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INTRODUCTION |
Prostasin is a serine protease discovered in ejaculated human
semen in 1994 (1). The molecular mass of prostasin is 40 kDa when
examined by SDS-polyacrylamide gel electrophoresis
(PAGE)1 under reducing
conditions. Prostasin displays trypsin-like enzymatic activities by
hydrolyzing peptidyl fluorogenic substrates such as
D-Pro-Phe-Arg-AMC. This trypsin-like enzymatic
activity can be inhibited by aprotinin, antipain, leupeptin, and
benzamidine. Prostasin is present at high levels in normal human semen
(8.61 ± 0.42 µg/ml) and in the prostate gland (143.7 ± 15.9 ng/mg). Lower amounts of prostasin can also be detected in other
tissues. In the prostate gland, the prostasin protein is present in the epithelial cells as well as in the secretion inside the lumen. The
full-length human prostasin mRNA has been deduced (2). The
predicted mature prostasin peptide sequence has a potential carboxyl-terminal hydrophobic membrane anchorage domain followed by a
short cytoplasmic tail. The translated amino acid residue sequence of
prostasin is similar to those of human prostase, testisin, plasma
kallikrein, coagulation factor XI, hepsin, plasminogen, and acrosin
(2-4). A membrane-bound Xenopus kidney epithelial cell
sodium channel-activating protease (CAP1) was found highly homologous
to human prostasin, sharing 53% sequence identity at the amino acid
level (5). Recently, the mouse counterpart of CAP1, mCAP1, has been
cloned from a cortical collecting duct cell line (6). mCAP1 shares 77%
amino acid sequence identity with human prostasin.
Serine proteases play important roles in a diverse range of the body's
normal physiological processes, and they are implicated in various
pathological processes such as cardiovascular disorders and cancers
(7). The prostate produces a number of serine proteases such as
prostate-specific antigen (8), human glandular kallikrein (9), and the
most recently discovered prostase (3). Some of these serine
proteases are suspected to affect fertility or semen liquefaction (10),
and others are implicated in normal prostate development or prostatic
diseases (11-14). For example, both prostate-specific antigen and
human glandular kallikrein have become important diagnostic and
prognostic markers for prostate cancer. Serine proteases are usually
regulated at the post-translational level in addition to the
transcriptional regulation at their gene level. The body's own
strategy of regulating the serine proteases is to bind the serine
proteases with a protein inhibitor such as the inhibitors of the serpin
class (15). These serpin-serine protease pairs are highly specific with
regard to the two molecules involved; examples include
1-antitrypsin and elastase (15), kallistatin and
kallikrein (16, 17),
1-antichymotrypsin and prostate-specific antigen (18). The mechanism of serpin inhibition of
serine proteases involves the formation of a covalently linked complex
at a 1:1 stoichiometry (19). Such a complex exhibits resistance to
treatment with SDS or boiling (16, 17).
The physiological functions of prostasin are not fully understood. In a
recent study (20) we showed that prostasin expression is significantly
down-regulated in high grade prostate tumors and is lost in highly
invasive human and mouse prostate cancer cell lines. Transfection of
two human prostate cancer cell lines DU-145 and PC-3 with human
prostasin cDNA reduced in vitro invasiveness of the
cells, suggesting an invasion suppressor role for prostasin. This
anti-invasion activity is apparently conferred by the cellular prostasin but not the secreted prostasin. In the present study, we
determined that prostasin is a GPI-anchored membrane protein in
addition to being a secreted protease. The subcellular localization of
prostasin was investigated in cells expressing native or recombinant prostasin. We have also identified a prostasin-binding protein (PBP), a
potentially serpin class serine protease inhibitor specific for
prostasin. We further demonstrated that the membrane-bound prostasin is
an active serine protease. These results will provide structural and
regulatory information for further investigation of the functions of
prostasin in normal prostate development, prostatic diseases, as well
as reproductive biology.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Plasmid DNA Transfection--
A human embryonic
kidney epithelial cell line, 293-EBNA (Invitrogen, Carlsbad, CA), was
maintained in Dulbecco's modified Eagle medium supplemented with 10%
fetal bovine serum. Human prostate cancer cell lines LNCaP, DU-145, and
PC-3 were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). The LNCaP and the DU-145 cells were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate; the PC-3 cells were maintained in F-12K
medium supplemented with 10% fetal bovine serum. All cells were kept
at 37 °C with 5% CO2. All tissue culture media, sera,
and supplements were purchased from Life Technologies, Inc.
A full-length human prostasin cDNA of 1,896 base pairs (including a
209-base pair 5'-untranslated region, 1,032 base pairs of the coding
region, and a 655-base pair 3'-untranslated region) was generated by
means of reverse transcription-polymerase chain reaction with the
following two primers, 5'-AGA CGG TGC TGG TGA CTC GT-3' and 5'-TGT GCT
CAA ACA TTT TAA TC-3', using the total RNA of LNCaP cells as template
(2). The amplified cDNA was cloned into a mammalian expression
vector, pREP-8 (Invitrogen), at its polylinker site. Transfection of
the prostasin cDNA plasmid into 293-EBNA cells was carried out
using electroporation. The electroporated cells were then subcultured
for selection of transfectants (293/Pro) using 5 mM
histidinol (Sigma) in the culture medium for 2 weeks. The pREP-8
vector plasmid was transfected into 293-EBNA cells and subjected to
histidinol selection to establish the control cells (293/Vec).
The DU-145 and the PC-3 cells, which do not express prostasin (20),
were also transfected using plasmids containing the full-length human
prostasin cDNA. The methods for plasmid engineering and
establishment of transfectants that express human prostasin have been
described previously (20). The resulting cell lines that express human
prostasin were designated DU-145/Pro and PC-3/Pro.
SDS-PAGE and Western Blot Analysis--
These procedures were
carried out for all experiments unless stated otherwise. Samples were
suspended in 1 × SDS sample buffer (62.5 mM Tris-HCl
at pH 6.8, 2% v/v glycerol, 2% w/v SDS, and 2%
-mercaptoethanol),
boiled for 5 min, and resolved in a 10% polyacrylamide gel. The
resolved proteins were then transferred to a nitrocellulose membrane.
The membrane was stained with India ink for 15 min (1:1,000 in TBS-T:
20 mM Tris at pH 7.6 containing 0.137 M NaCl
and 0.1% Tween 20), blocked in 5% non-fat milk for 1 h, and
incubated with the primary antibody for 30 min in a tray or a Surf-blot
apparatus (Idea Scientific, Inc., Minneapolis). After washing, the
membrane was incubated with a secondary antibody conjugated with
horseradish peroxidase (Sigma, used at a 1:10,000 dilution) for 30 min.
Signals were detected using ECL (enhanced chemiluminescence) with
WestPico reagents (Pierce) following the supplier's protocol. The
membrane was then exposed to Kodak x-ray film. The primary antibodies
used were as follows: polyclonal antibodies against prostasin
(recombinant or native, used at 1:1,000), a monoclonal antibody against
1-integrin (used at 1:1,000), and a monoclonal antibody
against poly(ADP-ribose) polymerase (used at 1:500). Antibodies
against
1-integrin and poly(ADP-ribose) polymerase were
from BD Transduction Laboratories (San Diego).
Purification of Recombinant Human Prostasin--
The 293/Pro
cells were grown to a confluent monolayer in Dulbecco's modified
Eagle's medium and 10% fetal bovine serum containing 5 mM
histidinol. Cells were then placed in Opti-MEM I serum-free medium
(Life Technologies, Inc.) for 72 h before collection of the
conditioned medium. The collected medium was tested for recombinant prostasin (r-hPro) by Western blot analysis using a prostasin-specific antibody (1). For purification of the secreted prostasin, the serum-free medium was centrifuged at 10,000 rpm for 20 min to remove
dead cells or debris and then passed through an aprotinin-agarose column (1.5 × 20 cm, Sigma) equilibrated with 25 mM
Tris-HCl at pH 7.6 at a flow rate of 25 ml/h. After extensive washing
to remove any unbound proteins, the bound prostasin was eluted with 0.1 M glycine (pH 3.0) containing 0.1 M NaCl at a
flow rate of 60 ml/h. The eluted prostasin was immediately neutralized
with appropriate amounts of 1 M Tris, combined,
concentrated with Centricon-10 concentrators (Amicon Inc., Beverly,
MA), and stored at
20 °C before use in other assays.
Preparation of a Polyclonal Antiserum against Recombinant
Prostasin--
250 µg of the purified r-hPro in 0.5 ml of
phosphate-buffered saline (PBS, pH 7.4) was emulsified with an equal
volume of complete Freund's adjuvant (Sigma) and was injected
subcutaneously into a 1.5-kg female New Zealand White rabbit (Charles
River Laboratories, Wilmington, MA). Booster injections with 100 µg
of r-hPro (emulsified with incomplete Freund's adjuvant, Sigma) were
performed three times at 3-week intervals. Preimmune rabbit serum was
collected before the initial immunization.
Immunocytochemistry--
The PC-3/Pro or LNCaP cells were seeded
on glass coverslips (Fisher Scientific) at a density of 5 × 104/coverslip and grown for 24-36 h prior to a double
immunostaining. Briefly, cells were rinsed three times in 1 × PBS, fixed in 4% paraformaldehyde, and permeablized with 0.18% Triton
X-100 in PBS for 10 min. After blocking in 10% normal goat serum (Life Technologies, Inc.) in 1 × PBS, cells were incubated with the primary antibodies for 45 min, washed, incubated with the appropriate secondary antibodies at room temperature for 30 min, and then washed
three times for 10 min each in 1 × PBS. A rabbit polyclonal antibody against prostasin was used at a dilution of 1:100. A monoclonal antibody against poly(ADP-ribose) polymerase was used as a
nuclear specific marker at a dilution of 1:75. A goat anti-rabbit IgG
conjugated with fluorescein (1:50, Life Technologies, Inc.) and a goat
anti-mouse IgG conjugated with Cy3 (1:800, Jackson ImmunoResearch,
West Grove, PA) were used as the secondary antibodies. The coverslips
were mounted with Gel/Mount (Fisher Scientific) and analyzed on a Carl
Zeiss LSM510 laser scanning microscope.
Subcellular Fractionation and Differential
Extraction--
Subcellular fractionation was performed as described
in Krajewski et al. (21) and Pemberton et
al. (22). Briefly, confluent cells in 4 × 150-cm2 flasks (estimated 5-10 × 107
cells/total) were washed three times with 1 × PBS and removed by
mechanical force for the 293/Pro cells or trypsin treatment (0.25%
with 1 mM EDTA) for the PC-3/Pro and LNCaP cells. The cells were resuspended in 7 ml of cold MES buffer (17 mM
at pH 7.4, 2.5 mM EDTA, and 250 mM sucrose)
containing protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml
antipain). The following steps were performed at 4 °C. Cell
suspension was homogenized with a Dounce homogenizer for 60 strokes
followed by centrifugation twice at 500 × g for 10 min, resulting in the crude nuclear fraction in the pellet. The
supernatant was centrifuged twice at 10,000 × g for 15 min, resulting in the heavy membrane fraction in the pellet containing mitochondria, lysosomes, and peroxisomes. The supernatant from the
10,000 × g centrifugation was subjected to an
ultracentrifugation at 100,000 × g for 60 min,
resulting in a light membrane fraction in the pellet containing the
plasma membrane, microsomes, and endoplasmic reticula. The supernatant
from the final centrifugation contains soluble or cytosolic proteins.
The pellets from each centrifugation were washed with 2 × 10 ml
of MES to eliminate carryovers.
Differential extraction of membrane fractions was carried out according
to Pei et al. (23). Briefly, pellet/membrane fractions were
divided equally into three portions and were extracted with 1% Triton
X-114 in Tris buffer (10 mM Tris-HCl, pH 7.5) or high salt
(350 mM NaCl in Tris buffer), or alkali (50 mM
glycine/NaOH, pH 11.0) for 1 h on ice. The samples were
centrifuged at 100,000 × g for 30 min. The resulting
pellet was dissolved in 1 × SDS sample buffer for gel
electrophoresis. The supernatant was subjected to a trichloroacetic
acid precipitation to recover proteins for gel electrophoresis.
Detergent Phase Separation and Phosphatidylinositol-specific
Phospholipase C (PI-PLC) Treatment--
The procedure was adapted from
those described by Bordier (24) and Rosenberg (25). Briefly, cells
(5 × 106) were lysed in 1 ml of ice-cold TBS (10 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA) containing 1% Triton X-114 (Sigma) and protease
inhibitors for 2 h with gentle shaking at 4 °C. The lysate was
then centrifuged at 14,000 rpm for 30 min. The supernatant (500 µl,
or 700-800 µg of total protein) was overlaid onto a 300-µl sucrose
cushion (6% w/v sucrose in TBS containing 0.06% Triton X-114). The
solution was incubated at 37 °C for 3 min and centrifuged at
300 × g for 3 min at room temperature to separate the
detergent phase (pellet) and the aqueous phase. The aqueous phase was
removed and extracted further with 0.5% Triton X-114 and 2% Triton
X-114. The aqueous phase after the final centrifugation contains the soluble proteins. The detergent phase (pellet) from the first centrifugation was resuspended in 500 µl of ice-cold TBS, incubated at 37 °C for 3 min, and centrifuged at 300 × g for
3 min at room temperature to ensure the purity of the detergent phase.
The detergent phase was resuspended in 100 µl of ice-cold TBS. 10 µl of the resuspended detergent phase was subjected to PI-PLC (Sigma)
digestion at 37 °C for 1 h with gentle shaking in a total volume of 100 µl of reaction buffer (10 mM Tris-HCl at pH
7.5, 144 mM NaCl). 100 µl of ice-cold TBS containing 2%
Triton X-114 was then added to the digestion mixture and subjected to
phase separation as described above. At the final step, both the
aqueous and detergent phases were precipitated with 6% w/v
trichloroacetic acid and 0.013% sodium deoxycholate. The precipitates
were resuspended in 30 µl of 1 × SDS sample buffer, neutralized
with ammonium hydroxide (microliter amounts), boiled, and subjected to
SDS-PAGE and Western blot analysis.
Human prostates removed by radical prostatectomy performed at Orlando
Regional Medical Center (Orlando, FL) were sectioned with a cryostat at
20-µm thickness. 80 sections were collected and rinsed with PBS twice
to remove prostatic fluid. The washed prostate sections were lysed in 1 ml of TBS containing 1% Triton X-114 at 4 °C overnight with
rocking. The lysed prostate tissues were centrifuged and subjected to
the same phase separation and PI-PLC treatment procedures as described
above. Several representative prostate sections (7 µm) cut at
intervals of the 80 20-µm sections were subjected to standard
hematoxylin and eosin staining for confirmation of benign prostate
morphology. The use of human tissues was approved by the Institutional
Review Boards of Orlando Regional Medical Center and the University of
Central Florida.
[3H]Ethanolamine Labeling, Immunoprecipitation, and
Fluorography--
PC-3/Pro (3 × 105) or LNCaP
(1 × 106) cells were seeded in a 35-mm dish in 1 ml
of Opti-MEM I serum-free medium. The next day, [1-3H]ethan-1-ol-2-amine hydrochloride (37 MBq, 1 mCi/ml,
and 30.0 Ci/mmol, Amersham Pharmacia Biotech) was added to the culture medium at a concentration of 100 µCi/ml, and the cells were cultured for another 24 h. Cells were washed once with PBS and lysed in 0.5 ml of RIPA buffer (PBS at pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors at 4 °C for
1 h. The lysate was centrifuged at 14,000 rpm for 30 min to remove
insoluble material. The supernatant was subjected to
immunoprecipitation with 2 µg/ml anti-prostasin IgG (purified using
Econo-Pac protein A cartridge, Bio-Rad) and protein A-Sepharose beads
(Sigma) at 4 °C overnight. The beads were washed with RIPA buffer
three times, resuspended in 2 × SDS sample buffer with
-mercaptoethanol, boiled, and analyzed by SDS-PAGE. The gel was
fixed in a solution of 2-propanol:water:acetic acid (12.5: 32.5: 5) for
30 min and soaked in Amplify fluorographic reagent (Amersham Pharmacia
Biotech) for another 30 min. The gel was dried, and
3H-labeled molecules were detected by exposure to an x-ray
film with an intensifying screen at
80 °C for 14 days.
Prostasin Binding Assay--
Seminal vesicle fluid was expressed
from one pair of mouse seminal vesicles (C57BL/6 mouse, Harlan,
Indianapolis) and mixed with 1 ml of 25 mM Tris-HCl, pH
7.6, and centrifuged at 14,000 rpm at 4 °C for 30 min. 5 µl of the
supernatant (50 µg of total protein) was incubated with either 0.5 µg of the purified recombinant human prostasin or subcellular
fractions of 293/Pro and PC-3/Pro cells (prepared in the absence of
serine protease inhibitors) at 37 °C for 60 min, or for various time
periods for a time course study. The binding reaction was stopped by
the addition of SDS sample buffer and heating at 100 °C for 5 min.
Mouse tissues were homogenized in PBS (1 g of tissue/5 ml) and
centrifuged in a microcentrifuge at 14,000 rpm for 30 min at 4 °C.
40 µg of total protein for each tissue extract was used in the
binding assay. 1 µl of human or mouse plasma was also subjected to
prostasin binding assay. The use of animals was approved by the IACUC
of the University of Central Florida. Human seminal vesicles were
obtained from radical prostatectomy performed at Orlando Regional
Medical Center. No seminal vesicle metastasis from prostate cancer was
found according to the pathology report. Human seminal vesicle fluid
was diluted with PBS at a ratio of 1:2, mixed by vortex, and spun. 10 µl of the diluted fluid was incubated with 0.5 µg of purified
prostasin at 37 °C for 60 min.
Membrane Overlay Zymography--
The membrane overlay zymography
was carried out using the protocols of Enzyme System Product
(Livermore, CA) and Beals et al. (26). Briefly, samples were
first resolved in a 10% polyacrylamide gel without SDS or
-mercaptoethanol. After electrophoresis, the gel was equilibrated in
a reaction buffer (50 mM Tris-HCl, pH 9.0) for 15 min.
Pre-wet acetate-cellulose membrane impregnated with the prostasin
substrate D-Pro-Phe-Arg-AFC (Enzyme System Product)
was then carefully laid over the gel without entrapping air bubbles.
The membrane-overlaid gel was placed in a moist chamber at 37 °C for
3-5 h. The reaction was monitored using an ultraviolet lamp and photographed.
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RESULTS |
Expression and Purification of Recombinant Human
Prostasin--
Serum-free conditioned medium from 293/Pro cell culture
was prepared and passed through an aprotinin-agarose column for a one-step affinity-chromatographic purification of the recombinant prostasin as described under "Experimental Procedures" (see also Ref. 1). A Coomassie Blue staining of the purified recombinant prostasin is shown in Fig. 1, left
panel (r-hPro). The r-hPro migrates at 40 kDa on an SDS-PAGE under
reducing conditions. Because of glycosylation of the prostasin molecule
(1, 2), it appeared as a rather diffused band on the gel. We prepared a
polyclonal antibody (r-Pro Ab) using the purified r-hPro as an antigen.
The r-Pro antibody recognized the purified recombinant prostasin
(secreted form), the recombinant prostasin in 293/Pro total cell lysate (nonsecreted form), and the native prostasin in ejaculated human semen
(obtained from healthy volunteers, Ref. 1) (Fig. 1, upper right
panel, lanes 1, 2, and 4). The
prostasin protein in the same set of samples was also recognized by a
prostasin-specific antibody referenced previously (1) (Fig. 1,
lower right panel). Neither antibody cross-reacts with any
nonspecific protein in the control 293/Vec total cell lysate (Fig. 1,
right panel, lanes 3). The results indicate that
the polyclonal antibody against the recombinant prostasin is specific
to prostasin and that the recombinant prostasin prepared using the
amplified cDNA has an immunological reactivity similar to that of
the native prostasin. The antibody against the recombinant prostasin
was used in the ensuing assays conducted in this study.

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Fig. 1.
SDS-PAGE and Western blot analysis.
Recombinant human prostasin purified from serum-free conditioned medium
of the 293/Pro cells was analyzed by 10% SDS-PAGE under reducing
conditions. The purified recombinant prostasin (3 µg) migrated at 40 kDa (left panel, r-hPro) and is recognized by an
antibody made against the purified recombinant prostasin (IB:
r-Pro Ab, lane 1) as well as a prostasin-specific
antibody made against purified native prostasin (IB: n-Pro
Ab, lane 1, Ref. 1). The quantity of purified prostasin
in lanes 1 is 0.5 µg. Samples from the 293/Pro cell lysate
(lanes 2, 20 µg), 293/Vec (lanes 3, 20 µg)
cell lysate, and human semen (lanes 4, 30 µg) were
immunoblotted with r-Pro antibody (upper right panel,
1:1,000 dilution) and n-Pro antibody (lower right panel,
1:1,000 dilution). Both antibodies recognize the recombinant prostasin
as well as the native prostasin but do not have cross-reactivity with
293/Vec proteins.
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Recombinant Prostasin Is a Membrane-bound Protein--
A
hydropathy plot of the translated prostasin amino acid sequence
indicated that the prostasin polypeptide has a potential membrane-anchorage domain at the carboxyl terminus (2). To confirm the
presence of a potentially membrane-anchored form of the prostasin
protein, the 293/Pro cells were subjected to subcellular fractionation
by differential centrifugation. Fig. 2
shows that prostasin was present in the crude nuclear fraction (P1,
500-g pellet), heavy membranes (P2, 10,000-g pellet), and light
membranes (P3, 100,000-g pellet), as determined by Western blot
analysis. Prostasin was also detected in the cytosol (S). The purified
r-hPro was used as positive control. Equal amounts of total protein (30 µg) from each membrane fraction and the cytosol were applied in each
lane.

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Fig. 2.
Analysis of prostasin in 293/Pro cell
fractions. The cells were fractionated through differential
centrifugation. An equal amount of protein (30 µg) from each
centrifugation step was resolved by 10% SDS-PAGE followed by
immunoblotting with a prostasin-specific polyclonal antibody (1:1,000
dilution). Prostasin (40 kDa) is detected in the nuclear fraction (P1),
heavy membrane fraction (P2, including mitochondria, lysosomes, and
peroxisomes), light membrane fraction (P3, including plasma membrane,
microsomes, and endoplasmic reticula) as well as the cytosol (S) of
293/Pro. Purified r-hPro (0.5 µg) was used as a positive
control.
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To show that the membrane-anchored prostasin is not a peculiarity in
the 293 cells, we performed similar subcellular fractionation analysis
on prostasin cDNA-transfected human prostate cancer cell line PC-3
(PC-3/Pro) and the human prostate cancer cell line LNCaP, which
expresses endogenous prostasin (2, 20). As shown in Fig.
3A, prostasin is detected in
P1, P2, and P3 fractions, but not in the cytosol (S) of PC-3/Pro. In
the LNCaP cells, endogenously expressed prostasin is detected only in
the P3 fraction. The membrane fractions from PC-3/Pro cells were then
immunoblotted with a monoclonal antibody against a nuclear protein
poly(ADP-ribose) polymerase or a monoclonal antibody against a plasma
membrane-bound protein
1-integrin to ensure the purity of each
fraction. The results showed that the prostasin protein exists in a
membrane-bound form in all cell lines tested. The cells transfected
with the vector DNA alone (293/Vec and PC-3/Vec) were subjected to the
same fractionation procedures followed by SDS-PAGE/Western blot
analysis. No prostasin was detected (data not shown). We further
subjected PC-3/Pro and LNCaP cells to a double immunostaining and
analyzed the subcellular localization of prostasin using confocal
microscopy. The confocal microscopic analysis of PC-3/Pro cells
localized prostasin (green) to the ER-Golgi complex
(Fig. 3B), consistent with the cell fractionation results
shown in Fig. 3A. Because the nuclear membrane is
practically a prominent component of the ER (27), it is not surprising
that this portion of prostasin appeared in the P1 fraction. The LNCaP cells, however, did not show punctate or nuclear-ER-Golgi complex staining, again, consistent with the cell fractionation results shown
in Fig. 3A.

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Fig. 3.
Analysis of prostasin in prostate
cancer cell lines. Panel A, Western blot analysis of
membrane-bound prostasin in PC-3/Pro and LNCaP cells. The experimental
procedures were the same as described in Fig. 2. Prostasin (40 kDa) is
detected in the nuclear fraction (P1), heavy membrane fraction (P2),
light membrane fraction (P3), but not in the cytosol (S) of PC-3/Pro.
In LNCaP cell fractions, prostasin is detected only in P3. Antibodies
against a nuclear protein, the poly(ADP-ribose) polymerase (PARP,
1:500, or 0.5 ng/ml), and a plasma membrane protein, 1-integrin
(1:1,000, or 0.25 ng/ml), were used as fractionation markers.
Panel B, confocal microscopic localization of prostasin. The
PC-3/Pro and LNCaP cells were fixed, permeablized, and subjected to a
double immunostaining. One focal plane for each cell type is presented
to show prostasin signals (green). Prostasin is detected
primarily at the nuclear-ER-Golgi complex as well as punctate regions
in the PC-3/Pro cell. In the LNCaP cells, no punctate prostasin can be
seen. The nuclear marker protein poly(ADP-ribose) polymerase
(red) is seen in both cell types. A merge image for either
cell type is presented to the right. The images were taken
after subtracting background signal on a preimmune serum-stained
control coverslip. Magnification, × 400. The antibody dilution ratios
are: anti-prostasin, 1:100; anti-poly(ADP-ribose) polymerase, 1:75 (or
3.3 ng/ml); goat anti-rabbit IgG conjugated with fluorescein
isothiocyanate, 1:50; and goat anti-mouse IgG-Cy3, 1:800.
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To test if prostasin is truly a membrane-anchored protein rather than a
membrane-associated protein, we subjected the P1, P2, and P3 fractions
of the PC-3/Pro cells to treatment with a detergent, high salt, or
alkali. As shown in Fig. 4,
membrane-bound prostasin (pellet) was released into the supernatant
only by the detergent treatment but not the high salt or alkali
treatment. The detergent released prostasin and the membrane-bound
prostasin had similar molecular weight. The results indicated that
prostasin is a true membrane-anchored protein.

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Fig. 4.
Recombinant prostasin is a true
membrane-bound protein. Approximately 80-100 µg of total
protein of each membrane fraction of PC-3/Pro cells (as described in
legend to Fig. 3A) was subjected to detergent
(TX), high salt (HiS), or alkali (Alk)
treatment followed by centrifugation to separate the supernatant and
the pellet for SDS-PAGE and Western blot analysis. Prostasin in all
fractions can only be released from the membrane (pellet) to the
supernatant (soluble protein) by the detergent treatment but not high
salt or alkali treatment.
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Membrane Prostasin Is GPI-anchored--
A comparison of the
potential carboxyl-terminal membrane-anchorage domain of prostasin (2)
with GPI-anchored proteins (28) predicts a GPI linkage for prostasin as
well (data not shown). Such a linkage may be susceptible to cleavage by
PI-PLC, GPI-specific phospholipase D, or nitrous acid (25). In our
studies, we first chose PI-PLC to test if prostasin is a GPI-anchored
membrane protein. The 293/Pro cells were lysed in TBS containing 1%
Triton X-114. After phase separation, the aqueous phase (Fig.
5, lane 1, 30 µg of total
protein) and the detergent phase (lane 2, 3 µg of total
protein) were analyzed by Western blot using the prostasin-specific antibody. The majority of the prostasin protein in 293/Pro cells is
associated with the membrane, which was retained in the detergent phase. The size difference between the soluble and the membrane-bound prostasin may be attributed to the GPI moiety that is linked to prostasin (Fig. 5, lanes 1 and 2). The detergent
phase was then subjected to PI-PLC digestion at various enzyme
concentrations followed by a second phase separation. In Fig. 5,
lanes 3-5 represent samples of the aqueous phases after
PI-PLC digestion. The PI-PLC treatment released prostasin from the
detergent phase in a dose-dependent manner (lane
3, 0.25 unit; lane 4, 0.125 unit, and lane
5, 0 units). The PI-PLC-released prostasin and the soluble
prostasin are similar in molecular mass as shown in Fig. 5. The
results support a GPI-anchoring mechanism for the membrane-bound
prostasin.

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Fig. 5.
Detergent phase separation of 293/Pro cells
and phospholipase C treatment. The 293/Pro cells were lysed in TBS
containing 1% Triton X-114 and subjected to phase separation (700-800
µg of total protein as the starting quantity). The detergent phase
containing membrane-associated proteins (equivalent to 1/10 of the
total starting membrane-associated proteins) was further treated with
PI-PLC followed by additional phase separation. The soluble proteins
(lane 1, 30 µg) and the detergent phase proteins
(lane 2, 3 µg) before PI-PLC treatment and the soluble
proteins extracted from the detergent phase after PI-PLC treatment were
subjected to SDS-PAGE and Western blot analysis using a
prostasin-specific antibody. The membrane-bound prostasin is released
from the membrane after PI-PLC digestion as it was detected in the
post-PLC soluble phase. The amounts of PI-PLC used in the reactions
are: lane 3, 0.25 unit; lane 4, 0.125 unit; and
lane 5, 0 unit in a total reaction volume of 100 µl. The
results indicate that prostasin is anchored to membrane via GPI. The
size difference between the membrane-bound prostasin and the soluble
prostasin (lanes 1 and 2) may be attributed to
the GPI moiety that is linked to prostasin.
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One question that remained unclear was whether the native prostasin in
the prostate tissue epithelial cells is membrane-bound by GPI anchorage
as well. We selected a panel of prostate cancer cell lines and 293/Pro
cells that express either recombinant or endogenous prostasin, and
normal human prostate tissues in our next experiment. Cell lines that
express recombinant prostasin were 293/Pro, PC-3/Pro, and DU-145/Pro.
The human prostate cancer cell line LNCaP and normal human prostate
tissues were used for testing native cellular prostasin. All samples
(300 µg of total protein as the starting quantity) were subjected to
detergent phase separation before and after PI-PLC digestion as
described under "Experimental Procedures." PC-3 transfected with a
vector plasmid (PC-3/Vec) was used as negative control. The results are presented in Fig. 6A. Without
PI-PLC treatment, both the recombinant and native prostasin are mainly
membrane-anchored (found in the detergent phase). Soluble prostasin is
detected in 293/Pro and prostate tissues. After PI-PLC treatment, the
membrane-anchored prostasin is released into the soluble fraction from
293/Pro, PC-3/Pro, and prostate tissues, but not from DU-145/Pro and
LNCaP. The results indicated that the native prostasin in normal human prostate tissue is also GPI-anchored. The membrane-bound prostasin in
DU-145/Pro and LNCaP is resistant to PI-PLC digestion. We further tested if prostasin can be labeled biosynthetically with
[3H]ethanolamine, which is specifically incorporated in
the GPI unit of GPI-anchored proteins (29). We chose the PC-3/Pro
(expressing recombinant prostasin) and LNCaP (expressing native
prostasin) cells for [3H]ethanolamine biosynthetic
labeling. As shown in Fig. 6B,
[3H]ethanolamine was incorporated into either recombinant
or native prostasin, demonstrating that both are truly
GPI-anchored.

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Fig. 6.
Prostasin is a GPI-anchored membrane
protein. Panel A, detergent phase separation and PI-PLC
treatment. Human prostate tissues and cell lines that express either
endogenous prostasin (LNCaP) or recombinant prostasin (293/Pro,
PC-3/Pro, and DU-145/Pro) were subjected to detergent phase
separation/PI-PLC digestion followed by prostasin immunoblotting (300 µg of total protein was used for each sample as the starting
quantity). Both the recombinant and native prostasin are mainly
membrane-anchored (pre-PLC, detergent phase or D). Soluble
prostasin is detected in 293/Pro and prostate tissues (pre-PLC, soluble
phase or S). After PI-PLC treatment, the membrane-anchored
prostasin is released into the soluble fraction from 293/Pro, PC-3/Pro,
and prostate tissues (post-PLC, S) but not from
DU-145/Pro and LNCaP (post-PLC, D). The PC-3/Vec
cells showed negative results in all fractions tested. Because of a
high level prostasin expression in the 293/Pro cells, a portion of
prostasin remained in the detergent phase after PI-PLC digestion.
Panel B, incorporation of [3H]ethanolamine
into prostasin. PC-3/Pro or LNCaP cells were incubated with 100 µCi
of [3H]ethanolamine in 1 ml of Opti-MEM I serum-free
medium for 24 h in 5% CO2 at 37 °C. The cell
lysates were subjected to immunoprecipitation using the prostasin
antibody (purified IgG fraction, 2 µg/ml) and protein A-Sepharose
beads as described under "Experimental Procedures." After SDS-PAGE
separation of the samples, the labeled protein was detected by
fluorography using Amplify fluorographic reagent and exposure to an
x-ray film at 80 °C with an intensifying screen for 14 days.
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Identification of a Prostasin-binding Protein--
An incubation
of the purified r-hPro with mouse or human seminal vesicle fluid
yielded a higher molecular weight form of prostasin-containing band as
analyzed by SDS-PAGE and immunoblotting. The result presented in Fig.
7A indicated that the purified
r-hPro formed an 82-kDa complex with a mouse seminal vesicle protein
(named as the mouse prostasin-binding protein, or mPBP). The complex
was apparently covalently linked and not via a disulfide bond because
it was SDS- and heat-stable and resistant to
-mercaptoethanol. The
complex formation was detected at as early as 1 min postincubation with a t1/2 of ~5 min and reached a plateau at
~20 min. Densitometry measurements of the complex bands in different
lanes were performed using the LabWork 3.0 software (Ultra-Violet
Products, Upland, CA) (data not shown). Mouse plasma and various tissue
extracts including the prostate, coagulating gland, testis, epididymis, vas deferens, adrenal gland, pituitary, thymus, liver, lung, kidney, spleen, heart, brain, uterus, pancreas, and salivary glands were subjected to the same prostasin-binding assay procedures. No SDS- and
heat-stable complex was detected under the same experimental conditions
(Fig. 7B). A PBP in human seminal vesicle fluid was also
identified. As shown in Fig. 7C, lane 2, a higher
molecular mass complex (82 kDa) was detected after an incubation of
r-hPro with the human seminal vesicle fluid. The complex formation
between prostasin and the human seminal vesicle PBP was inhibited by
heparin (Fig. 7C, lane 3). Incubation of mouse or
human plasma with r-hPro did not result in formation of any SDS- and
heat-stable complex (Fig. 7D, lane 1, mouse
plasma; lane 3, human plasma). In control assays, mouse
plasma was incubated with prostasin for 30 min before seminal vesicle
fluid was added for another 30 min of incubation to demonstrate the
binding activity of the prostasin being tested in the presence of
plasma (Fig. 7D, lane 2). Or, human plasma was
incubated with purified human tissue kallikrein and subjected to a
Western blot analysis using a human kallikrein-specific antibody (30)
(Fig. 7D, lane 4). The control binding assay
showed a 92-kDa kallikrein-kallistatin complex as described previously
(16), demonstrating the quality of the human plasma being tested.

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Fig. 7.
Complex formation between prostasin and its
binding protein. Panel A, 0.5 µg of purified
recombinant prostasin was incubated with mouse seminal vesicle fluid at
37 °C for various time periods as indicated. The samples were
subjected to SDS-PAGE under reducing conditions followed by
immunoblotting with a prostasin-specific antibody. Prostasin forms an
82-kDa complex (upper arrow) with mPBP in mouse seminal
vesicle fluid. The complex formation can be detected as early as 1 min
postincubation. Excess unbound prostasin is indicated by the
lower arrow. Prostasin alone without incubation with mouse
seminal vesicle fluid is labeled as 0 min. Panel
B, various mouse tissue extracts were analyzed in a prostasin
binding assay and prostasin immunoblotting as described.
SVF, mouse seminal vesicle fluid; Coag. gl.,
coagulating gland; Vas def., vas deferens; Adrenal
gl., adrenal gland; Salivary gl., salivary glands.
Panel C, 0.5 µg of purified recombinant prostasin was
incubated with human seminal vesicle fluid at 37 °C for 60 min. Similarly, prostasin forms an 82-kDa complex with the hPBP in
human seminal vesicle fluid (lane 2), and the complex
formation was inhibited by 1 unit of heparin (lane 3).
Purified recombinant prostasin alone was used as a control (lane
1). Panel D, human or mouse plasma was tested in a
prostasin binding assay. HUK, human urinary (tissue)
kallikrein (0.5 µg); Pro, purified recombinant
prostasin (0.5 µg); SVF, mouse seminal vesicle fluid (5 µl); MP, mouse plasma (1 µl); HP, human
plasma (1 µl). Lanes 1-3, probed with prostasin antibody
(1:1,000); lane 4, probed with a human kallikrein antibody
(1:1,000).
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The complex formation between prostasin and mPBP was investigated
further by incubating the purified r-hPro with serine protease inhibitors (Fig. 8, lanes
2-5) or the prostasin antibody (lanes 7 and
8) for 15 min at room temperature before an incubation with mouse seminal vesicle fluid for another 60 min at 37 °C. Or, mouse seminal vesicle fluid was first incubated with heparin before the
addition of prostasin (Fig. 8, lane 6). The complex
formation between prostasin and mPBP was inhibited by serine protease
inhibitors such as aprotinin at dosages of 1 µg/ml and 5 µg/ml,
phenylmethylsulfonyl fluoride at dosages of 1 mM and 5 mM, and prostasin antibody at 0.1 µl and 0.5 µl. The
amount of the complex was either reduced or absent in the corresponding
lanes of Fig. 8. Heparin (1 unit, lane 6) inhibited complex
formation. Complex formation between prostasin and mPBP without
additional reagents was used as the binding reaction control (Fig. 8,
lane 1). The results suggested that mPBP interacts with
prostasin at the serine active site and that heparin may alter mPBP
binding property. The properties displayed by mPBP are shared by the
serpin class serine protease inhibitors. We have observed similar
properties for the serpin, kallistatin (16, 17). The predicated
molecular mass of PBP (mouse or human) is estimated at ~47 kDa, given
the 40-kDa apparent molecular mass of prostasin and considering the
fact that serpin molecules lose a carboxyl-terminal fragment of ~5
kDa when complexed with a serine protease (31).

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Fig. 8.
Inhibition of complex formation between
prostasin and mPBP. 0.5 µg of Purified recombinant prostasin was
incubated with 5 µl of mouse seminal vesicle fluid in the presence of
aprotinin (lanes 2 and 3), phenylmethylsulfonyl
fluoride (PMSF; lanes 4 and 5), heparin
(lane 6), prostasin antibody (lanes 7 and
8) at 37 °C for 1 h. All samples were subjected to
SDS-PAGE under reducing conditions followed by immunoblotting with a
prostasin-specific antibody. The complex formation (upper
arrow) between prostasin and mPBP in mouse seminal vesicle fluid
is inhibited by aprotinin, phenylmethylsulfonyl fluoride, heparin, and
the antibody against prostasin. The asterisk (*) indicates
the IgG heavy chain and light chain recognized by the goat anti-rabbit
secondary antibody used in the Western blot analysis. Excess unbound
prostasin is indicated by the lower arrow. Complex formation
between r-hPro and mPBP in mouse seminal vesicle fluid without any
other reagent was used as positive control (lane 1).
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mPBP Inhibits the Serine Protease Activity of Prostasin--
We
performed a membrane overlay zymography analysis to test if mPBP
inhibits prostasin activity in vitro. The prostasin binding assay was carried out by incubating the purified r-hPro with mouse seminal vesicle fluid in the absence of the serine protease inhibitor aprotinin (Fig. 9, lane 3) or
in the presence of aprotinin (lane 4). Each sample was then
divided into two equal portions and subjected to a native PAGE analysis
(i.e., SDS and
-mercaptoethanol were not included in the
gel solution or the samples, and the samples were not heated before
loading) followed by membrane overlay zymography (left
panel) or Western blot analysis using the prostasin antibody (right panel). Mouse seminal vesicle fluid proteins alone
(Fig. 9, lane 1) displayed no enzymatic activities toward
the synthetic substrate D-Pro-Phe-Arg-AFC (left
panel) nor cross-reactivity with the prostasin antibody
(right panel). The purified r-hPro alone (lane 2)
demonstrated enzymatic activity toward D-Pro-Phe-Arg-AFC (left panel) and was recognized by the prostasin antibody
(right panel). When prostasin formed a complex with mPBP in
the mouse seminal vesicle fluid, it no longer cleaves
D-Pro-Phe-Arg-AFC because no fluorescence is present at the
complex band location in lane 3 of the left
panel, whereas the complex is identified by the prostasin antibody
as the upper band in lane 3 of the right panel. The remaining unbound prostasin yielded, expectedly, lesser fluorescence (left panel, lower band in
lane 3 compared with lane 2) and was recognized
by the prostasin antibody (right panel, lane 3).
When the purified r-hPro was preincubated with the serine protease
inhibitor aprotinin before incubation with the mouse seminal vesicle
fluid, no complex was detected (right panel, lane 4). A reduced level of fluorescence appeared at the prostasin band
in lane 4 of the left panel because of the
presence of aprotinin. Because the binding of aprotinin to prostasin
was reversible while proteins were being resolved in the gel, the
inhibition of prostasin activity seen in lane 4 was not
complete. The results suggested that mPBP not only binds to prostasin
at the serine active site but also inhibits the serine protease
activity of prostasin in vitro. Two bands were observed in
the prostasin alone sample in the immunoblot (Fig. 9, right
panel, lane 2), the differential mobility may be caused
by differential glycosylation (1). Aprotinin binding to prostasin
changes the charge/mass ratio of the protein; therefore, migration of
the aprotinin-bound prostasin in a native PAGE could change as well,
potentially causing the multiple banding pattern seen in lane
4 of the immunoblot (Fig. 9, right panel).

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Fig. 9.
Membrane overlay zymography. Samples
from a prostasin binding assay were resolved on a 10% native
acrylamide gel without SDS/boiling or -mercaptoethanol. The gel was
then either overlaid with a membrane impregnated with a prostasin
substrate (D-Pro-Phe-Arg-AFC) (left panel) or
transferred for prostasin immunoblotting (right panel).
Lane 1, 5 µl of mouse seminal vesicle fluid alone;
lane 2, 0.5 µg of purified r-hPro alone; lane
3, mixture of r-hPro and mouse seminal vesicle fluid; lane
4, same as lane 3 except r-hPro was preincubated with 5 µg/ml aprotinin for 15 min before the addition of mouse seminal
vesicle fluid. The fluorogenic substrate impregnated in the membrane
was hydrolyzed by prostasin in the gel, and isoprostasin patterns in
the membrane appear as fluorescent bands. The results suggest that mPBP
not only binds to prostasin at the serine active site but also inhibits
prostasin's serine protease activity in vitro.
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Membrane Prostasin Binds to mPBP--
To test if the
membrane-bound prostasin has binding activity toward mPBP, the 293/Pro
and PC-3/Pro cells were subjected to differential centrifugation as
described under "Experimental Procedures" except that no protease
inhibitors were added during membrane fractionation. Immediately after
centrifugation, an aliquot of each membrane fraction (30-40 µg of
total protein) was incubated with an aliquot of mouse seminal vesicle
fluid (5 µl) at 37 °C for 1 h. The binding mixture was then
analyzed by Western blot analysis using a prostasin-specific antibody.
In Fig. 10, the left panel
shows that the membrane-bound prostasin in 293/Pro cells (P2 and P3
fractions) formed an 82-kDa complex when incubated with mouse seminal
vesicle fluid. The prostasin protein in the crude nuclear fraction (P1)
and the cytosol (S) did not form any detectable complex. The purified
r-hPro from the conditioned medium (i.e. the secreted
prostasin) was used as positive control for the binding assay. In Fig.
10, right panel, we show that the membrane-bound prostasin
in PC-3/Pro cells also formed a complex with mPBP, and this reaction
was inhibited by the serine protease inhibitor aprotinin.

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Fig. 10.
Membrane-bound prostasin forms a complex
with mPBP. Membrane fractions of 293/Pro and PC-3/Pro cells
(30-40 µg of total protein) were incubated with 5 µl of mouse
seminal vesicle fluid at 37 °C for 1 h. Right panel,
samples where indicated, were preincubated with aprotinin before the
addition of mouse seminal vesicle fluid. All sample mixtures were
analyzed on an SDS-PAGE under reducing conditions followed by prostasin
immunoblotting. P1, nucleus fraction; P2, heavy membranes; P3, light
membranes; S, cytosolic proteins; and r-hPro, purified recombinant
prostasin. The membrane-bound prostasin in heavy or light membrane
fractions formed a complex (upper arrow) when incubated with
mouse seminal vesicle fluid, whereas prostasin in P1 and cytosolic
fractions showed no complex formation. r-hPro was used as a positive
control in the in vitro binding assay. Excess unbound
prostasin is indicated by the lower arrow. The addition of
aprotinin at 5 µg/ml inhibited complex formation as shown in the
right panel.
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DISCUSSION |
The prostasin serine protease is predominantly synthesized in the
prostate in human (1). Recently our laboratory demonstrated that
prostasin expression is significantly down-regulated in high grade
prostate tumors and absent in invasive human and mouse prostate cancer
cell lines. We have also shown, in an in vitro Matrigel invasion assay, that cellular prostasin may be an invasion suppressor of prostate cancer (20). In the present study, we intended to investigate the intracellular distribution of prostasin and to determine whether the cellular prostasin is an active serine protease, to provide clues to the potential mechanisms of prostasin's cellular function.
We first established a mammalian expression system to produce a
recombinant human prostasin. The purified secreted recombinant prostasin displayed biochemical characteristics similar to those of the
native prostasin (purified from human semen, Ref. 1), such as the
molecular mass on SDS-PAGE, immunological reactivity (Fig. 1),
enzymatic activity toward the synthetic substrate
D-Pro-Phe-Arg-AFC (Fig. 9), and responsiveness to serine
protease inhibitors (Figs. 8 and 9). We then used a polyclonal antibody
specific for human prostasin to determine whether prostasin can exist
in a membrane-bound form because its predicted structure suggested this
possibility (2). By means of sequential centrifugation of the 293/Pro
and PC-3/Pro cell components (as shown in Figs. 2 and 3A),
we were able to identify prostasin in various subcellular compartments such as the crude nuclei, heavy membranes (including mitochondria, lysosomes, and peroxisomes), and light membranes (including plasma membrane, microsomes, and endoplasmic reticula). A confocal microscopy analysis of the PC-3/Pro cells (Fig. 3B) revealed
prostasin's subcellular localization to be primarily at the
nuclear-ER-Golgi complex (27). The immunofluorescently localized
prostasin at the nuclear-ER-Golgi complex is believed to be that
identified in the Western blot analysis of the nuclear fraction P1. The
endogenously expressed prostasin in the LNCaP cells, however, was only
detected in the light membrane fraction P3 (Fig. 3A) not at
the nuclear-ER-Golgi complex (Fig. 3B). The different
subcellular localization of prostasin between the recombinant
expression system and the endogenous expression system may be caused by
expression level differences. Cells expressing recombinant prostasin
produced high amounts of prostasin with the 293/Pro being the highest
followed by PC-3/Pro and DU-145/Pro. The prostasin expression level in
the LNCaP cells was considerably lower than that in these transfected
cell lines. The expression levels were determined by a semiquantitative
Western blot analysis (data not shown). Alternatively, different cell
lines may have different protein sorting mechanisms, leading to
different subcellular localization patterns (32). On the other hand,
GPI-anchored prostasin might be associated with sterols and therefore
can be found in many compartments of the cell including the plasma
membrane, the Golgi apparatus, ER, nucleus, lysosomes and mitochondria, and in lipid particles (33). Prostasin in the nuclear fraction (P1) did
not show binding activity to PBP (Fig. 10). It is presently unclear why
prostasin in this fraction was unable to form a complex with mPBP. The
functional significance of prostasin in the nuclear-ER-Golgi complex is
also unclear at present and will be investigated in the future.
Despite the apparently different subcellular localization of prostasin
in overexpressing cells versus endogenously expressing cells, prostasin is found in an membrane-bound form in all cell lines
tested as well as in normal human prostate tissues (Fig. 6A). The membrane-bound prostasin was released when
extracted with a detergent but remained membrane-bound when treated
with high salt or alkali (Fig. 4), ruling out the possibility that prostasin is associated with another membrane-bound protein via noncovalent linkages. We also demonstrated that the membrane-bound and
the detergent-released prostasin have similar molecular mass (Fig. 4),
ruling out the possibility that prostasin is covalently linked to
another membrane-bound protein.
The native prostasin in normal prostate tissue and the recombinant
prostasin in 293/Pro and PC-3/Pro cells were easily released from the
membrane with PI-PLC treatment (Figs. 5 and 6A), suggesting that prostasin is bound to the membrane via a GPI anchor rather than
through a true transmembrane domain. The membrane-bound prostasin in
LNCaP cells (native) or DU-145/Pro cells (recombinant), however, was
resistant to PI-PLC treatment (Fig. 6A). As reported in
Englund (34) and Hiroshi et al. (35), not all GPI-anchored
proteins are susceptible to PI-PLC digestion. The membrane-anchored
prostasin in LNCaP and DU-145/Pro could potentially be susceptible to
other phospholipases such as GPI-phospholipase D (34, 35). Our results from the [3H]ethanolamine biosynthetic labeling
experiment with PC-3/Pro and LNCaP cells, however, provided direct
evidence that in the prostate epithelial cells recombinant or native
prostasin is GPI-anchored, regardless of its sensitivity to PI-PLC
treatment (Fig. 6B).
Among all four human cell lines that express either recombinant or
native prostasin, as well as normal human prostate tissue, prostasin
exists mainly as a membrane-bound protein (Fig. 6A). A small
portion of prostasin in the 293/Pro cells is in the cytosolic fraction.
This cytosolic prostasin could be a misprocessed or misfolded form that
was exported from the ER before GPI anchor attachment, a mechanism
documented previously (36). The presumably misfolded prostasin in the
cytosol had no binding activities when it was incubated with mouse
seminal vesicle fluid, possibly because of the misfolding. The secreted
recombinant prostasin, when purified from the 293/Pro culture medium,
however, is enzymatically active and able to form a complex with mPBP
(Figs. 9 and 10), indicating that the cytosolic prostasin is not the
source of secreted prostasin. The soluble fraction of prostasin seen in
the human prostate tissues (Fig. 6A) before PI-PLC treatment
may also be a misfolded form by the same mechanism described above or
may be attributed to residual prostatic fluid caused by possible
incomplete washing before tissue lysis.
We identified a PBP in mouse and human seminal vesicles (Fig. 7).
Prostasin forms an 82-kDa, SDS- and heat-stable complex when incubated
with seminal vesicle fluid as determined by SDS-PAGE under reducing
conditions followed by prostasin immunoblotting. This complex is
apparently covalently formed between prostasin and PBP and not via a
disulfide linkage. We have chosen to use mouse seminal vesicles for an
in-depth analysis of PBP because of easier availability. Complex
formation between prostasin and mPBP was inhibited by the polyclonal
prostasin antibody, heparin, and serine protease inhibitors. In a
membrane overlay zymography analysis (Fig. 9), the prostasin-mPBP
complex showed no activities to a synthetic substrate
D-Pro-Phe-Arg-AFC, whereas unbound prostasin was active.
These results suggest that mPBP may be a serpin class serine protease
inhibitor. The true nature of the mechanism of prostasin inhibition by
mPBP will be investigated upon purification and sequence analysis of
this protein. An incubation of mouse or human plasma with r-hPro did
not result in formation of any covalently-bound complex. This result
would rule out the possibility of mPBP being one of the known members
of the serpin family present normally in the blood, such as
1-antitrypsin,
1-antichymotrypsin, kallistatin, plasminogen activator inhibitor, and protein C
inhibitor. At present, the functional significance of PBP with respect
to prostate biology is unclear. Future studies will be aimed at
determining the prostasin binding site in PBP, which could potentially
reveal clues on prostasin's natural protein substrate.
One of our goals for the present study was to determine whether the
membrane-anchored prostasin is an active serine protease. To accomplish
this, we needed a prostasin-specific enzymatic activity assay that is
applicable for membrane-bound prostasin because this form of prostasin
exists in a complex mixture. The membrane overlay zymography assay was
not applicable for the membrane-anchored prostasin because
lipid-associated proteins cannot be well resolved in nondenaturing
native gel electrophoresis. The identification of mPBP offered us an
indirect but prostasin-specific assay to address this question. As
presented in Fig. 10, the membrane-bound human prostasin also displayed
binding activity to mPBP, and the binding is inhibited by a serine
protease inhibitor (aprotinin) that competes for the serine active
site, suggesting that the membrane-bound prostasin is likely an active
serine protease. Demonstration of membrane-bound prostasin being an
active serine protease will provide clues for investigating the signal
transduction pathway(s) involved in the anti-invasion activity of
prostasin because this anti-invasion activity is conferred by the
cellular prostasin but not the secreted prostasin (20).
Prostasin is made in the prostate and secreted as an active serine
protease (1), whereas PBP is made in the seminal vesicles. The fact
that prostasin forms a complex with PBP suggests that the two proteins
interact with each other when semen is ejaculated, thereby implicating
a role for both proteins in semen coagulation and liquefaction.
Prostasin and PBP in male reproductive tracts may serve together in a
partnership to affect fertility. Investigating the prostasin-PBP
partnership could also lead to a better understanding of the various
factors affecting fertility or, causing infertility. Overall,
prostasin, as a GPI-anchored or a secreted active serine protease, may
have multiple physiological functions, depending on the
localization of the prostasin protein, whether it is membrane-bound or secreted.