to detect and quantify the KLK14 protein in healthy and cancerous tissues and fluids via a sandwich-type ELISA consisting of mouse and rabbit polyclonal antisera, generated against recombinant KLK14 containing two fusion tags (10). Given the unexpectedly low KLK14 protein levels measured with this KLK14-ELISA, partic-ularly in tissues and corresponding secretions that express abundant KLK14 mRNA (e.g. central nervous system-related tissues and fluids, including the brain, cerebellum, spinal cord and cerebrospinal fluid, respectively) (10), an additional survey of KLK14 content in tissues was required to clarify such discrepancies. Here, we describe the development of an improved KLK14-ELISA based on a monoclonal/polyclonal antibody configuration and re-examine KLK14 expression. As our new ELISA is generated against the native form of recombinant KLK14 (i.e. without fusion tags) and can effectively and specifically detect both recombinant mature and pro-KLK14, a more reliable and accurate measure of the total KLK14 content in the tissues, fluids, and cell lines was attained. KLK14 levels were highest in the normal skin/HaCaT keratinocyte cell line, breast/breast milk, and prostate/seminal plasma, in accord with prior studies. The concentrations of KLK14 in these tissues/fluids were comparable with those obtained by our previous ELISA (10). As evidenced by undetectable amounts of KLK14 in the majority of central nervous system tissues assayed (with the exception of minor levels in the medulla and spinal cord), our results confirm the previous observation that the KLK14 protein is generally absent in the central nervous system, despite the relative abundance of KLK14 transcripts in similar central nervous system tissues (7).
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 rep-  Table 5). M, molecular mass standards in kDa. 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-S4Ј and P4-P4Ј can also be critical (59). To elucidate the primary and extend substrate specificity of KLK14, we have examined its optimal occupancy across S4-S4Ј subsites based on the P4-P4Ј 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 Tyr 528 -Ser 529 in vitro. 3 With respect to its extended specificity, we uncovered relatively modest amino acid preferences for KLK14 at S4-S2 and S1Ј-S4Ј. Although a variety of P4, P3, P2, P1Ј, P2Ј and P3Ј 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, P1Ј, P2Ј, and P3Ј present in ϳ20% of all substrates are Thr, Gly, Leu, Ser, Ser, and Thr, respectively. A similar preference for P1Ј-Ser has also been documented for other KLKs, including KLK1 and KLK2 (60). With the exception of P2Ј-Tyr/Phe and P3Ј-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 P4Ј, as this residue was present in 30% of all substrates, indicating that P4Ј 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 ϳ25% activation of pro-KLK14 by KLK5, which increased to ϳ80% 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
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 Zn 2ϩ (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 zincbinding 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 (Arg 64 -Gly 65 ), 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 Lys 96 -Lys 97 and Arg 549 -Lys 550 , and may release a fibronectin fragment containing anastellin by cleavage at Arg 290 -Ala 291 and Arg 903 -Ser 904 . 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 Arg 290 -Ala 291 and Arg 903 -Ser 904 , 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 possi-bly 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 Arg 64 -Gly 65 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.