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The Prostate 39:28–35 (1999)
Characterization of the Enzymatic Activity of
PSM: Comparison With Brain NAALADase
Carol W. Tiffany, Rena G. Lapidus, Aviva Merion, David C. Calvin, and
Barbara S. Slusher*
Guilford Pharmaceuticals, Inc., Baltimore, Maryland
BACKGROUND. The prostate cancer marker prostate-specific membrane antigen (PSM) is
highly homologous to the brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is known to cleave terminal carboxy glutamates from both the
neuronal peptide N-acetylaspartylglutamate (NAAG) and folate polyglutamate. In this report, we compare the NAAG hydrolyzing activity of NAALADase and the prostate enzyme
PSM.
METHODS. Using a NAAG hydrolytic radioenzymatic assay, we compared the pharmacological and kinetic properties of the brain and prostate enzymes.
RESULTS. Eight normal prostate tissues from different species exhibited NAAG hydrolyzing
activity. Among 14 cancer cell lines examined, activity was observed in human LNCaP, PC-82,
and rat Dunning G and AT-1 cells. Brain exhibited membrane-localized activity exclusively,
while the prostate enzyme had activity in both membrane and cytosolic fractions. The only
observed pharmacological difference was the sensitivity to their putative substrates, folate
polyglutamate and NAAG. Kinetically, the soluble form of the prostate enzyme had two
catalytic sites, while the membrane-bound form exhibited single site kinetics with a lower
Vmax than the brain enzyme, which may suggest a less active hydrolase in the prostate.
CONCLUSIONS. The brain enzyme NAALADase and the prostate enzyme PSM are remarkably similar. The importance of the differences in substrate specificities and kinetic parameters remains to be elucidated. Prostate 39:28–35, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS:
Dunning; folic acid; LNCaP; prostate cancer
INTRODUCTION
Recently, rat brain N-acetylated-alpha-linked acidic
dipeptidase (NAALADase) was shown to have 86%
DNA sequence homology to the prostate cancer
marker, prostate-specific membrane antigen (PSM) [1]
(reviewed by Heston [2] and Fair et al. [3]). PSM was
cloned from the LNCaP prostate cancer cell line [4]. It
is a 110-kDa glycoprotein containing a short cytoplasmic region, a transmembrane domain, and large extracellular region. An alternatively spliced form of the
protein, termed PSM⬘, has been cloned from normal
prostate [5]. PSM⬘ lacks 266 nucleotides of the amino
terminus which comprises the ATG and the transmembrane domain, making it a cytosolic protein.
PSM⬘ mRNA is primarily expressed in normal prostate tissue, while PSM mRNA is more prevalent in
© 1999 Wiley-Liss, Inc.
prostate cancers. In fact, PSM/PSM⬘ mRNA ratios
have been reported to be 100 times greater in cancer
than in normal prostate tissues, suggesting that these
ratios may be utilized to indicate stage of disease [5].
Also, the presence of PSM mRNA, not PSM⬘, as determined by in situ hybridization, may be indicative of
hormone-refractory and metastatic disease [6]. PSM
has been reported to be measurable by RT-PCR and in
some cases by Western blot analysis in the serum of
patients with benign prostatic hyperplasia and prostate cancer [7,8].
Purified to homogeneity, rat brain NAALADase
*Correspondence to: Barbara S. Slusher, Guilford Pharmaceuticals,
Inc., 6611 Tributary St., Baltimore, MD 21224. E-mail: slusher b@
guilfordpharm.com
Received 28 May 1998; Accepted 20 August 1998
Characterization of PSM Enzyme Activity
has been extensively characterized and localized to
brain, kidney, and testes [9–11]. In the brain, NAALADase is known to hydrolyze the neuronal dipeptide
N-acetyl-aspartate-glutamate [NAAG]. The reaction
product, glutamate, is implicated in neuronal transmission [12,13]. Brain-localized NAALADase is a
membrane-bound, chloride-dependent zinc metalloproteinase which requires a divalent cation cofactor; it
is inhibited by ␮M amounts of quisqualic acid and
phosphate and mM amounts of EDTA [10].
PSM cDNA has been transiently transfected into
two PSM-negative human prostate cancer cell lines,
PC-3 and Du145. Cell lysates from the two transfected
cell lines and the PSM-positive LNCaP cell line hydrolytically liberate glutamate from NAAG, and their enzymatic activities appear to have similar sensitivities
to those of known NAALADase inhibitors [1]. More
recently, Pinto et al. [14] stably transfected PSM into
PC-3 cells and characterized it as a folate hydrolase
with high affinity for folate polyglutamate, cleaving
the glutamyl bond of the terminal carboxy glutamates.
In this report, we pharmacologically and kinetically
characterize the enzymatic activity of prostate PSM in
both mammalian tissues and prostate cancer cell lines
and compare it to rat brain NAALADase.
MATERIALS AND METHODS
Materials
The human prostate cancer cell lines, Du145, PC-3,
and NCI H660, were obtained from the American
Type Culture Collection (Rockville, MD). The human
prostate cancer line LNCaP was a gift from Dr.
William Nelson at Johns Hopkins Medical School (Baltimore, MD). The human prostate cancer cell lines
Tsu-PR1 and DuPRO, and the rat Dunning R2337
prostate cancer lines G, H, AT-1, AT-2, AT-3, MatLu,
and MatLyLu, were a kind gift of Dr. John Isaacs at the
Johns Hopkins Oncology Center (Baltimore, MD). The
PC-82 human prostate xenograft, also a kind gift of Dr.
Isaacs, is a serially transplanted human prostate xenograft derived from a primary human prostate carcinoma [15]. Interestingly, this tumor maintains many of
the important physiological properties (e.g., differentiation status and androgen sensitivity) of clinical
prostate cancer when serially transplanted in nude
mice. When passaged, the PC-82 tumor is assessed
histologically to ensure that no changes have occurred.
Cell growth media and reagents were purchased
from Paragon Biotech (Baltimore, MD). Frozen rat
brains as well as frozen rabbit, cat, and dog prostates
were purchased from Pell-Freeze (Rogers, AK). Frozen
guinea pig prostates were obtained from Rockland
29
(Gilbertsville, PA). Nude mice and Sprague Dawley
rats used for in-house prostate dissection were purchased from Harlan Sprague Dawley (Indianapolis,
IN). Human prostates collected postmortem from
three Caucasian men aged 89, 70, and 62 were prepared as membrane and soluble fractions by Analytical Biological Services, Inc. (Wilmington, DE). Tissues
were assayed separately and reported as mean ± SD.
The chemiluminescent detection agent ECL was
purchased from Pierce Chemical Co. (Rockford, IL),
and Kodak X-ray film was purchased from Amersham
Life Science, Inc. (Arlington Heights, IL). Radiolabelled NAAG was purchased from NEN™ Life Science Products (Boston, MA), and scintillation cocktail
UltimaGold was from Packard Instrument Co. (Meridan, CT). Baker Ultra-Pure water was obtained from
VWR Scientific Products (Bridgeport, NJ). Folate polyglutamates and methotrexate polyglutamates were
purchased from Schirck’s Laboratories (Jona, Switzerland). All other chemicals were purchased from
Sigma-Aldrich Corp. (St. Louis, MO).
Tissue Preparations
Prostate tissue. Tissue was prepared by homogenizing with a Polytron威 P10 (Brinkman, Westbury, NY) in
10 volumes (weight/volume) of ice-cold Baker UltraPure water, sonicating twice for 30 sec each with a
point sonicator, and passing the homogenate through
a 100-mesh sieve. The resultant homogeneous solution
was centrifuged at 50,000g for 20 min. Supernatant
was removed and adjusted to 50 mM TrisCl pH 7.4 at
37°C (soluble fraction). Pellets were homogenized and
sonicated in half the original water volume, using 50
mM TrisCl buffer (membrane). All fractions were aliquoted and stored at −80°C until assayed.
Rat brain. Synaptic membrane fractions from frozen
rat brains were prepared as previously described [10].
Briefly, brains were homogenized in 10 volumes
(weight/volume) of 0.32 M sucrose and centrifuged at
800g for 10 min. The supernatant was removed and
centrifuged at 20,000g for 20 min. The pellet was suspended in 10 volumes (weight/volume) of Baker UltraPure water using a Polytron and then centrifuged at
8,000g for 20 min. The supernatant and buffy coat
were removed and centrifuged at 35,000g for 10 min.
The resulting pellet was resuspended in 20 volumes of
50 mM TrisCl, pH 7.4, at 37°C, incubated for 30 min at
37°C, and then centrifuged at 35,000g for 10 min. After
washing the pellet in 20 ml buffer and centrifuging
twice more, the membranes were suspended in TrisCl
buffer to a final concentration of approximately 5 mg/
ml protein.
30
Tiffany et al.
Prostate cancer cell lines. Prostate cancer cell lines
were maintained in 95% air, 5% CO2 at 37°C in a humidified incubator in RPMI plus 10% heat-inactivated
fetal calf serum and 100 units of penicillin and 100
␮g/ml streptomycin. Dexamethasone (1 × 10−8 M)
was added to the growth media for the all the Dunning cell lines except G. Cells were grown to confluency in t-75 flasks, scraped into ice-cold phosphatebuffered saline (PBS), and centrifuged at 1000g to obtain a pellet. The cell pellets were sonicated in 0.5 ml of
50 mM TrisCl pH 7.4 and then centrifuged at 100,000g
for 10 min. Supernatants (soluble fraction) were removed to clean tubes, and pellets (membrane fraction)
were resonicated in 0.25 ml of TrisCl buffer. All
samples were stored at −80°C until use.
TABLE I. Enzyme Activity in Mammalian
Prostate Tissues*
Prostate
tissues
Specific activity (pmol/min/mg protein)
Mouse
Rat
Guinea pig
Cat
Rabbit
Dog
Human
Rat brain
Membrane
Soluble
1.87 ± .38
0.25 ± .04
4.01 ± 2.2
1.01 ± .30
0.66 ± .02
0.66 ± .16
6.15 ± 1.28
5.18 ± .21
0.38 ± .04
ND
0.53 ± .22
0.64 ± .30
0.54 ± .02
0.38 ± .05
3.16 ± .32
ND
*ND, not detectable.
Antibody Production
A polyclonal antibody, GP-02, was produced by
Covance Research Products (Denver, PA) against the
external amino acid sequence position 390–409,
SGAAVVHEIVRSFGTLKKEG [4]. The peptide was
synthesized by Princeton Biomolecules (Columbus,
OH), and 0.25 mg were injected subcutaneously dorsally into 2 New Zealand white female rabbits. The
animals were boosted with 0.25 mg or 0.4 mg of antigen every 3 weeks. Sera were collected 10 days postinjection and analyzed by Western blotting. Visualization of immunopositive bands from LNCaP and rat
brain lysates at the approximate size of previously
published reports of PSM/NAALADase was considered a positive response [8–10]. Production bleed and
exsanguination followed analysis of the sixth test
bleed.
Western Blot Analysis
Total cellular proteins (150 ␮g) were resolved on a
7.5% SDS-PAGE gel and proteins were electroblotted
to nitrocellulose membranes at 325 mA for 1 hr. Immunoblot analysis with the PSM antibody GP-02 was
performed using standard protocols. Briefly, the blot
was incubated overnight at 4°C in TBS-TM buffer (100
mM Tris, pH 7.6, 0.7 M NaCl, 1% Tween-20, and 10%
evaporated milk) and then for 2 hr with a 1:1,000 dilution of PSM Ab GP-02 in TBS-TM. The membrane
was washed six times for 5 min each in TBS-T (TBSTM without the milk) and then incubated for 1 hr with
a 1:25,000 dilution of a horseradish peroxidase goat
anti-rabbit antiserum in TBS-TM. The blot was
washed five more times, and then Western blot reactions were detected by a chemiluminescent-based
photoblot system.
Protein Assay
Proteins were assayed using the BioRad DC (BioRad Laboratories, Inc., Hercules, CA) procedure.
NAAG Hydrolyzing Assay
NAAG hydrolysis was performed essentially as described elsewhere [9], with minor modifications.
Briefly, 50 mM TrisCl, pH 7.4, at 37°C, 1 mM CoCl2,
and 10–100 ␮g of tissue protein were preincubated for
10 min at 37°C; 50 ␮l of 0.6 ␮M 3H-NAAG radiolabelled on the terminal glutamate were added to each
tube, and the incubation continued for 15 min. The
reaction was stopped with 1 ml of ice-cold 100 mM
NaPO4, and the cleaved glutamate was separated
from unreacted substrate by ion exchange chromatography. When the effect of CoCl2 or NaCl was tested,
the tissue was extracted and the reactions performed
in 50 mM Tris-acetate.
Kinetic Analysis
Enzyme kinetics were examined using hot saturation of substrate, with 3H-NAAG concentrations from
10 nM to 2 ␮M. Saturation isotherms and EadieHofstee graphs of kinetic data were generated in the
Prism™ program from GraphPad (San Diego, CA).
Statistical Analysis
Statistical analysis was performed using a one-way
ANOVA in the Origin graphics program from Microcal™ (Northampton, MA).
RESULTS
Prostate Tissues
As shown in Table I, human prostate and each animal prostate assayed had measurable amounts of
NAAG hydrolyzing activity. Significant amounts of
activity were observed in mouse, guinea pig, and human prostate tissue, with specific activities of 1.87,
Characterization of PSM Enzyme Activity
TABLE II. Enzyme Activity in Prostate Cancer
Cell Lines*
Specific activity (pmol/min/mg)
Prostate cell line
Human I
LNCaP
PC-82
DuPRO
NCI H660
Du145
PC-3
TsuPR1
Rat Dunning
G
H
At-1
At-2
At-3
MatLu
MatLyLu
Rat brain
Membrane
Soluble
12.5 ± 2.8
58.0 ± 5.2
ND
ND
ND
ND
ND
4.3 ± 1.3
21.9 ± 1.3
ND
ND
ND
ND
ND
0.11 ± .04
ND
0.19 ± .02
ND
ND
ND
ND
5.2 ± 0.2
ND
ND
ND
ND
ND
ND
ND
ND
*ND, not detectable.
4.01, and 6.15 pmol/min/mg, respectively, in the
membrane fraction, while cat, rabbit, dog, and rat had
activities of 1.0 pmol/min/mg or less. Only the human tissue displayed appreciable enzyme activity in
the soluble fraction, 3.16 pmol/min/mg, while the enzymes from the other species had activities less than
1.0 pmol/min/mg. Activity was undetectable in cytosol from rat prostate.
Prostate Cancer Cell Lines
Four of the 14 prostate cancer cell lines analyzed
displayed NAAG hydrolyzing activity (Table II). The
human cell line LNCaP and the PC-82 xenograft had
high specific activities in the membrane fractions (12.5
and 58.0 pmol/min/mg, respectively) and soluble
fractions (4.3 and 21.9 pmol/min/mg, respectively).
The human prostate cancer cell lines DuPRO, Du145,
PC-3, and Tsu-PR1 displayed no detectable enzymatic
activity in either the membrane or cytosolic fractions.
Rat Dunning G and AT-1 cells exhibited low activity
levels in the membrane fraction (0.11 and 0.19 pmol/
min/mg) and no detectable activity in the soluble fraction. Rat Dunning lines H, AT-2, AT-3, MatLu, and
MatLyLu had no detectable hydrolyzing activity.
31
brain and human prostate cancer cell lines. The tissues
and cell lines containing enzyme activity (i.e., rat
brain, LNCaP, and Dunning G and AT-1 cells) exhibited positive bands ranging from 90–98 kDa, as shown
in Figure 1. The human and rat cell lines which were
negative for enzymatic activity (i.e., DuPRO, Du145,
PC-3, Tsu-PR1, AT-2, AT-3, MatLu, and MatLyLu) had
no detectable immunopositive proteins in this range
as measured by Western blot analysis. Specifically, the
immunopositive protein in rat brain was 92 kDa, in
human prostate cancer 98 kDa, and in rat Dunning
prostate cancer 90 kDa. A separate immunoreactive
band at 105 kDa was apparent in three of the rat Dunning lines and is currently under investigation.
Pharmacological Characterization of PSM
Enzyme Activity
The PSM enzyme activity observed in prostate cancer cell lines had similar sensitivities to those of three
structurally distinct NAALADase inhibitors including
NaPO4, quisqualate, and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) [16]. Enzyme activity in PC82 tissue, Dunning G, AT-1, and LNCaP cells was inhibited by NaPO4, quisqualate, and 2-PMPA, with Ki
values ranging from 170–312 ␮M, 1.5–6.3 ␮M, and 0.6–
4.6 nM, respectively (Table III).
Because LNCaP cells exhibited significant enzyme
activity and were easily grown in cell culture, we used
LNCaP cells as a prototype to fully characterize the
enzymatic activity of the membrane and soluble forms
of PSM. A list of inhibitors and cofactors that were
evaluated is provided in Table IV. The prostate enzyme, like brain NAALADase, was found to be chloride- and cobalt-simulated (2-fold and 10-fold stimulation at 10 mM, respectively). Both tissue sources exhibited inhibition of enzyme activity by quisqualate
(Ki, 3–5 ␮M), NAAG-related peptides (Ki, 27–38 ␮M),
2-PMPA (Ki, 1–5 nM), and EDTA (Ki, 600–1,000 ␮M).
With regard to substrates, both rat brain NAALADase and the two forms of PSM showed nM sensitivities to methotrexate triglutamate, folate pentaglutamate, and NAAG; however, there were two statistically significant differences. First, the brain enzyme
was less sensitive to folate pentaglutamate as compared to the membrane-bound prostate enzyme (884
nM vs. 196 nM, P < 0.04) and the soluble prostate
enzyme (884 nM vs. 174 nM, P < 0.04). Secondly, the
membrane form of PSM was more sensitive than the
soluble form of the enzyme to NAAG (0.67 ␮M vs. 1.69
␮M, P < 0.02), although neither was statistically different from the brain enzyme (1.09 ␮M).
Western Blot Analysis
Kinetic Analysis
Immunopositive proteins were detected by antibody GP-02 on Western blot using extracts from rat
The kinetic analysis of LNCaP PSM enzyme activity
is shown in Figure 2. Rat brain enzyme displayed
32
Tiffany et al.
Fig. 1. Western blot analysis of PSM antibody GP-02 in rat brain and human and rat prostate cancer cell lines. The antibody recognized
immunoreactive proteins in rat brain (92 kDa), LNCaP cells (98 kDa), and rat Dunning G and AT-1 cells (90 kDa), consistent with the
molecular weight range previously reported for PSM/NAALADase. An unexplained 105-kDa immunoreactive positive protein was also
found in AT-1, MatLu, and MatLyLu and is currently under investigation.
TABLE III. Comparison of Enzyme Activity in Prostate Cancer Cell Lines and
Rat Brain
Kivalues
Tissue
source
Rat brain
LNCaP
Dunning G
Dunning AT-1
PC-82
Membrane
Membrane
soluble
Membrane
Membrane
Membrane
soluble
NaPO4
Quisqualate
2-PMPA
170 ␮M ± 12
253 ␮M ± 22
298 ␮M ± 35
210 ␮M ± 60
312 ␮M ± 54
175 ␮M ± 27
171 ␮M ± 12
6.3 ␮M ± 2.0
3.6 ␮M ± 1.4
5.8 ␮M ± 2.1
2.3 ␮M ± 1.0
1.7 ␮M ± 0.9
2.2 ␮M ± 0.9
1.5 ␮M ± 0.5
1.1 nM ± 0.2
1.9 nM ± 0.4
2.4 nM ± 0.6
3.7 nM ± 1.2
4.6 nM ± 2.0
1.0 nM ± 0.5
0.6 nM ± 0.2
single-site kinetics with a Km of 0.5 ␮M and Vmax at
200 pmol/min/mg (Fig 2A); these data are similar to
previously reported values [10]. LNCaP membrane
PSM also displayed single-site kinetics, with a Km of
0.2 ␮M and a Vmax of 80 pmol/min/mg (Fig. 2B). Interestingly, the soluble prostate enzyme exhibited two
enzyme sites with Km values of 0.2 ␮M and 1.2 ␮M
and Vmax values of 40 and 125 pmol/min/mg, respectively (Fig. 2C).
DISCUSSION
The prostate cancer marker prostate-specific membrane antigen (PSM) was recently cloned and reported
to have significant DNA homology (86%) to the rat
brain carboxypeptidase termed NAALADase [1]. NAALADase was identified over 15 years ago and has
been extensively characterized. It is a type II integral
membrane protein and a zinc metalloproteinase which
exhibits Cl− dependence, Co2+ stimulation, and inhibition by ␮M phosphate, ␮M quisqualate, and nM
2-PMPA. It is localized in the brain, kidney, prostate,
and brush border membrane of the intestine [9–11,13].
In this paper, we pharmacologically and kinetically
characterized the ability of the prostate enzyme to hydrolyze NAAG and compared this enzyme activity to
that of rat brain NAALADase.
Normal prostate tissues from eight mammalian
species examined displayed NAAG hydrolyzing in
both membrane and cytosolic fractions. The only exception was rat prostate, which exhibited no detectable activity in the cytosol. Of the malignant cell lines
examined, only the human LNCaP, PC-82, and rat
Dunning G and AT-1 cell lines had measurable enzyme activity; only the human lines possessed both
membrane and soluble forms of the enzyme. These
Characterization of PSM Enzyme Activity
33
TABLE IV. Pharmacological Comparison of Enzymatic Activity of LNCaP Cells and Rat Brain
Kivalues (␮M)
LNCaP
Inhibitors
Reducing agents
Dithiothreitol
Chelators
EDTA
EGTA
Membrane
Soluble
Rat brain membrane
>1,000
>1,000
>1,000
667 ± 110
847 ± 250
Peptides
NAAG
Asp-glu
Glu-glu
Glutamate
NAA
0.670 ± 0.110**
32.1 ± 11.0
38.6 ± 13.7
132 ± 33.0
>1,000
Chemotherapeutics
Methotrexate Triglutamate
Methotrexate
1,020 ± 150
872 ± 125
775 ± 264
411 ± 48.0
1.69 ± 0.360
27.0 ± 6.00
34.4 ± 7.30
132 ± 28.0
>1,000
1.09 ± 0.170
32.5 ± 9.0
22.0 ± 7.50
125 ± 63.0
>1,000
0.196 ± 0.059
30.3 ± 6.70
0.170 ± 0.008
38.2 ± 2.60
0.190 ± 0.046
42.0 ± 2.0
Folate derivatives
Folate pentaglutamate
Folic acid
0.196 ± 0.029*
22.7 ± 8.30
0.174 ± 0.007*
25.1 ± 4.50
0.884 ± 0.209
27.7 ± 4.70
Miscellaneous
Quisqualate
2-PMPA
3.60 ± 1.40
0.002 ± 0.0004
5.80 ± 2.10
0.002 ± 0.0006
5.50 ± 1.70
0.001 ± 0.0002
1
2.31 ± 0.4 stim@ 10 mM
13.2 ± 4.1 stim@ 10 mM
1.9 ± 0.2 stim@ 10 mM
8.9 ± 1.7 stim@ 10 mM
1.3 ± 0.2 stim@ 10 mM
3.0 ± 0.7 stim@ 10 mM
Activators
Metal ions
NaCl
CoCl2
*P 艋 .04 vs. rat brain.
**P = .02 vs. supernatant.
1 = stimulation of enzymatic activity.
data are in agreement with previously published reports that PSM is expressed in normal and prostate
cancer tissues [17]. Additionally, it has been reported
that increased PSM gene expression is observed in
prostate cancer [5,6]. We corroborate these results by
demonstrating an increase in enzymatic activity in
two human prostate cancer cell lines as compared to
normal human prostate tissues.
Using a newly developed antibody to PSM, GP-02,
we performed Western blot analysis on rat brain tissue
and prostate cancer cell lines. Consistent with the enzymatic activity reported, only rat brain, LNCaP, and
Dunning G and AT-1 cell lines displayed reactive
bands in the size range previously reported for PSM/
NAALADase [8–10,18]. Our antibody reacted with a
92-kDa rat brain protein, a 98-kDa species in the LNCaP cells, and a 90-kDa species in the rat Dunning cell
lines. The varying molecular size of PSM may be due
to different posttranslational modifications, as has
previously been described [18]. It has been reported
that the 750-amino-acid PSM protein before posttranslational modification is 84 kDa, but with the addition
of N-linked sugar complexes the molecular weight is
110 kDa [18]. A larger molecular weight protein that
was reactive to GP-02 was also identified in the Dunning AT-1, MatLu, and MatLyLu cell lines. These cell
lines did not have PSM enzyme activity but it is possible that they contain a nonfunctional form of the
enzyme or a protein highly homologous to PSM. This
is currently under investigation.
We used the LNCaP cell line to pharmacologically
characterize the prostate enzyme and compare it to rat
brain NAALADase. The two enzymes were found to
be remarkably similar in their sensitivities to metal
ions, EGTA, PO4, quisqualate, the potent NAALADase inhibitor 2-PMPA [16], and the chemotherapeutic agent methotrexate and its polyglutamated form,
methotrexate-triglutamate. The similarities observed
34
Tiffany et al.
Fig. 2. Saturation isotherm of NAAG hydrolyzing activity in (A)
rat brain membranes (Km, 0.5 µM; Vmax, 200 pmol/min/mg), (B)
LNCaP membranes (Km, 0.2 µM; Vmax, 80 pmol/min/mg), and (C)
LNCaP supernatants (Km1, 0.2 µM; Vmax1, 40 pmol/min/mg; Km2,
1.2 µM; Vmax2, 125 pmol/min/mg). Analysis was performed with
3
H-NAAG concentrations from 10 nM to 2 µM. Graphs are representative of independent experiments performed in duplicate.
are remarkable because the enzymes compared were
from two tissue sources (brain vs. prostate) and two
different species (human vs. rat). The two enzymes
were divergent only with regard to substrate specificity. LNCaP enzyme activity, both membrane and cytosolic, had a statistically significant higher affinity for
folate pentaglutamate than brain NAALADase. These
Ki values support the theory of different functional
roles of brain and prostate enzymes. Specifically, the
brain enzyme may be primarily NAAG-hydrolyzing,
while prostate PSM may hydrolyze folate polyglutamate as part of its functional role.
Pinto et al. [14] hypothesized that PSM carboxypeptidase activity is necessary for folate polyglutamate
hydrolysis. Since folic acid is necessary for cell growth
and polyglutamated folic acid is not transported into
the cell, a glutamate-hydrolyzing enzyme would be
necessary to internalize folic acid. Given that folic acid
metabolism plays a large role in cell division, folate
polyglutamate hydrolysis, and thus PSM enzymatic
action, may play an important role in the progression
of prostate cancer. However, the high expression of
functional PSM activity present in normal prostate tissue from postmortem subjects suggests that there is a
role for PSM enzymatic action in normal prostate tissue as well. It may be reasonable to propose that the
liberation of glutamate acts as a signal in prostate tissue metabolism, similar to the action of glutamate as a
transmitter in neuronal tissue. In fact, Heston et al.
recently reported the presence of ionotropic glutamate
receptors in prostate tissue [19].
Rat brain NAALADase also differs from PSM and
PSM⬘ in its enzyme kinetics. The rat brain enzyme
displayed single-site kinetics, with Km of 0.5 ␮M and
Vmax of 200 pmol/min/mg, as previously described
[10]. The prostate membrane PSM kinetics were also
single-site; the Km was 0.2 ␮M and the Vmax was 80
pmol/min/mg, possibly suggesting that the prostate
enzyme may be a less active hydrolase than the brain
enzyme. With regard to the prostate soluble enzyme,
PSM⬘, we report for the first time that the soluble enzyme had two catalytic sites. The first site was comparable to membrane PSM (Km = 0.2 ␮M and Vmax =
40 pmol/min/mg), while the second site appeared
more similar to the brain enzyme (Km = 1.2 ␮M and
Vmax = 125 pmol/min/mg). The significance of this
difference and the relationship, if any, between the
prostate-localized peptidase and brain NAALADase
remain to be discovered. It is also important to note
that the soluble form of the enzyme in LNCaP cells
displayed nearly identical Ki values to those of the
membrane enzyme in LNCaP cells. We believe it is the
cytosolic form of PSM or PSM⬘. However, this has not
been determined conclusively, and we admit the possibility that the activity may be due to an as-yet uncharacterized enzyme or variant of PSM.
It is noteworthy that other metalloproteinases, such
as angiotensin I converting enzyme (ACE) and neutral
endopeptidase (enkephalase), were originally thought
to be brain-specific and were later discovered to have
widespread tissue distribution. Both ACE [20] and
neutral endopeptidase (NEP) [21], like PSM, are abun-
Characterization of PSM Enzyme Activity
dant in prostate tissue. Interestingly, NEP activity is
associated with androgen-dependent prostate cancer
cell lines, and loss of NEP activity can be correlated
with both androgen-insensitivity and metastatic prostate cancer cells [22].
8.
CONCLUSIONS
PSM displayed NAAG hydrolyzing activity in all
normal prostate tissues examined and several prostate
cancer cell lines. Pharmacologically, PSM enzyme activity was remarkably similar to that of the rat brain
enzyme NAALADase. Ions, chelators, and known inhibitors and cofactors of brain NAALADase had similar effects on the prostate enzyme. Although the two
enzymes were found to be highly homologous, several
differences were discovered. First, unlike the brain enzyme which is exclusively present in the membrane
fraction, the prostate enzyme has both membrane and
cytosolic forms, both of which display functional enzyme activity. Second, the enzymes differ pharmacologically in regard to substrates. The prostate enzyme
exhibited a higher affinity for folate polyglutamate
than the brain enzyme, which may suggest unique
functional roles. Last, variations in kinetic patterns
were also noted. The brain enzyme had a lower Km for
NAAG and a higher Vmax than the prostate enzyme;
these may also relate to differences in their function.
The apparent role of the metalloproteinase PSM remains to be elucidated.
REFERENCES
1. Carter RF, Feldman AR, Coyle JT. Prostate-specific membrane
antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase. Proc Natl Acad Sci USA 1996;
93:749–753.
2. Heston WDW. Characterization and glutamyl preferring carboxypeptidase function of prostate-specific membrane antigen:
a novel folate hydrolase. Urology [Suppl]1997;49:104–112.
3. Fair WR, Israeli RS, Heston WDW. Prostate-specific membrane
antigen. Prostate 1997;32:140–148.
4. Israeli RS, Powell CT, Fair WR, Heston WDW. Molecular cloning of a complementary DNA encoding a prostate-specific
membrane antigen. Cancer Res 1993;53:227–230.
5. Su SL, Huang IP, Fair WR, Powell CT, Heston WDW. Alternatively spliced variants of prostate-specific membrane antigen
RNA; ratio of expression as potential measure of progress. Cancer Res 1995;55:1441–1443.
6. Kawakami M, Nakayama J. Enhanced expression of prostatespecific membrane antigen gene in prostate cancer a revealed by
in situ hybridization. Cancer Res 1997;57:2321–2324.
7. Loric S, Dumas F, Eschwege P, Blanchet P, Benoit G, Jardin A,
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
35
Lacour B. Enhanced detection of hematogenous circulating
prostate cells in patients with prostate adenocarcinoma by using
nested reverse transcription polymerase chain reaction assay
based on prostate-specific membrane antigen. Clin Chem 1995;
41:1698–1704.
Rochon YP, Horoszewicz JS, Boynton AL, Holmes EH, Barron
RJ III, Erickson SJ, Kenny GM, Murphy GP. Western blot assay
for prostate-specific membrane antigen in serum of prostate
cancer patients. Prostate 1994;25:219–223.
Slusher BS, Robinson MB, Tsai G, Simmons ML, Richards SS,
Coyle JT. Rat brain N-acetylated alpha-linked acidic dipeptidase
activity. J Biol Chem 1990;265:21297–21301.
Robinson MB, Blakely RD, Couto R, Coyle JT. Hydrolysis of the
brain dipeptide N-acetyl-aspartyl-L-glutamate. J Biol Chem
1987;262:14498–14506.
Slusher BS, Tsai G, Yoo G, Coyle JT. Immunocytochemical localization of the N-acetyl-aspartyl-glutamate hydrolyzing enzyme N-acetylated alpha-linked acidic dipeptidase [NAALADase]. J Comp Neurol 1992;315:217–229.
Hollmann M, Heinemann S. Cloned glutamate receptors. Annu
Rev Neurosci 1994;17:31–108.
Stauch BL, Robinson MB, Forloni G, Tsai G, Coyle JT. The effects
of N-acetylated alpha-linked acidic dipeptidase [NAALADase]
inhibitors on [3H]NAAG catabolism in vivo. Neurosci Lett 1989;
100:295–300.
Pinto JT, Suffoletto BP, Berzin TM, Qiao CH, Lin S, Tong WP,
May F, Mukherjee B, Heston WDW. Prostate-specific membrane
antigen: a novel folate hydrolase in human prostatic carcinoma
cells. Clin Cancer Res 1996;2:1445–1451.
Hoehn W, Schroeder FH, Riemann JF, Joebsis AC, Hermanek P.
Human prostatic adenocarcinoma: some characteristics of a serially transplantable line in nude mice (PC-82). Prostate 1980;1:
95–104.
Jackson PF, Cole DK, Slusher BS, Stetz SL, Ross LE, Donzanti
BA, Trainor DA. Design, synthesis, and biological activity of a
potent inhibitor of the neuropeptidase N-acetylated ␣-linked
acidic dipeptidase. J Med Chem 1996;39:619–622.
Silver DA, Pellicer I, Fair WR, Heston WDW, Cordon-Cardo C.
Prostate-specific membrane antigen expression in normal and
malignant tissues. Clin Cancer Res 1997;3:81–85.
Holmes EH, Greene TG, Tino WT, Boynton AL, Aldape HC,
Misrock SL, Murphy GP. Analysis of glycosylation of prostate
specific membrane antigen derived from LNCaP cells, prostatic
carcinoma tumors, and serum from prostate cancer patients.
Prostate [Suppl] 1996;7:25–29.
Heston WDW, Silver DA, Pellicer I, Fair WR, Cordon-Cardo C.
Ionotrophic glutamate receptor distribution in prostate tissue. J
Urol 1996;155:513.
Corvol P, Michaud A, Soubrier F, Williams TA. Recent advances
in knowledge of the structure and function of the angiotensin I
converting enzyme. J Hypertens 1995;13:53–60.
Krongrad A, Atochina E, Ryan JW, Roos BA. Endopeptidase
24.11 activity in the human prostate cancer cell lines LNCaP and
PPC-1. Urol Res 1997;25:113–116.
Papandreou CN, Usmani B, Geng Y, Bogenreider T, Freeman R,
Wilk S, Finstad CL, Reuter VE, Powell CT, Scheinberg D, Magill
C, Scher HI, Albino AP, Nanus DM. Neutral endopeptidase
24.11 loss in metastatic human prostate cancer contributes to
androgen-independent progression. Nat Med 1998;4:50–57.
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