Post on 16-May-2023
Correspondence to: Lana Grauer, Hybritech Incorporated, P.O. Box
269006, San Diego, California 92196-9006.
Received for publication December 5, 1995; accepted for publication
February 29, 1996.
353
Journal of Anthology. Vol. 17, No. 4, July/August 1996
Copyright © American Society of Andrology
Identification of Human Glandular Kallikrein hK2From LNCaP Cells
LANA S. GRAUER, M. CRISTINE CHARLESWORTH, MOHAMMAD S. SAEDI, JUDITH A. FINLAY,
RU-SHYA LIU, KRISTINE KUUS-REICHEL, CHARLES Y.-F YOUNG, AND DONALD J. TINDALL
From Hybritech Incorporated, San Diego, California and Department of Urology, Mayo Clinic/Foundation,
Rochester, Minnesota.
ABSTRACT: Based on studies indicating that human glandular kal-likrein (hK2) mANA is present in the prostate, we prepared a mono-clonal antibody to a synthetic peptide corresponding to the 41-56region of hK2 to try to identify the hK2 protein. Although prostate-specific antigen (PSA) and hK2 share 80% homology, the 41-56
amino acid sequence of hK2 is only 50% homologous with PSA. Amonoclonal antibody, HK1A523, was identified that demonstrateshigh specificity for hK2. In western blot analysis, the antibody has a1 .000-fold greater sensitivity for the detection of hK2 than for PSA.
The antibody was used to probe spent media from the prostate car-
cinoma cell line, LNCaP. An irnmunoreactive species was N-termin-ally sequenced and identified as mature hK2. HK1 A523 was alsoutilized to probe prostate tumor cytosols and seminal fluid whereputative forms of hK2 were also identified. The hK2 protein thereforeis expressed and secreted from prostate carcinoma cells.
Key words: Prostate carcinoma, rnonoclonal antibody, seminalfluid.
J Androl 1996;17:353-359
In 1987 a new human glandular kallikrein gene was iso-
lated (Schedlich et al, 1987). The predicted proteinproduct of this gene was named human glandular kalli-
krein or hGK-1 and later renamed hK2 (Berg et al, 1992).
The glandular kallikreins are serine proteases believed to
have a role in activation of growth factors and peptidehormones (Murray et al, 1990). Members of the kallikrein
family include prostate-specific antigen, PSA (hK3)
(Wang et al, 1979), and pancreatic/renal tissue kallikrein
(hKl) (Clements, 1989). The hK2 transcript was identi-
fied in prostate epithelial cells by Chapdelaine et al (1988)
and Young et al (1992) by in situ hybridization. The ex-
pression of hK2 mRNA was reported to be restricted to
prostate tissue (Morris, 1989; Henttu et al, 1990). Al-
though the mRNA for hK2 has been isolated, the protein
has yet to be identified. Early efforts to identify the pro-
tein proved difficult due to its similarity to PSA (Paradis
et al, 1989). The protein sequence for hK2 was deduced
from the cDNA sequence and shown to share 80% se-
quence homology with PSA (Schedlich et al, 1987). One
region of sequence diversity occurred at amino acids 41-
56. In this stretch of 16 residues there were 8 amino acid
differences between the two proteins. In addition, PSA
contained a putative glycosylation site in this region atAsn-45 (Lundwall and Lilja, 1987) that was not present
in hK2.
We have developed a monoclonal antibody (mab),
HK 1A523, against the 41-56 region of hK2 that reactswith recombinant forms of hK2 and shows minimal cross-
reactivity to PSA. The mab was used as a probe to iden-
tify hK2 in LNCaP spent media. The immunoreactive
band was sequenced and identified as hK2. Prostate car-
cinoma cytosol and seminal fluid were also probed with
the hK2-specific antibody. Both of these sources con-
tained HK1A523 immunoreactive protein.
Materials and Methods
Peptide Synthesis
The peptide corresponding to amino acids 41-56 of the hK2
protein was synthesized using standard F-moc solid-phase chem-
istry on an Applied Biosystems 431A peptide synthesizer. Pep-tides were purified by reverse-phase high-performance liquid
chromatography (HPLC) and analyzed by mass spectrometryand analytical HPLC prior to immunization and assay develop-
ment.
Cloning and Expression of Recombinant Constructs
PreprohK2 (pphK2) was prepared in bacteria as explained in
Saedi et al (1995). The hK2 cDNA fragment encoding the entiresequence of pphK2 was cloned into a T7 expression vector underthe control of the T7 promoter. The plasmid (pBpphK2) was
transformed into Escherichia coli BL21, and pphK2 expressionwas induced by the addition of isopropyl-3-D-thiogalactopyra-
noside. Bacterial pellets were lysed and the inclusion bodieswere subjected to SDS-PAGE. The pphK2 was purified by gel
354 Journal of Andrology . July/August 1996
elution. The preproPSA (ppPSA) was generated in a similar
fashion.ProhK2 was purified from the AV12-664 hamster tumor cell
transfected with pphK2 cDNA, and mature hK2 was obtained
from the recombinant prohk2 preparation following mild tryp-sinization (Mikolajczyk, unpublished data).
Antibodies
Six-week-old A/J mice obtained from Jackson Laboratories (BarHarbor, Maine) were immunized intraperitoneally with 50 pg of
hK2 41 -56-KLH. After two injections of immunogen, positive
titers were obtained and mice were final boosted intravenously
with 25 ig of the same immunogen. Hybridomas were producedby fusion of mouse splenocytes with P3.653 myeloma cells
(Kohler, 1975) obtained from American Type Culture Collection
in Bethesda, Maryland. Monoclonal antibodies were selectedbased on reactivity with peptide as well as binding to recombi-
nant pphK2 and recombinant prohK2, and failure to react with
recombinant ppPSA and native PSA purified from seminal fluid.Monoclonal antibodies were screened by indirect enzyme-linked
immunosorbent assay (ELISA) on biotinylated protein captured
on avidin-coated microtiter plates and detected by horse radishperoxidase (HRP)-conjugated goat anti-mouse immunoglobulin,
followed by color development with o-phenyl-aminediamine
(Sigma, St. Louis, Missouri). Ascites was produced in nude mice
and the mabs were purified by protein A chromatography.
Affinity-purified goat anti-protein C inhibitor (PCI) was pur-
chased from Enzyme Research Labs (South Bend, Indiana). The
anti-PSA mab P5M773 was previously prepared from a mouse
immunized with purified PSA. The polyclonal rabbit anti-PSA
was prepared previously by immunizing a rabbit with purified
PSA. All horseradish-conjugated secondary antibodies were ob-
tained from Jackson ImmunoLabs (West Grove, Pennsylvania).
LNCaP Cell Line and Mibolerone Induction
The prostate carcinoma cell line, LNCaP.FGC was obtained fromthe American Type Culture Collection (Bethesda, Maryland).The cells were maintained in RPMI media with 10% horse serum
at 37#{176}Cand 5% CO2. After cells had reached 80% confluency,
they were incubated in serum-free media containing 10 nM mi-
bolerone (Amersham, Arlington Heights, Illinois) for 48 or 100
hours under the same growth conditions. Supernatants were har-vested, concentrated by Amicon ultrafiltration to 50X concen-
trates, and stored at - 20#{176}C.
Western Blot Analysis of hK2
Reduced and denatured proteins were electrophoresed on 4-20%
polyacrylamide gels and blotted onto nitrocellulose (Towbin et
al, 1979). Successful transfer was checked by staining blots with
Ponceau S protein stain. Blots were blocked in 5% nonfat dry
milk in phosphate-buffered saline (PBS) and then incubated withantibodies at 4#{176}Covernight. Following washes, secondary anti-
bodies conjugated to HRP were incubated with the blots for 1
hour and again washed with PBS. Antibody reactivity was de-
tected by the addition of 10 mg/mI diaminobenzidine, 0.1% hy-
drogen peroxide in PBS, or using the enhanced chemilumines-
cence (ECL) reagent (Amersham, Arlington Heights, Illinois) ac-
cording to manufacturer’s instructions. In experiments where
41 58
hK2 HCLKKNSQVWLGRHNL
PSA HCIRNK SVI LLGRHS L
FIG. 1. The amino acid sequences of hK2 and PSA at residues 41-56.
ECL detection was used, blots were incubated with antibodies
for 1 hour at room temperature and exposed to X-ray film, typ-
ically for 30 seconds.
Partial Purification of hK2 from LNCaP Supernatants
Concentrated spent media from mibolerone-stimulated LNCaP
cells was chromatographed by anion exchange and hydrophobic
interaction to yield a partially purified preparation of hK2 and
PSA. These proteins were separated and identified by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and western blot analysis with HK 1 A523 and PSA-specific rab-
bit polyclonal antisera (Towbin et al, 1979). The proteins were
electrophoresed and blotted onto high-density polyvinylidene di-
fluoride (PVDF) membranes. The hK2 and PSA bands were cut
out and sequenced on an Applied Biosystems (ABI) 492 Procise
Automated Sequencer using pulse liquid mode. Data analysis
was performed using the ABI 610A sequence analysis software.
Cytosol PreparationSpecimens of prostate carcinoma obtained at surgical resection
were immediately frozen and stored at - 80#{176}C.Frozen sections
of the specimens were examined microscopically and those spec-imens containing greater than 90% tumor cells were selected for
homogenization. The tissue was cut into small pieces and then
homogenized in 4 volumes of 0.1 M tris-HCI pH 7.5, 100 mM
NaCI, 2 mM CaCI, 1 mM Phenylmethyl sulfonyl fluoride using
a Dounce homogenizer. Nuclei and intact cells were removed by
centrifugation at 500 x g for 5 minutes. The supernatant was
centrifuged at 39,000 X g for 30 minutes at 4#{176}C.The supernatant
was removed and frozen and used as the cytosol source in further
experiments.
PSA Quantitation MethodPSA levels were determined by an immunoradiometric assay,
Tandem#{174}-R PSA (Hybritech, San Diego, California).
Results
The 4 1-56 amino acid sequences of hK2 and PSA contain
significant amino acid diversity (50%) (Fig. l).We syn-thesized this region of hK2 as an immunogen for the pro-
duction of hK2-specific antibodies. HK1A523 (IgG2a)was selected based on its strong reactivity with recom-
binant pphK2 and limited binding to recombinant ppPSA
in ELISA. HK1A523 was examined more extensively bywestern blot analysis for potential cross-reactivity to PSA
purified from seminal fluid and recombinant hK2. Incu-
bation of HK1A523 with increasing amounts of purified
recombinant hK2 demonstrated that the antibody can de-
tect as little as 15 ng of the recombinant (Fig. 2).
Grauer et al - Human Glandular Kallikrein 355
A. hK2
B. PSA
19 ng 78 ng 312 ng 1.25 .tg 5 g 20 p.g
33 kDa
20 kDa
15 kDa
33 kDa
20 kDa
19.5 kDa
FIG. 2. Reactivity of HK1A523 with hK2 and PSA on western blot. Increasing amounts of hK2 or PSA were electrophoresed under reducingconditions on SDS-PAGE on a 4-20% gel and blotted to nitrocellulose. The blots were reacted with 10 /rnl of HK1A523 followed by goat anti-mouse lgG-HRP and detection with diaminobenzidine. (A) hK2; (B) PSA.
1 2 3 4
356 Journal of Andrology . July/August 1996
98kDa
64
50
36
30
16
6
FIG. 3. Induction of hK2 with mibolerone in LNCaP cells. Media from LNCaP cells grown in the presence or absence of 10 nM mibolerone wasconcentrated 50x and electrophoresed under reducing conditions on SDS-PAGE 4-20% gels (30 il/lane). Following transfer to nitrocellulose, theblot was probed with HK1A523 (10 jig/mI), followed by goat anti-mouse lgG-HRP and detection with diaminobenzidine. Lane 1, molecular weightmarkers; lane 2, 50x LNCaP spent media; lane 3, 50X LNCaP media + 10 nM mibolerone, 48 hours; lane 4, 50x LNCaP media + 10 nM mibolerone,100 hours.
HK1A523 detects the predominant form of hK2 at 33
kDa. At higher levels of hK2, in addition to the 33-kDa
form, two smaller species at approximately 20 kDa and
15 kDa are also detected with the mab. In contrast, in-
cubation of HK 1 A523 with increasing amounts of PSA
demonstrated that the antibody detects PSA only at the
highest level of (20 rag). An anti-PSA mab PSM773 stains
all levels of PSA (data not shown). Therefore, HK1A523
has a 1,000-fold greater sensitivity for the detection of
hK2 than for PSA in this format. The predominant cross-
reactivity seen with HK1A523 at the highest level of PSA
(20 p.g) is not with intact PSA at 33 kDa but rather with
two smaller bands at approximately 20 kDa and 19.5 kDa.
These results suggest that HK 1A523 can be used to detect
hK2 specifically by western blot with minimal cross-reac-
tivity to PSA.
In order to determine if the hK2 protein is expressed
in LNCaP cells and regulated by the androgen, mibole-
rone, concentrated media from LNCaP cells grown in the
presence or absence of androgen was probed with
HK1A523 in western blots. HK1A523 reacts with a band
at 33 kDa in LNCaP spent growth media (Fig. 3). The
addition of 10 nM mibolerone to the growth media for
48 hours increases the expression of the protein approx-
imately twofold (lane 3) and incubation for 100 hours
increases expression levels approximately fivefold (lane
4). The control LNCaP media (50X concentrate) contains
7 ag/ml PSA or 140 hg PSA/lane and the 100-hour mi-
bolerone-induced LNCaP media (50X concentrate) con-
tains 33 p.g/ml PSA or 660 ng PSA/lane. Because the
amount of PSA loaded per lane is significantly below the
detection level for HK1A523 (i.e., 20 pjg), the 33-kDa
immunoreactive bands are deduced to be hK2.In order to confirm that the antibody-reactive band is
hK2, the protein was partially purified and sequenced.
Mibolerone-induced LNCaP spent media was concentrat-
ed and fractionated by anion exchange and hydrophobicinteraction chromatography. The immunoreactive en-
riched material was separated by gel electrophoresis and
blotted to PVDF membranes. The blot was stained with
Coomassie blue and probed with the anti-hK2 mab. The
Coomassie blue-stained band that comigrated with the
HK1A523 immunoreactive band was N-terminal se-
quenced through 20 residues and shown to be mature
hK2. A second band of slightly higher molecular weight
was N-terminal sequenced through 20 residues and iden-
tified as PSA.
Prostate carcinoma cytosols prepared from surgicalspecimens were examined for the presence of hK2.
HK 1 A523 reacts with three protein bands migrating at 62
1 2 3 4 5
Grauer et al - Human Glandular Kallikrein 357
98 kDa
64
50
36
30
16
6
FIG. 4. Reactivity of HK1A523 with prostate carcinoma cytosol. Prostate carcinoma cytosol (50 jig/lane) and PSA (1 jig/lane) were electrophoresedunder reducing conditions on an SDS-PAGE 4-20% gel. Following transfer to nitrocellulose, the blot was probed with HK1A523 or anti-PSA mabfollowed by goat anti-mouse lgG-HRP and ECL detection. Lane 1, molecular weight markers; lane 2, prostate carcinoma cytosol specimen #1, probedwith HK1A523 (10 jig/mI); lane 3, prostate carcinoma cytosol specimen #2, probed with HK1A523 (10 jig/mI); lane 4, 2 jig of PSA probed withHK1A523 (10 jig/mI); lane 5, 2 jig of PSA probed with anti-PSA mab (0.2 jig/mI).
kDa, 33 kDa, and approximately 20 kDa in both prostate
carcinoma cytosol preparations (Fig. 4, lanes 2 and 3). In
lane 2 the cytosol loaded onto the gel contains 880 ng of
PSA and lane 3 contains 280 ng of PSA. Lanes 4 and 5
are controls showing that 2 g of purified PSA cannot be
detected by HK1A523 (lane 4) while the anti-PSA mab,
PSM773 binds the 2 p.g of PSA (lane 5). These data sug-
gest that western blot detection is specific for hK2 and
that hK2 exists as multiple forms in prostate tissue.
Seminal fluid was also evaluated by western blot anal-
ysis with the anti-hK2 mab. HK1A523 reacts with four
bands in seminal fluid at 85 kDa, 33 kDa, and approxi-
mately 20 kDa and 15 kDa (Fig. 5, lane 2). An 85-kDa
band is also seen when seminal fluid is probed with an
anti-protein C inhibitor (PCI) mab (lane 3). Because both
antibodies bound an 85-kDa protein, this protein appears
to represent an hK2-PCI complex. The lower band (55
kDa) reacting with anti-PCI antibody in lane 3 most likely
represents free PCI in the seminal fluid. The 33-kDa band
in seminal fluid comigrates with recombinant mature hk2
(lane 4). Each lane of seminal fluid (50 p.g protein) con-
tains 2 g of PSA determined by the PSA-specific im-
munoassay. This level of PSA was previously shown to
be too low to be detected with the anti-hK2 mab (Fig. 4).
Discussion
This report describes the first identification of the hK2
protein in prostate cancer cells. The 33-kDa protein was
identified as hK2 by its reactivity with the anti-hK2-spe-
cific mab, HK1A523, followed by N-terminal sequencing
of the immunoreactive band blotted to PVDF membranes.Amino acids at positions 17 and 19 in hK2 are different
from corresponding residues in PSA and serve as a means
to distinguish hK2 from structurally similar PSA. The ex-
pression of the protein in LNCaP cells was up-regulated
by the synthetic androgen, mibolerone. This amplification
of the hK2 protein in LNCaP cells is consistent with pre-
viously reported increases in hK2 mRNA in LNCaP cells
following androgen treatment (Young et al, 1992).A high-affinity mab, HK1A523, was identified, which
exhibited a 1,000-fold increased sensitivity to hK2 than
to PSA by western blot analysis. The cross-reactivity seen
at high levels of PSA (20 p.g) may represent an hK2 con-
taminant in the PSA preparation, which copurified with
PSA. When recombinant ppPSA was examined under
identical conditions even at the highest levels of ppPSA
(20 rig), no cross-reactivity was detected (data not
shown). The high specificity of the antibody for hK2 and
2
i., -
358 Journal of Andrology . July/August 1996
98 kDa
64
50
36
30
16
6
1 2 3 4
FIG. 5. Reactivity of HK1 A523 with seminal fluid. Pooled seminal fluid(50 jig/lane) was electrophoresed under reducing conditions on an SDS-PAGE 4-20% gel. Following transfer to nitrocellulose, the blot wasprobed with HK1 A523 and anti-PCI antibody followed by appropriate sec-ondary antibodies labeled with HAP and ECL detection. Lane 1, molec-ular weight markers; lane 2, 50 jig seminal fluid, probed with HK1A523(10 jig/mI); lane 3, 50 jig seminal fluid, probed with anti-PCI (0.5 jig/mI);lane 4, 20 ng recombinant hK2, probed with HK1A523 (10 jig/mI).
minimal cross-reactivity with PSA made this antibody a
useful tool to probe prostate carcinoma tissue and seminal
fluid for the presence of hK2.
Multiple forms of hK2 were detected on western blots
of prostate carcinoma cytosols and seminal fluid with
HK1A523. In seminal fluid, a band at 85 kDa appeared
to be a complex of hK2 and protein C inhibitor (PCI)because antibodies to both proteins reacted with the same
molecular weight band on western blots. PCI is known to
exist in seminal fluid at high concentrations (220 mgfL)
(Laurell et al, 1992) and PSA has been shown to bind to
PCI and form a complex with a molecular weight of 90
kDa (Espana et al, 1991). Identification of hK2 in seminal
fluid by sequence analysis was recently reported (Deper-
thes et al, 1995). This group also described multiple
forms, including a 55-kDa complex of hK2 and a frag-
ment of PCI.
A 62-kDa immunoreactive form was detected in pros-
tate carcinoma cytosols, which may also represent a com-
plex of hK2 with an inhibitor. The exact nature of this
complex will require additional experimentation. Com-
plex formation between proteases and inhibitors often re-sults in loss of proteolytic activity and thereby serves as
a means of regulation. These high molecular weight forms
may serve to regulate the proteolytic activity of hK2.
Low molecular weight immunoreactive forms were
also detected in prostate carcinoma cytosols and seminal
fluid, which may represent breakdown products of the
mature protein. Immunoreactive bands were seen at ap-
proximately 20 kDa in prostate carcinoma cytosols and
at approximately 20 kDa and 14 kDa in seminal fluid.
The recombinant form of hK2 shown in Figure 2 also
contained two smaller molecular weight species at ap-
proximately 20 and 14 kDa. The exact nature of these
smaller forms will be determined by sequence analysis.
The physiological role of hK2 is currently unknown,
Due to its high sequence homology with PSA it may have
similar functions. It has been accepted that PSA facilitates
male fertility by aiding in the proteolytic liquefaction of
seminal coagulum (Lilja et al, 1987). Lilja (1985) and
Watt et al (1986) suggested that hK2 may in fact be the
protease responsible for the clipping and inactivation of
PSA and thereby serve as a regulator of PSA activity.
Final elucidation of the role of hK2 awaits additional in
vitro and in vivo studies.
The hK2-specific mab HK1A523 described in this re-
port has been used to identify hK2 in the spent media
from the prostate carcinoma cell line, LNCaP. N-terminal
sequence analysis of the immunoreactive band from
LNCaP cells confirmed its identity as mature hk2. Human
glandular kallikrein represents a potential new marker for
prostate cancer that may be useful in augmenting the clin-
ical information derived from PSA assays.
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