The extracellular matrix is a complex array of secreted molecules, including collagens

47 Pages·2006·1.49 MB·English

JBC Papers in Press. Published on November 16, 2006 as Manuscript M608348200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M608348200
1
EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF THE CANCER-
RELATED SERINE PROTEASE, HUMAN TISSUE KALLIKREIN 14

Carla A. Borgoño1, Iacovos P. Michael1, Julie L.V. Shaw1, Liu-Ying Luo1, Manik C.
Ghosh1, Antoninus Soosaipillai1, Linda Grass1, Dionyssios Katsaros2 and Eleftherios
P. Diamandis1. From the 1Department of Pathology and Laboratory Medicine,
Mount Sinai Hospital and the Department of Laboratory Medicine and
Pathobiology, University of Toronto and Toronto, ON, M5G 1X5, Canada and the
2Department of Gynecology, Gynecologic Oncology Unit, University of Turin,
Turin, Italy 10126.

Running Title: Enzymatic action of KLK14
Address correspondence to: Eleftherios P. Diamandis, MD, PhD, Department of Pathology and
Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada
M5G 1X5; Tel: [phone]; Fax: [phone]; email: [email protected]

Human tissue kallikrein 14 (KLK14) is results indicate that KLK14 may be
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a novel extracellular serine protease. implicated in several facets of tumour w
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Clinical data links KLK14 expression to progression, including growth, invasion and lo
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several diseases, primarily cancer, however, angiogenesis, as well as in arthritic disease ed
little is known of its (patho)physiological via digestion of cartilage. These findings fro
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role. In order to functionally characterize may have clinical implications for the h
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KLK14, we expressed and purified management of cancer and other disorders ://w
recombinant KLK14 in mature and pro- in which KLK14 activity is elevated. w
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enzyme forms, determined its expression .jb
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pattern, specificity, regulation and in vitro Proteases comprise a group of enzymes .o
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substrates. Using our novel immunoassay, that catalyze peptide bond hydrolysis. Serine b/
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the normal and/or diseased skin, breast, proteases (SP), characterized by the presence g
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prostate and ovary contained the highest of a nucleophilic serine residue at the active st o
concentration of KLK14. Serum KLK14 site, account for 30% of all proteases within n A
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KLK14 displayed trypsin-like specificity the most highly populated being clan PA(S), 1
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with high selectivity for P1-Arg over Lys. and are further classified into families that
KLK14 activity could be regulated by: 1) share high sequence similarity (2).
autolytic cleavage leading to enzymatic Human tissue kallikreins (KLK, EC
inactivation, 2) the inhibitory serpins α - 3.4.21) form a subgroup of 15 secreted
1
antitrypsin, α -antiplasmin, antithrombin (chymo)tryptic-like SP within the S1A family
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III and α -antichymotrypsin with second- of clan PA(S) and are encoded by the largest
1
order rate constants (k /K) of 49.8, 23.8, contiguous cluster of protease genes in the
+2 i
1.48 and 0.224 µM-1min-1 respectively, as entire genome, located on chromosome
well as plasminogen activator inhibitor-1 19q13.4 (1;3;4). Since KLKs are widely
and 3) citrate and zinc ions, which exerted expressed, KLK activity is implicated in an
stimulatory and inhibitory effects on assorted array of normal physiological
KLK14 activity, respectively. We also processes (e.g. blood pressure regulation, skin
expanded the in vitro target repertoire of homeostasis, semen liquefaction) and in the
KLK14 to include collagens I-IV, pathobiology of several diseases (e.g. cancer,
fibronectin, laminin, kininogen, fibrinogen, neurodegenerative disorders, dermatoses) (3-
plasminogen, vitronectin and insulin-like 5). Due to the frequent dysregulation of KLK
growth factor binding proteins 2 and 3. Our expression in malignancy, KLKs have been
Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Enzymatic action of KLK14 2

intensely studied in terms of their clinical nomenclature (20) is used to describe the
applicability as cancer biomarkers. In addition interaction between protease subsites (Sn-
to human kallikrein 3 [KLK3, also known as S1;S1’-Sn’) and corresponding substrate
prostate-specific antigen (PSA)], the premier residues (Pn-P1;P1’-Pn’), where P1-P1’
biomarker in clinical medicine for prostate denotes the scissile bond]. However, we (21)
cancer (6), most other KLKs have emerged as and others (19), through the use of phage-
promising markers of diagnosis, prognosis, display technology and chromogenic
prediction of therapeutic response and substrates, respectively, have recently
monitoring for several cancer types, demonstrated that KLK14 can accommodate
particularly ovarian carcinoma (5). both basic (Arg, Lys) and hydrophobic (Tyr)
Along with seven other KLK genes, P1 residues at the S1 subsite with a preference
human tissue kallikrein gene 14 (KLK14) was for Arg. Hence, KLK14 manifests dual,
independently identified in our laboratory by trypsin-like and chymotrypsin-like, substrate
positional candidate cloning in 2001 (7;8). specificity.
Subsequent studies demonstrated that the To date, putative biological substrates and
KLK14 gene is under steroid-hormone (patho)physiological roles for KLK14 have
regulation (9;10) and is most highly expressed been inferred from its expression pattern,
in the glandular epithelia of the breast (10;11) substrate specificity and the known function of
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and prostate (8;10) and in the epidermis (i.e. co-localized KLKs. Phage-displayed w
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stratum granulosum) and appendages (i.e. hair pentapeptide motifs preferred by KLK14 were lo
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follicular epithelium, eccrine sweat glands) of identified within several intact proteins, ed
the skin (10;12;13). Consequent to its including the extracellular matrix (ECM) fro
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secretion, KLK14 forms a constituent of molecules laminin, collagen type IV and h
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seminal plasma (10) and sweat (14) and is matrilin-4 (21). The latter may support a role ://w
present in its catalytically active form in the for KLK14 in cancer progression via ECM w
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stratum corneum of the skin (13-15). digestion, particularly in breast, ovarian and .jb
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Furthermore, aberrant KLK14 expression has prostate tumours that bear elevated KLK14 .o
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been detected in patients with breast levels. Due to its presence and prominent b/
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(7;10;11;16), ovarian (7;9;10), prostate (7;17) activity in the stratum corneum of the skin g
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and testicular (7) cancers and peeling skin (13-15), KLK14 is also implicated in st o
syndrome (18), at the tissue and/or serum epidermal desquamation (i.e. shedding) via n A
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aggressive forms of breast (11;16) and link adjacent corneocytes (19). More recently, 1
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prostate cancer (17), and with the prognosis of our group has reported that KLK14 may also
breast (16) and ovarian (9) cancer patients. mediate pleiotropic effects by signaling
Thus, KLK14 represents a potential biomarker through proteinase-activated receptors (PAR)
and therapeutic target for several pathologic 1, 2 and 4 (22). KLK14 may exercise its
conditions. functions alone or in a proteolytic cascade
As with all S1A family SP, KLK14 is pathway, as KLK5 may activate pro-KLK14
synthesized as an inactive precursor or and vice versa (19).
zymogen (herein denoted pro-KLK14), Collectively, studies on KLK14 reveal that
containing a 6-aa N-terminal pro-peptide that this novel SP may play important
maintains its latency (7;8). Proteolytic (patho)physiological roles at the main sites of
removal of the pro-peptide at Lys24-Ile25 is its expression and bears clinical utility. In
required to generate active KLK14; a process order to gain further insights into the
that may be performed by KLK5 (19). Due to biological significance of KLK14 action, the
the presence of Asp198 in its S1 binding pocket present study examines KLK14 expression,
(7;8), KLK14 is predicted to exert trypsin-like specificity, activity against candidate
substrate specificity with a preference for substrates and several modes of regulation.
basic P1 residues [Schechter and Berger
Enzymatic action of KLK14 3

Experimental Procedures using Biomax-10 Ultrafree®-15 Centrifugal
Filter Device (Millipore Corp., Bedford, MA).
Cloning, expression and purification of The concentrated fractions were diluted 1:4 in
recombinant KLK14 – Mature KLK14. Mature 100mM Tris-HCl (pH 7.8) binding buffer and
recombinant KLK14 was produced in the incubated with 1ml of soybean trypsin
EasyselectTM P. pastoris expression system inhibitor agarose beads (Sigma) overnight at
(Invitrogen, Carlsbad, CA), as previously 4°C. The beads were then packed into an
described (21). Briefly, PCR-amplified KLK14 Econo-Pac open column (Bio-Rad, Hercules,
cDNA encoding the mature enzyme form of CA) and washed three times with binding
KLK14 [amino acids 25-251 based on buffer. KLK14 was eluted with 0.1M glycine
Genbank accession no. AAK48524] was buffer (pH 3.0).
cloned into P. pastoris expression vector Pro-KLK14. First-strand cDNA synthesis
pPICZαA (Invitrogen) at EcoRI and XbaI was performed by reverse transcriptase using
restriction enzyme sites between the 5' the SuperscriptTM preamplification system
promoter and the 3' terminator of the AOX1 (Gibco BRL, Gaithersburg, MD) with 2 µg of
gene, in-frame with the yeast α-mating factor total human cerebellum RNA (Clontech, Palo
(for secretion), using standard techniques. The Alto, CA) as a template. The cDNA encoding
PmeI-linearized pPICZαA-KLK14 construct prepro-KLK14 [amino acids 1-251 based on D
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was transformed into chemically competent P. Genbank accession no. AAK48524] was PCR- w
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pastoris yeast strains X-33, GS115 and KM71. amplified in a 50 µL reaction mixture loa
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A stable X-33 transformant was selected and containing: 1 µL of cerebellum cDNA as a ed
recombinant KLK14 expression was induced template, 100 ng primers [Forward: 5’ CACC from
with 1% methanol/day for 6 days at 30°C in a ATG TTC CTC CTG CTG ACA GCA CTT; http
shaking incubator (250rpm). Reverse: 5’ AGA CCA TCA TTT GTC CCG ://w
Mature recombinant KLK14 was purified CAT CGT TTC CT, containing CACC w
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to homogeneity from culture supernatant by a sequence required for TOPO® cloning .jb
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two-step procedure consisting of cation- (underlined) and the native KLK14 stop codon .org
exchange and affinity chromatography. (italics)], 10 mM Tris-HCl (pH 8.3), 50 mM by/
Typically, 1L of culture supernatant was KCl, 1.5 mM MgCl , 200 µM gu
clarified by centrifugation and concentrated deoxynucleoside triphosphate2s (dNTPs) and est o
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20-fold by positive pressure ultrafiltration in 0.75 µL (2.6 U) of Expand Long Template A
an AmiconTM stirring chamber (Millipore PCR polymerase mix (Roche Diagnostics, pril 2
Corporation, Bedford, MA) with a 10-kDa Mannheim, Germany), on an Eppendorf , 2
0
cutoff nitrocellulose membrane (Millipore). 1
master cycler (Eppendorf, Westbury, NY). 9
The concentrated supernatant was diluted 1:4
PCR conditions were 94°C for 2 min,
in 10mM MES (pH 5.3) and fractionated on a
followed by 94°C for 30 s, 60°C for 30 s,
pre-equilibrated 5ml cation exchange
72°C for 30 s for 40 cycles, and a final
HiTrap™ SP HP column (GE Healthcare,
extension at 68°C for 7 min. The PCR product
Piscataway, NJ), with the automated
was cloned into the mammalian expression
ÄKTAFPLC chromatography system
vector pcDNA3.1TMD/V5-His-TOPO®
(Amersham Biosciences/GE Healthcare,
(Invitrogen) using the pcDNA3.1TM
Piscataway, NJ). Adsorbed KLK14 was eluted
Directional TOPO® Expression Kit
with a multi-step salt gradient using 1M KCl
(Invitrogen), according to the manufacturer’s
in 10mM MES (pH 5.3) at a flow-rate of
protocol. The KLK14 sequence within the
3ml/min as follows: a) continuous linear
construct, denoted pcDNA3.1-KLK14, was
gradient of 0-0.3M KCl for 17 min, b)
confirmed with an automated DNA sequencer
maintenance at 0.3M KCl for 20 min followed
using vector-specific primers in both
by c) a continuous linear gradient from 0.3-
directions.
1M KCl for 50 min. Elution fractions (3ml)
Human embryonic kidney (HEK)-293
containing KLK14 were identified (as
cells [American Type Culture Collection
described below), pooled and concentrated
Enzymatic action of KLK14 4

(ATCC), Rockville, MD] were stably described herein) and by detection methods
transfected with 3 µg of PmeI-linearized mentioned below. Fractions containing pro-
pcDNA3.1-KLK14 by lipofection using KLK14 were then pooled, concentrated and
PolyFect transfection reagent (Qiagen, supplemented with a final concentration of 1%
Valencia, CA), according to the trifluoroacetic acid (TFA), and loaded in 0.1%
manufacturer’s instructions. Transfected TFA in H O onto a 1 ml Vydac C4 reversed-
2
HEK-293 cells were incubated in Dulbecco's phase column (The Separations Group, Inc.,
modified Eagle's medium (DMEM; Hesperia, CA) connected to an Agilent series
Invitrogen) supplemented with 10% fetal 1100 HPLC chromatography system equipped
bovine serum (FBS), in a humidified with a diode array detector (Agilent
atmosphere containing 5% CO . After 48 hrs, Technologies, Inc, Palo Alto, CA). Elution
2
cells were re-plated into 35mm dishes, and the was performed with a multistep acetonitrile
selection agent, Geneticin (G418, 300µg/ml (ACN) gradient at a flow-rate of 0.8 ml/min
final concentration; Invitrogen) was added. consisting of 5 min linear gradients of 20-25%,
Resistant clones were chosen over 14 days 25-30%, 30-32%, 32-35%, 35-40% and 40-
under continuous G418 selection and cultured 100% ACN intervened by 15 min steps at 25,
separately in 24-well followed by 6-well plates. 30, 32 and 35% ACN. ACN was removed
One clone, denoted P1-4, was selected for from each fraction by evaporation with
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large-scale expression of pro-KLK14 and nitrogen gas at a pressure of 15 psi. w
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grown in DMEM with 10% FBS until 80% For both recombinant KLK14 proteins: lo
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confluency. At this stage, the media was purity was assessed on silver-stained SDS- ed
replaced with serum-free CD CHO media PAGE gels (described below), concentration fro
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supplemented with GlutaMaxTMI (8mM final was determined by the bicinchoninic acid h
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concentration; Invitrogen). Conditioned media (BCA) method (Pierce Chemical Co., ://w
was collected by centrifugation after 10 days Rockford, IL), protein identity was confirmed w
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of incubation. b y t a n d e m m a s s s p e c t r o m e t r y , a s d e s c r i b e d .jb
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Recombinant pro-KLK14 was purified to previously (23) and proteins were aliquoted .o
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homogeneity from the conditioned serum-free and stored at -80°C in 0.1M sodium acetate b/
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media of HEK-293 cells stably transfected buffer (pH 5.0). g
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with pcDNA3.1-KLK14 sequentially by st o
cation-exchange and reversed-phase Detection of recombinant KLK14 - To monitor n A
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yeast culture supernatant. Concentrated CM SDS-PAGE, western blot and zymography. 1
9
was diluted 1:4 in 10mM MES (pH5.3) and SDS-PAGE. SDS-PAGE was performed
applied to a previously equilibrated 5ml SP using the NuPAGE Bis-Tris electrophoresis
HP Sepharose column (GE Healthcare), system and precast 4–12% gradient
previously equilibrated with 10mM MES (pH polyacrylamide gels at 200 V for 30 min
5.3), on the ÄKTAFPLC Chromatography (Invitrogen). Proteins were visualized with a
System at a flow rate of 1 mL/min. Pro- Coomassie G-250 staining solution,
KLK14 was eluted with a multistep salt SimplyBlueTM SafeStain (Invitrogen), and/or
gradient of 1M KCl in 10mM MES (pH 5.3) at by silver staining with the Silver XpressTM kit
a flow-rate of 3ml/min as follows: a) (Invitrogen), according to the manufacturer’s
continuous linear gradient of 0-0.25M KCl for instructions.
17 min, b) maintenance at 0.25M KCl for 33 Western blots. For immunodetection of
min, c) linear gradient of 0.25-0.35M KCl for KLK14, proteins resolved by SDS-PAGE
8.3 min, d) maintenance at 0.35M KCl for 33 were subsequently transferred onto a Hybond-
min, followed by e) a linear gradient of 0.35- C Extra nitrocellulose membrane (GE
0.8M KCl for 33 min. Elution fractions (3ml) HealthcareJ) at 30 V for 1 h. The membrane
were analyzed by our newly developed was blocked with Tris-buffered saline-Tween
enzyme-linked immunosorbent assay (ELISA; [0.1 mol/liter Tris- HCl buffer (pH 7.5)
Enzymatic action of KLK14 5

containing 0.15 mol/liter NaCl and 0.1% technology. The splenocytes from pre-selected
Tween 20] supplemented with 5% nonfat dry immunized mice were fused with the Sp2/0
milk overnight at 4°C and probed with an myeloma cells using polyethylene glycol
KLK14 polyclonal rabbit antibody (produced 1500. The resultant hybridoma cells were
in-house; diluted 1:2000 in Tris-buffered cultured in 96-well plates in DMEM
saline-Tween) for 1 h at room temperature. (Invitrogen) containing 20% FBS, 200 mM
The membrane was washed three times for 15 glutamine, 1% OPI (oxaloacetic acid, pyruvic
min with Tris-buffered saline-Tween, and acid, insulin), and 2% HAT (hypoxanthine,
treated with alkaline phosphatase (ALP)- aminopterin, thymidine; Sigma-Aldrich) for
conjugated goat anti-rabbit antibody (1:10000 selection at 37°C, 5% CO for 10–14 days.
2
in Tris-buffered saline-Tween; Jackson Hybridoma supernatants were collected and
Immunoresearch Laboratories, West Grove, screened by a previously described
PA) for 1 h at room temperature. Finally, the immunofluorometic technique (24). Positive
membranes were washed again as above, and clones were then expanded sequentially in 24-
fluorescence was detected on X-ray film using well and 6-well plates in complete media
chemiluminescent substrate (Diagnostic (reducing the FBS to 15% and changing the
Products Corp., Los Angeles, CA). HAT to HT). Antibody isotyping was
Zymography. The proteolytic activity of subsequently performed using a mouse
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recombinant KLK14 proteins was visualized immunoglobulin subtype identification kit ow
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by gelatin zymography (Invitrogen). (Sigma) to isolate clones producing anti- lo
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Recombinant KLK14 was diluted 1:1 in Tris- KLK14 monoclonal antibodies of the IgG ed
glycine SDS sample buffer and class. One positive hybridoma clone, denoted fro
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electrophoresed on precast Novex® 10% 2E9, was expanded in flasks in serum-free CD h
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gelatin zymogram gels (Invitrogen) at 125V CHO media (Invitrogen) containing 200 mM ://w
for 2.5 hours at 4°C. The gels were glutamine, for large scale antibody production. w
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subsequently incubated in zymogram Anti-KLK14 monoclonal antibody 2E9 was .jb
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renaturing buffer (Invitrogen) for two 30 min purified from the culture supernatant using the .o
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intervals at room temperature, followed by Affi-Gel Protein-A MAPS II Kit (Bio-Rad b/
y
incubation in zymogram developing buffer Laboratories, Hercules, CA), according to g
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(Invitrogen) for 3 hrs at 37°. Gels were stained manufacturer’s instructions, and used in st o
with SimplyBlueTM SafeStain and destained subsequent immunoassay development. n A
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uarnetails othfe p rwotheiates e layctitciv ibtyan wdes rec ovrirseibspleo.n ding to Development of an enzyme-linked ril 2, 2
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immunosorbent assay (ELISA) for KLK14 - A 1
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Generation of antibodies against KLK14 - "sandwich-type" ELISA, with a mouse
New Zealand white female rabbits and Balb/C monoclonal/rabbit polyclonal configuration
female mice were repeatedly immunized with was developed as follows: the KLK14-specific
purified recombinant mature KLK14 (100 µg) monoclonal antibody (clone 2E9; developed
for polyclonal and monoclonal antibody in-house) was first immobilized on a 96-well
development, respectively. KLK14 was white polystyrene plate (500ng/well) by
diluted 1:1 in complete Freund’s adjuvant for incubating in coating buffer [50mmol/L Tris,
the first injection and in incomplete Freund’s 0.05% sodium azide (pH 7.8)] overnight at
adjuvant for subsequent injections. Injections room temperature. The plate was then washed
were repeated three times for mice and six three times with washing buffer [50 mmol/L
times for rabbits at 3-week intervals. The Tris, 150 mmol/L NaCl, 0.05% Tween 20 (pH
polyclonal sera were tested every 2 weeks by 7.8)]. KLK14 standards or samples were then
an immunofluorometric method described in pipetted into each well [100 µl/well, diluted
detail elsewhere (10), until the highest 1:1 in assay buffer (50 mmol/L Tris, 6%
antibody titers against KLK14 were detected. bovine serum albumin, 10% goat IgG, 2%
Monoclonal antibodies against KLK14 mouse IgG, 1% bovine IgG, 0.5 mol/L KCl,
were produced by standard hybridoma 0.05% sodium azide, pH 7.8)], incubated for 2
Enzymatic action of KLK14 6

hrs with shaking, and then washed six times, hippocampus, medulla, midbrain, occipital
as above. Subsequently, 100 µl of rabbit anti- cortex, pituitary, pons, temporal lobe cortex,
KLK14 polyclonal sera (developed in-house) and spinal cord were prepared as described
diluted 1000-fold in assay buffer was added elsewhere (10). Cancerous ovarian tissues,
and incubated for 1 hr. After incubation, the containing >80% tumor cells, were obtained
plate was washed as above, and ALP- from patients with primary epithelial ovarian
conjugated goat anti-rabbit IgG (Jackson cancer who underwent surgery and treatment
ImmunoResearch), diluted 3000-fold in assay for ovarian cancer at the Department of
buffer, was applied and plates were incubated Gynecology of the University of Turin, Turin,
for 45 min. After washing as above, the ALP Italy between July of 1991 and April of 1999.
substrate diflunisal phosphate (100µL of a Ovarian tumour cytosolic extracts were
1mM solution) in substrate buffer (0.1 mol/L prepared as previously described (26).
Tris, pH 9.1, 0.1 mol/L NaCl, and 1 mmol/L Total protein content of the extracts was
MgCl ) was added to each well and incubated determined using the bicinchoninic acid
2
for 10 min followed by addition of developing method, with bovine serum albumin as a
solution (100 µL, containing 1 mol/L Tris base, standard (Pierce Chemical Co.).
0.4 mol/L NaOH, 2 mmol/L TbCl , and 3 Biological fluids. Cerebrospinal fluid,
3
mmol/L EDTA) for 1 min. The resultant breast milk, amniotic fluid, seminal plasma, D
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fluorescence was measured by time-resolved ascites fluid from ovarian cancer patients, w
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fluorometry on the Cyberfluor 615 breast cancer cytosolic extracts and sera from loa
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Immunoanalyzer (MDS Nordion, Kanata, ON). males and females without known disease and ed
Calibration and data reduction were performed of prostate cancer patients were residual from
automatically, as described elsewhere (25). samples submitted previously for routine http
The optimal assay configuration and biochemical testing and stored at -80°C. ://w
conditions described above were obtained by Investigations were carried out in w
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testing several monoclonal antibody and accordance with the ethical standards of the .jb
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polyclonal sera dilutions and diluents, as well Helsinki Declaration of 1975, as revised in .org
as different incubation times for each step of 1983, and have been approved by the b/
y
the procedure. Assay sensitivity (detection Institutional Review Board of Mount Sinai gu
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limit), specificity (including cross-reactivity to Hospital, Toronto, Canada and the Institute of st o
other KLK family members), linearity, Obstetrics and Gynecology, Turin, Italy. n A
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rimecporveecriysi,o na,n dw ewrei thdient-errumni naendd asb eptwreeveionu-rsulyn 1088C (eHllT lBin1e2s)., T bhree aasstt rcoacnycteorm cae lcle lliln leisn eM, SDWA-- ril 2, 2
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described (10). MB-468 (HTB132), BT-474 (HTB-20), T- 19
47D (HTB-133), MCF7 (HTB-22), and ZR-75,
Tissue extracts, biological fluids and cancer MFM223, MDA-MB-231 (HTB-26), MDA-
cell lines - The concentration of KLK14 in MB-453 (HTB-131), MDA-MB-361 (HTB27),
normal and cancerous tissue extracts, BT-20 (HTB19); the colorectal
biological fluids and conditioned media of adenocarcinoma cell line, COLO 320HSR
various cancer cell lines was determined with (CCL-220.1); the ovarian cancer cell lines
the newly developed KLK14-specific ELISA. Caov-3 (HTB-75), MDAH 2774 (CRL-10303),
Tissue extracts. A panel of 40 normal TOV-21G (CRL-11730), TOV112D (CRL-
tissue extracts including adrenal gland, 11731), OVCAR-3 (HTB-161), OV-90 (CRL-
axillary lymph node, bone, breast, colon, 11732), ES2 (CRL-1978); the cervical cancer
endometrium, esophagus, fallopian tube, cell lines HeLa (CCL-2), C-4 I (CRL-1594),
kidney, liver, lung, mesenteric lymph node, ME-180 (HTB-33), HT-3 (HTB-32);
skeletal muscle, ovary, pancreas, prostate, neuroblastoma cell lines SK-N-DZ (CRL-
seminal vesicle, skin, small intestine, spleen, 2149) and IMR-32 (CCL-127) and
stomach, testis, thyroid, trachea, ureter, uterus, neuroepithelioma cell line Sk-N-MC (HTB-
salivary gland, pharyngeal tonsil, placenta, 10); the pancreatic cancer cell line, MIA
cervix, cerebellum, frontal lobe cortex, PaCa-2 (CRL-1420), and the prostate cancer
Enzymatic action of KLK14 7

cell line LNCaP (CRL-1740) were purchased 380nm and 480nm, respectively, at 1 min
from ATCC. The BG-1 ovarian cancer cell intervals for 20 min at 37°C. KLK14-free
line was kindly provided by Dr. Henri reactions, for each peptide concentration, were
Rochefort (Montpellier University, used as negative controls and background
Montpellier, France), and the PC-3 cell line, counts obtained were subtracted from each
stably transfected with androgen receptor [PC- value. A standard curve was constructed
3 (AR) ] was provided by Dr. Theodore using known concentrations of AMC in order
6
Brown (Samuel Lunenfeld Research Institute, to calculate the rate of free AMC release. The
Mount Sinai Hospital, Toronto, Canada). Cells slope of the resultant AMC standard curve was
were cultured to near confluency in RPMI 19.184 AMC fluorescence counts/nM free
medium (Invitrogen) supplemented with AMC. The steady-state (Michaelis-Menten)
glutamine (200 nM) and FBS (100 mL/L) in kinetic constants (kcat/Km) were then
plastic flasks. calculated by non-linear regression analysis
using Enzyme Kinetics Module 1.1 (Sigma
Protease assays and kinetic constant Plot, SSPS, Chicago, IL). All experiments
determination - 7-Amino-4-methylcoumarin were performed in triplicate and repeated at
(AMC) was purchased from Sigma. The least twice.
following synthetic AMC substrates were
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obtained from Bachem Bioscience (King of Pro-KLK14 activation studies - The possible ow
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Prussia, PA): Boc-Phe-Ser-Arg-AMC (FSR- activation of pro-KLK14 by recombinant lo
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AMC), Boc-Val-Pro-Arg-AMC (VPR-AMC), mature KLK5 [produced in-house in P. ed
H-Pro-Phe-Arg-AMC (PFR-AMC), Z-Gly- pastoris as previously described (27)] was fro
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Gly-Arg-AMC (GGR-AMC), Boc-Leu-Gly- monitored via complex formation between h
Arg-AMC (LGR-AMC), Boc-Leu-Lys-Arg- KLK14 and the serine protease inhibitor ttp://w
AMC (LKR-AMC), Boc-Leu-Arg-Arg-AMC (serpin) α -antitrypsin (AT), an inhibitor of w
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(LRR-AMC), Boc-Gln-Arg-Arg-AMC (QRR- KLK14, but not of KLK5 (27). Pro-KLK14 .jb
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AMC), Boc-Gln-Ala-Arg-AMC (QAR-AMC), was pre-incubated with KLK5 in a final .o
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Boc-Gln-Gly-Arg-AMC (QGR-AMC), Tos- volume of 10µl at different molar ratios and b/
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Gly-Pro-Arg-AMC (GPR-AMC), Tos-Gly- incubation times. AT was then added at a 2 g
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Pro-Lys-AMC (GPK-AMC), Boc-Glu-Lys- fold molar excess to a final volume of 15µl. st o
Lys-AMC (EKK-AMC), Boc-Val-Leu-Lys- The reaction was incubated for 30 min at room n A
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APhMe-CA M(VC L(KA-AAPMFC-A),M aCn)d. SSuucc--LAelua--LAelua--VPraol-- taelomnpee raantudr ew. iAths rceocnotmroblsin, aAntT mwaatus rein cKuLbKat1e4d ril 2, 2
Tyr-AMC (LLVY-AMC) was purchased from and pro-KLK14 and with mature KLK5. 01
9
the Peptide Institute Inc. (Osaka, Japan). All Reaction mixtures were resolved by SDS-
substrates were diluted in dimethyl sulfoxide PAGE under reducing conditions and resultant
(Me2SO) to a final concentration of 80 mM gels were silver-stained.
and stored at -20°C. Inhibition assays – Serpins. Serine protease
Individual AMC-peptide substrates (final inhibitors (serpins), α -antitrypsin (AT;
1
concentrations ranging from 0.004 – 2mM) SERPINA1), α -antichymotrypsin (ACT;
1
were incubated with recombinant mature SERPINA3), antithrombin III (ATIII;
KLK14 (final concentration of 12nM) at a SERPINC1), α -antiplasmin (AP; SERPINF2)
2
final volume of 100µL under optimal buffer and plasminogen activator inhibitor-1 (PAI-1;
conditions within 96-well white polystyrene SERPINE1) were purchased from Calbiochem
microtitre plates. Reaction mixtures contained (San Diego, CA). Inhibitors displayed > 95%
less than 5% (v/v) DMSO. Initial rates of purity on Coomassie-blue stained
KLK14-mediated peptide hydrolysis was polyacrylamide gels, were diluted in water to a
monitored by measuring free AMC final concentration of 1 mg/ml and stored at –
fluorescence on the Wallac 1420 Victor2™ 20°C. In order to calculate second-rate
fluorometer (Perkin-Elmer, Wellesley, MA) constants, the inhibition of KLK14 by serpins
with excitation and emission filters set at was performed under pseudo-first order
Enzymatic action of KLK14 8

conditions, as previously described (28-30). 0.5 mM) was applied to each well. AMC
KLK14 was pre-incubated with each serpin fluorescence was measured on the Wallac
for various lengths of time (0-5 min for AT, 1420 Victor2™ fluorometer, as described
AP & PAI-1); 0-10 min for ATIII; 0-60 min above. KLK14-free reactions were used as
for ACT) and at different KLK14:serpin molar negative controls and background
ratios at room temperature (for AT, AP, PAI-1 fluorescence was subtracted from each value.
& ATIII) or 37°C (for ACT) with gentle All experiments were performed in triplicate.
agitation. The reaction was quenched by
diluting 10 µl in 190 µl of optimal buffer In vitro proteolysis experiments - The
containing 0.2mM QAR-AMC. AMC following substrates were incubated with
fluorescence was measured on the Wallac KLK14: mouse sarcoma collagen type I,
1420 Victor2™ fluorometer, as described chicken sternal cartilage collagen type II, calf
above. All experiments were performed in skin collagen type III, human placenta
triplicate. collagen type IV, human foreskin fibroblast
To monitor KLK14-serpin complex fibronectin, human plasma vitronectin, laminin,
formation, KLK14 (0.5µg; 1µM final low molecular weight kininogen were
concentration) was incubated with each serpin purchased from Sigma. Plasminogen from
at molar ratios of 1:1, 1:2, 1:5 and 1:10 at human plasma and recombinant human D
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room temperature for 30 min (for AT, AP, insulin-like growth factor binding proteins 2 w
n
PAI-1 & ATIII) or at 37 °C for 1hr (for ACT). and 3 (IGFBP-2, IGPBP-3) were obtained loa
d
Reaction mixtures were resolved by SDS- from R&D Systems Inc. (Minneapolis, MN). ed
PAGE under reducing conditions; gels were Collagen I, II, III (2 µg), collagen IV, fro
m
stained with SimplyBlueTM SafeStain. laminin, fibronectin, kininogen (5 µg), h
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fibrinogen, plasminogen (10 µg), vitronectin, ://w
Effect of ions on KLK14 activity - KLK14 was IGFBP-2 and IGFBP-3 (300ng) were w
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incubated with each cation and citrate incubated with specific amounts of KLK14 .jb
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[solutions prepared from salts of ZnCl2, (6ng - 1µg) in optimal assay buffer over time .org
ZnSO , Zn(CH COO) , CaCl , MgCl , NaCl, at room temperature or 37°C with gentle b/
4 3 2 2 2 y
KCl, sodium citrate, at final concentrations of agitation. KLK14-free reactions were included gu
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0, 12, 60, 120, 1200, and 12000 nM] in as negative controls. Reactions were st o
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optimal assay buffer at a final volume of 100 terminated by freezing in liquid nitrogen and A
p
µl for 10 min at 37°C with gentle agitation. At resolved by reducing SDS-PAGE, as ril 2
this point, QAR-AMC (at a final concentration described above. Gels were stained with , 2
of 0.5mM) was applied to each reaction SimplyBlueTM SafeStain or with the Silver 019
mixture and AMC fluorescence was measured XpressTM kit (Invitrogen).
on the Wallac 1420 Victor2™ fluorometer, as Furthermore, KLK14 (0.4 and 0.8µg) was
described above. Residual KLK14 activity incubated with fluorescent conjugates of
against QAR-AMC after incubation with collagen types I and IV (2.5 µg) and
individual cations/citrate was calculated. fibrinogen (3.75 µg) (Molecular Probes,
Eugene, OR) in optimal assay buffer (final
Reversal of Zn2+ inhibition of KLK14 by volume 200 µl) at room temperature.
EDTA - KLK14 (12 nM final concentration) Fluorescence was measured every 10 min for
was incubated alone or with: a) Zn2+ (1.2 µM 2 h on the Wallac 1420 Victor2™ fluorometer
final concentration ZnCl ), b) EDTA (1mM set at 492 nm for excitation and 535 nm for
2
final concentration), c) Zn2+ and EDTA and d) emission. Enzyme-free reactions were used as
Zn2+ followed by the addition of EDTA after negative controls and background
20 min, at a final volume of 100µl in optimal fluorescence was subtracted from each value.
assay buffer in microtitre plate wells for 10 All experiments were performed in triplicate.
min at 37°C with gentle agitation. After
incubation, QAR-AMC (final concentration of
Enzymatic action of KLK14 9

N-terminal sequencing - N-terminal sequence polyacrylamide gels (Fig. 1A). The yield of
analysis was performed on KLK14-generated purified mature and pro-KLK14 from 1L
fragments derived from recombinant mature culture supernatants was in the range of 1.5-
KLK14, fibronectin, collagen IV, plasminogen, 3mg and 25-50µg, respectively, as determined
kininogen, fibrinogen and vitronectin. KLK14 by ELISA and total protein assays.
was incubated alone (3µg) or with each intact Pre-pro-KLK14 is a 251-aa polypeptide
protein (2.5-5µg) for different time points as comprised of a signal sequence (Met1-Ser18),
specified by the proteolysis experiments, followed by a 6-aa pro-peptide (Gln19-Lys24)
separated by reducing SDS-PAGE and and a 227-aa trypsin-like serine protease
transferred to a polyvinylidene difluoride domain (Ile25-Lys251) with a predicted
(PVDF) membrane (Amersham Biosciences, zymogen activation site at Lys24-Ile25 (7;8).
Piscataway, NJ), previously immersed in The resultant masses of both recombinant
100% methanol, at 30V for 1 hr. After the proteins were consistent with those inferred
transfer, the membrane was removed and from the primary KLK14 sequence, indicating
rinsed with de-ionized water, stained with a lack of glycosylation or other post-
Coomassie blue R-250 (0.1 % solution in 40% translational modifications. Purified mature
methanol) for 5 min and de-stained for 5 min KLK14 appeared as an enzymatically active
in 50% methanol. The membrane was then intact band of ~25 kDa along with 8 less D
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thoroughly washed with de-ionized water and abundant lower molecular weight bands, w
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air-dried. Fragments were excised and identified as products of autodegradation (Fig. loa
d
subjected to automated N-terminal Edman 1, lane 1; Fig. 2). Purified pro-KLK14 was ed
degradation (31) consisting of 5 cycles of visualized as a single, enzymatically inactive fro
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Edman chemistry on a ABI 492 Procise cLC band of ~27 kDa, as expected (Fig.1, lane 2). h
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sequencer (Applied Biosystems, Foster City, The identity of both recombinant KLK14 ://w
CA), followed by analysis of resultant proteins was verified by western blotting (Fig. w
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phenylthiohydantoin (PTH)-amino acid 1B) and confirmed by tandem mass .jb
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residues on an HPLC column. spectrometry. In the case of recombinant .org
KLK14, partially sequenced tryptic peptides b/
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RESULTS IITGGHTCTR, QVTHPNYNSR and gu
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QACASPGTSCR corresponded to amino acid st o
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Recombinant mature and pro-KLK14 - sequences 25-33, 97-106 and 134-144, A
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Recombinant mature and pro-KLK14 proteins respectively, of the KLK14 protein sequence. ril 2
were produced in their native forms, without Likewise, sequenced peptide fragments , 2
0
fusion tags, in yeast (P. pastoris) and obtained from the pro-KLK14 sample 1
9
mammalian (HEK-293) expression systems, included IIGGHTCTR,
respectively. Secreted expression was SSQPWQAALLAGPR, QVTHPNYNSR,
achieved by cloning KLK14 cDNA in-frame AVRPIEVTQACASPGTSCR and
with the yeast α-factor for mature KLK14 and DSCQGDSGGPLVCR, which matched
with the native KLK14 signal sequence in the precisely with KLK14 amino acid sequences
case of pro-KLK14. As monitored by SDS- 25-33, 34-47, 97-106, 126-144, 198-211,
PAGE, western blot analysis and/or ELISA, respectively (numbering is based on Genbank
mature KLK14 was detected in the yeast accession no. AAK48524).
culture supernatant after 1 d of methanol
induction, with the highest levels produced Distribution of KLK14 in tissues, fluids and
after 6 d; maximal pro-KLK14 levels were cell lines - A novel KLK14-specific ELISA
attained after a 10 d incubation period in was established in order to quantify KLK14
serum-free media (data not shown). After their levels in normal and cancerous tissue extracts,
respective two-step chromatographic biological fluids and cell lines. The optimized
purification procedures, recombinant mature KLK14-ELISA, comprised of monoclonal
and pro-KLK14 were obtained with > 95% antibody 2E9 (capture antibody) and
purity, as verified by silver-stained polyclonal rabbit anti-serum (detection
Enzymatic action of KLK14 10

antibody) generated against recombinant (TOV-21G, MDAH 2774, OV-90, TOV112D;
mature KLK14, is sensitive (detection limit of 0.103-0.604µg/L) cell lines.
0.05 µg/L), specific for KLK14 (<0.1% cross-
reactivity with other KLKs), recognizes both Autodegradation of KLK14 in vitro - Purified
mature and pro-KLK14 recombinant proteins recombinant mature KLK14 was comprised of
and is linear from 0.05 to 10 µg/L with a mixture of peptides visualized as multiple
between-run and within-run coefficients of bands under reducing SDS-PAGE (Fig.4A).
variation of <10%. Western blot (Fig.1B) and N-terminal
Among the 40 normal adult human tissues sequence analysis confirmed the identity of
screened for KLK14 content, 18 contained the low molecular weight (<25kDa) protein
detectable levels of KLK14 (Fig. 2). The bands as internal fragments of mature KLK14
highest KLK14 levels were found in skin (245 likely arising from auto-proteolytic cleavage
ng /g total protein), followed by breast (224 (Fig.4). All KLK14 cleavage sites occurred
ng/g total protein) and prostatic (131 ng/g total after P1-Arg residues (Fig. 4B) and the
protein) tissue extracts, in general accord with majority reside in exposed, solvent accessible
previous studies (7;8;10;11;13-15;17). surface loops, which are most susceptible to
Comparatively lower levels were observed in proteolytic cleavage (Fig.4C). Among KLK14
axillary lymph nodes (62.8 ng/g total protein) autodegradation fragments, only fragment I D
and medulla (59.2 ng/g total protein) extracts retains all residues of the catalytic triad (H67, ow
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with minor amounts (<50 ng/g total protein) in D111, S204; Fig. 4B) and may exhibit residual lo
a
d
the remaining tissues. serine protease activity. However, prolonged ed
The concentration of KLK14 in biological incubation (24-hrs) of mature KLK14 at 37°C fro
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fluids from healthy was quantified (Table 1). resulted in complete hydrolysis of the intact h
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In agreement with our prior study (10), the 25kDa band and a corresponding decrease in ://w
highest levels of KLK14 were measured in enzymatic activity (Fig.4D and E). Auto- w
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seminal plasma (15.8±16.0µg/L), followed by inactivation of KLK14 by autolytic cleavage .jb
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amniotic fluid (8.1±6.0µg/L) and saliva may represent a potential regulatory .org
(4.4±2.1µg/L). Follicular fluid and breast milk mechanism. b/
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contained lower concentrations (~1.2µg/L). gu
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KLK14 was not detected in the cerebrospinal Optimal pH and buffer composition for st o
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fluid samples examined. Compared to normal, KLK14 activity - Two buffer systems, 100mM A
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KLK14 levels were elevated in the serum of Na2HPO4 (pH 7-10) and 100mM Tris-HCl (pH ril 2
patients with prostate cancer (Fig. 3); a likely 6-9), were chosen to determine the optimal pH , 2
0
consequence of KLK14 overexpression and for KLK14 activity. Although KLK14 was 19
secretion by prostate tumour tissues (17). We most active at a pH of 8.0 in both systems,
also detected KLK14 in ascites fluids from KLK14 activity was 1.4-fold higher in
phosphate vs. Tris buffer. Furthermore,
ovarian cancer patients (0.34±0.7µg/L) as well
KLK14 displayed enhanced activity (13%
as in cytosolic extracts of ovarian
increase) in the presence of 0.01% Tween-20
(1.77±5.13µg/L) and breast (0.26±1.17µg/L)
compared to 0.2% bovine serum albumin
tumours.
(BSA) in which activity decreased 30%
Among the cell lines studied, KLK14 was
(Tween-20 and BSA prevent adsorption of
only detected in the conditioned media of a
KLK14). Hence, the buffer composition
non-tumorigenic keratinocyte cell line
utilized in subsequent studies was: 100mM
(HaCaT; 0.275µg/L) and a subset of
Na HPO , 0.01% Tween-20, pH 8.0.
neuroblastoma (Sk-N-MC, IMR32; 0.949 and 2 4

0.193µg/L, respectively), breast cancer
Activation of pro-KLK14 - As with other S1A
(MCF7, MDA-MB-468; 0.116 and 0.497µg/L,
family SP, pro-KLK14 requires the removal of
respectively), cervical cancer (C-4 I, ME-180,
the inhibitory N-terminal pro-peptide
HT-3; 0.158-0.456µg/L) and ovarian cancer
sequence at K24-I25 for conversion into its
enzymatically active form. Autoactivation is