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INTRODUCTION |
The low density lipoprotein receptor-related protein-2/megalin
(LRP-2)1,2
has been shown to be widely expressed by cells lining the reproductive tracts of male and female rodents (1-3). For example, it is expressed by epithelial cells of the efferent ducts, epididymis, and vas deferens
of the male and by the germinal layer and epithelium of endometrium and
oviduct of the female (1-4). The function of LRP-2 in the reproductive
tracts is not yet known, but an endocytic function can be inferred from
its apical surface localization and presence within endocytic
compartments including coated pits, endocytic vesicles, and early
endosomes of efferent duct cells and principal cells of the epididymis
(2).
During transit through the epididymis, spermatozoa undergo a
maturational process that includes morphological and physiological changes (e.g. acquisition of sperm motility and alterations
in the properties of the plasma membrane). A widely held view is that
the secretory and absorptive activity of the epithelial cells lining
the efferent ducts, epididymis, and accessory glands (e.g. seminal vesicles) profoundly influences sperm maturation (5-7). Given
the prominent expression of LRP-2 by efferent duct and epididymal epithelial cells, it has been hypothesized that LRP-2-mediated catabolism of proteins present in the epididymal fluid and/or associated with spermatozoa surfaces may be a contributing factor to
spermatozoa maturation (2). Indeed, many LRP-2 ligands are found in the
epididymis, seminal vesicle, and seminal plasma such as apolipoprotein
J/clusterin (2), apolipoprotein B (8), apolipoprotein E (9),
tissue-type plasminogen activator, urokinase-type plasminogen
activator, and plasminogen activator inhibitor-I (10) and have been
speculated to be involved in sperm maturation and fertilization.
We report here that LRP-2 is also expressed by epithelial cells of the
seminal vesicles. In the rat, the seminal vesicles are two coiled
tubular glands connected by a short duct to an excretory tubule that is
anatomically equivalent to the ampulla of humans and other primates and
connected to the ejaculatory duct (11). The seminal vesicles produce
proteins that become covalently cross-linked by a transglutaminase and
form the fibrin-like seminal coagulum. In rodents, the seminal coagulum
forms the copulatory plug, whereas in humans, seminal coagulum is
degraded within the female reproductive tract by proteases of prostatic
origin, such as prostate-specific antigen (PSA), a member of the
kallikrein family of serine proteinases (12, 13). The major components of the seminal coagulum belong to a multigene family (14, 15) that
includes proteins such as rat seminal vesicle secretory proteins II,
IV, and V (SVS-II, IV, and V, respectively) (16, 17), mouse
seminoclotin (14), and human seminogelins I and II (18). In the
following study, we present evidence indicating that a major rodent
seminal vesicle secretory protein and transglutaminase substrate,
SVS-II, is a novel LRP-2 ligand.
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EXPERIMENTAL PROCEDURES |
Proteins--
LRP-2 was purified from porcine kidney using
RAP-Sepharose affinity chromatography as described previously (19).
Human 39-kDa receptor-associated protein (RAP) was expressed as a
glutathione S-transferase fusion protein in bacteria and was
purified according to Williams et al. (20). SVS-II was
purified from rat seminal vesicle as described by Wagner and Kistler
(16). Subfragments of rat SVS-II corresponding to the N-terminal domain
(from residues 25 to 107), 13-residue-repeat domain central domain
(from residues 108 to 311), and C-terminal domain (from residues 312 to
414) were expressed in bacteria as fusion proteins with GST using the pGEX2T vector (Amersham Pharmacia Biotech) using primers described in
Table I. Fusion proteins were purified by
glutathionine-Sepharose (Amersham Pharmacia Biotech) chromatography as
described by the manufacturer. Fibulin-1 was purified from human
placenta as described in Argraves et al. (21). LRP-2 and
SVS-II were labeled with [125I]iodine (Amersham Pharmacia
Biotech) using Iodogen (Pierce Chemical Co., Rockford, IL) to specific
activities of 7-8 and 2-10 µCi/µg, respectively (~0.222
MBq/µg and ~0.279 MBq/µg, respectively).
Antibodies--
LRP-2-Sepharose affinity chromatography was used
to isolate anti-LRP-2 IgG from a polyclonal serum (rb6286) derived from
a rabbit immunized with porcine kidney LRP-2 prepared as described previously (22). The bound IgGs were eluted using both high and low pH
buffers and dialyzed against TBS (150 mM NaCl, 50 mM Tris, pH 7.4). After dialysis, the IgG preparation was
applied to a column of RAP-Sepharose (1.0 mg of protein/ml of resin)
and the unbound IgG selected on protein G-Sepharose. Control IgG was purified from normal rabbit serum using protein G-Sepharose.
Mouse monoclonal antibody 3C3 to human LRP-2 was prepared by fusing NS1
myeloma cells with spleen cells from Balb/c mice immunized with human
LRP-2 purified from urine (23). Hybridomas were selected based on
production of immunoglobulin G antibodies reactive with both human and
rat LRP-2 by both enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
Isolation of Rat Seminal Vesicle Fluid--
Sexually
inactive, male Sprague-Dawley rats (age 6-8 months) were killed
by CO2 inhalation, and their seminal vesicles were removed.
The fluid contents of the seminal vesicles were released immediately
into an ice-cold solution of spermine and spermidine (Sigma) (each at 5 mM in water). The fluid-containing solution was centrifuged
for 10 min at 10,000 × g, and the supernatant was
recovered. Protein concentration in the clarified fluid was estimated
using a commercial protein assay system (Bio-Rad).
Cells--
Mouse embryonic F9 teratocarcinoma cells (ATCC CCL
185) were obtained from American Type Culture Collection and grown on
176-cm2 plates (Corning, Corning, NY) coated with 0.1%
gelatin (Sigma) in Dulbecco's modified Eagle's medium (DMEM, Life
Technologies, Inc.) with 10% bovine calf serum (HyClone Laboratories,
Logan, VT), penicillin, and streptomycin (Mediatech, Herndon, VA). To augment LRP-2 expression, the cells were treated with 0.1 µM RA (Calbiochem, San Diego, CA) and 0.2 µM Bt2cAMP (Sigma) for 6-7 days without a
change of medium (24).
Immunohistochemistry--
Isolated rat seminal vesicles were
dissected into four parts along their proximal to distal axis. Each
segment was placed in Bouin's fixative (EM, Diagnostic Systems,
Gibbstown, NJ) for 4 h at room temperature and then placed in 70%
ethanol for 18 h at 4 °C. The fixed tissue specimens were
dehydrated, embedded in paraffin, and sectioned at 5 µM
thickness. Immunohistochemical staining was carried out using a
Sequenza coverplate system (Shandon Lipshaw, Pittsburgh, PA) with
polyclonal LRP-2 IgG (rb6286) and normal rabbit IgG (each at 2 µg/ml). An Elite Vectastain ABC kit (Vector Laboratories, Burlingame,
CA) and the chromogenic substrate DAB (Sigma) were used for antibody
detection. Sections were counter-stained with hematoxylin (Shandon Lipshaw).
Gel-blot Overlay Assay--
Aliquots of rat seminal vesicle
fluid, rat SVS-II, GST-SVS-II fusion proteins, GST, RAP, or fibulin-1
were electrophoresed with fibulin-1 and RAP on 4-12% polyacrylamide
gels (Novex, San Diego, CA) in the presence of SDS and under
non-reducing conditions. The separated proteins were
electrophoretically transferred onto nitrocellulose membranes
(Schleicher and Schuell), and unoccupied sites were blocked with 3%
nonfat milk in TBS (pH 8.0) (blocking buffer). The membranes were
incubated with 125I-LRP-2 (1 nM) in blocking
buffer containing 5 mM CaCl2 and 0.05% Tween
20. To block the function of LRP-2, radiolabeled LRP-2 solution was
preincubated with RAP (1 µM) for 1 h at room
temperature prior to incubation with the membranes. Following the
incubation, the membranes were washed with TBS containing 0.05% Tween
20 and exposed to Kodak BioMax MR film (Eastman Kodak Company,
Rochester, NY) at room temperature.
For non-radioactive probing, filters were incubated with unlabeled
LRP-2 (1 nM) in blocking buffer containing 5 mM
CaCl2 and 0.05% Tween 20 for 3 h at room temperature.
Bound receptor was detected using mouse anti-LRP-2 IgG (1 µg/ml),
anti-mouse IgG-HRP, and the ECL Plus Western blotting detection system
(Amersham Pharmacia Biotech).
Protein Sequence Analysis--
Seminal vesicle fluid, isolated
in the presence of spermine and spermidine, was electrophoretically
separated on a 4-12% acrylamide gel in the presence of SDS, under
non-reducing conditions. The gel was stained with 0.2% Coomassie
Brilliant Blue R-250 (Bio-Rad) in 45% methanol, 10% acetic acid. The
polypeptide band having an Mr corresponding to
that identified in seminal fluid by gel-blot overlay assay using
radioiodinated LRP-2 as probe was excised. The gel slice was minced,
and the pieces were washed and destained in 50% methanol for 18 h. The gel pieces were dehydrated in acetonitrile and rehydrated in 10 mM dithiothreitol, 0.1 M ammonium bicarbonate and reduced at 55 °C for 1 h. The dithiothreitol solution was removed, and the sample was alkylated in 50 mM
iodoacetamide, 0.1 M ammonium bicarbonate for 1 h at
room temperature in the dark. The gel pieces were washed with 0.1 M ammonium bicarbonate and dehydrated in acetonitrile for 5 min. The acetonitrile was removed, and the gel slices were rehydrated
in 0.1 M ammonium bicarbonate. The pieces were again
dehydrated in acetonitrile and dried by vacuum centrifugation. The gel
pieces were rehydrated in a solution of trypsin (12.5 ng/µl) in 0.1 M ammonium bicarbonate and incubated on ice for 45 min. The
trypsin-containing solution was removed, 0.05 M ammonium
bicarbonate was added, and the slices were incubated for 18 h at
37 °C. The proteolytic fragments were then extracted using 50%
acetonitrile, 5% formic acid and dried to <20 µl. The sample was
then analyzed by capillary high performance liquid
chromatography-electrospray tandem mass spectrometry using a
Finningan-MAT TSQ7000 located in the W. M. Keck Biomedical Mass Spectrometry Laboratory (University of Virginia, Charlottesville, VA).
Solid Phase Binding Assay--
Both homologous and heterologous
ligand displacement assays were carried out as described previously
(25). Radioiodinated proteins (1-2 nM) in TBS, 3% bovine
serum albumin (U. S. Biochemical Corp.), 5 mM
CaCl2, 0.05% Tween 20 were incubated for 3 h at room temperature in breakaway microtiter wells (Dynatech, Chantilly, VA)
coated with LRP-2, SVS-II, or ovalbumin (each coated at 3 µg/ml) in
the presence of increasing concentrations of the competitor. The wells
were washed with TBS, 5 mM CaCl2, 0.05% Tween
20, and bound radioactivity was determined using a gamma counter. The computer program Ligand (26) was used to analyze the competition data
and to determine dissociation constants (Kd) for receptor-ligand interactions.
Assay For Cellular Internalization and Degradation of125I-SVS-II--
Ligand internalization and degradation by
LRP-2 expressing cells was performed according to previously published
methods (24, 27). Briefly, RA- and Bt2cAMP-treated F9 cells
were plated into 3.83-cm2 wells at 1.5-1.75 × 105 cells/well and allowed to grow for 18 h at
37 °C, 5% CO2. Prior to the addition of radioactive
ligands, the cells were washed with DMEM and incubated in the assay
medium (DMEM, 20 mM HEPES, 1% Nutridoma serum substitute
(Boehringer Mannheim), penicillin/streptomycin, and 1.2% bovine serum
albumin) containing either SVS-II (1 µM), RAP (1 µM) or IgGs (200 µg/ml) for 0.5 h at 37 °C, 5%
CO2. To inhibit the lysosomal protease activity, the cells
were pretreated with 0.1 mM chloroquine (Sigma) for
0.5 h at 37 °C, 5% CO2. The pre-incubation medium
was removed, and medium containing the radioiodinated proteins (1 nM) plus competitors was added and incubated for 5 h
at 37 °C, 5% CO2. The conditioned culture medium was
treated with trichloroacetic acid (final concentration 10%) and
centrifuged at 10,000 × g for 10 min. The amount of
radioactivity present in the supernatant was taken to represent the
amount of degraded SVS-II (28). The cell layer was washed three times
with cold (4 °C) Dulbecco's phosphate-buffered saline and then
treated with 0.5 mg/ml trypsin, 50 µg/ml proteinase K, and 0.5 mM EDTA (all from Sigma) in Dulbecco's phosphate-buffered
saline for 2-4 min at 4 °C. The released cells were pelleted by
centrifugation at 1,300 × g for 15 min, and the amount
of radioactivity in the cell pellet was measured.
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RESULTS |
Immunohistological Analysis Reveals that LRP-2 Is Expressed by
Seminal Vesicle Epithelial Cells Located Proximal to the Duct--
We
have recently found that LRP-2 was expressed on apical surfaces of
epithelial cells lining the efferent ducts and in the intermediate
zone, proximal head, corpus, and caudal regions of the epididymis (2).
In the present study, we have examined LRP-2 expression in the
accessory sex gland, the seminal vesicle. As shown in Fig.
1, using affinity-selected LRP-2
antibodies, pronounced immunoperoxidase staining was observed on the
apical regions of epithelial cells covering the duct of the gland. In addition, LRP-2 staining was also strong in the apical portion of the
epithelial cells lining the ampulla (Fig. 1C). This staining also appeared to occur in budding vesicles (Fig. 1C,
arrow). Very weak LRP-2 staining was seen in the underlying
stroma lying adjacent to the LRP-2-expressing epithelial cells.
Examination of distal segments of the gland showed negligible LRP-2
staining in both the epithelial cells and the underlying stromal cells
(data not shown). The results indicate that LRP-2 is expressed by
epithelial cells lining the duct of the seminal vesicles and may be
present within secreted vesicular particles.

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Fig. 1.
Immunohistochemical localization of LRP-2
within rat seminal vesicle epithelial cells located proximal to the
duct. Rat seminal vesicle (panels A-D) and rat kidney
tissue sections (panels E-F) were incubated with affinity
purified rabbit anti-LRP-2 IgG (panels A, C, and E) or
pre-immune rabbit IgG (panels B, D, and F) followed by goat
anti-rabbit-horseradish peroxidase and the chromogenic substrate DAB.
The tissue sections were counter-stained using hematoxylin.
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LRP-2 Binds to a 100-kDa Protein Present in Seminal Vesicle
Fluid--
The expression of LRP-2 in the seminal vesicle prompted us
to examine seminal vesicle fluid for the presence of LRP-2-binding proteins. Seminal vesicle fluid collected in the presence of the polyamines spermine and spermidine, to prevent coagulation, was electrophoretically separated by SDS-polyacrylamide gel electrophoresis (under non-reducing conditions) and transferred to nitrocellulose membranes. The membrane-bound proteins were probed using
125I-LRP-2. As shown in Fig.
2, the radiolabeled receptor bound to a
polypeptide with an Mr of 100,000 (Fig.
2A). Other minor LRP-2-binding bands were also apparent in
the profile. As a control, the 125I-LRP-2 was shown to bind
to a known LRP-2 ligand, the 39-kDa RAP (29-31). Fibulin-1, a
non-LRP-2-binding protein did not exhibit binding to the radiolabeled
receptor in this assay.

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Fig. 2.
Radiolabeled LRP-2 binds to a 100-kDa
polypeptide present in seminal vesicle fluid. Rat seminal vesicle
fluid proteins (7 µg; lane 1), human fibulin-1 (3 µg;
lane 2), and human RAP (1 µg; lane 3) were
electrophoretically separated on SDS-containing 4-12% polyacrylamide
gradient gels in the absence of reducing agent. Separated proteins were
either stained with Coomassie Blue (A) or transferred to
nitrocellulose membranes and probed with 125I-LRP-2 (1 nM; B) and 125I-LRP-2 plus RAP (1 µM), an antagonist of the ligand binding activity of
LRP-2 (C).
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To evaluate the specificity of the binding interaction between the
receptor and the 100-kDa protein, duplicate filters were incubated with
125I-LRP-2 in the presence of RAP, an antagonist of the
ligand binding activity of LRP-2 (19). As shown in Fig. 2C,
RAP completely blocked binding of radioiodinated LRP-2 to the 100-kDa
protein. The results indicate that seminal vesicle fluid contains a
100-kDa protein that binds to LRP-2. Consistent with all other LRP-2
ligands (19), the binding of the 100-kDa protein to LRP-2 was inhibited by RAP.
Identification of the 100-kDa LRP-2-binding Protein as
Seminal Vesicle Secretory Protein II by Protein Sequence
Analysis--
To identify the 100-kDa LRP-2-binding protein, amino
acid sequence analysis was performed. Initial attempts to sequence the N terminus of the LRP-2-binding protein using Edman degradation did not
yield a sequence. Assuming that the protein might be N-terminal blocked, it was subjected to limited proteolysis, and the resulting fragments were evaluated by liquid capillary mass spectrometry and high
performance liquid chromatography-tandem mass spectrometry. As a
result, sequences were obtained from 13 peptides (Table
II). Data base analysis of these
sequences revealed that eleven corresponded to sequences contained
within rat seminal vesicle secretory protein (SVS-II) (32). SVS-II is a
48-kDa seminal fluid protein that forms a 100-kDa disulfide-linked
homodimer (16, 17, 32). Sequences from two of the peptides were
unidentifiable. The results indicate that the 100-kDa-LRP-2-binding
protein most likely corresponds to SVS-II.
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Table II
Mass spectroscopic sequence analysis of trypsin-generated proteolytic
fragments of 100-kDa seminal vesicle fluid protein
X designates isoleucine or leucine which cannot be distinguished by low
energy collisionally activated dissociation; M(o) designates oxidized
methionine; lowercase letters designate tentative assignments; _
designates an unknown amino acid; - - - designates an unknown number
of unknown amino acids; b2 = refers to the mass (in Da) of the
first two amino acid residues; UI indicates that the peptide sequence
obtained did not correspond to any sequence in the database.
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SVS-II Binds with High Affinity to LRP-2 in Solid Phase
Assay--
To confirm that LRP-2 binds to SVS-II, solid phase binding
assays were performed using purified components. SVS-II was purified from rat seminal vesicle fluid according to previously published methods (16). As shown in Fig.
3A, 125I-LRP-2
bound to purified SVS-II immobilized on microtiter well plastic but not
to ovalbumin-coated wells. The binding to SVS-II could be inhibited by
incubation with unlabeled LRP-2. A dissociation constant
(Kd) of 10.4 ± 4.9 nM
(n = 6) was determined for the binding of radiolabeled
LRP-2 to SVS-II from the best fit of the data to a single class of
binding sites model. Similarly, 125I-SVS-II was shown to
bind to wells coated with LRP-2, but not to ovalbumin-coated wells
(Fig. 3B). A Kd of 5.8 ± 2.5 nM (n = 5) was determined for the binding
of SVS-II to immobilized LRP-2. The differences in the observed
Kd values derived from the two types of assays may
reflect conformational differences between the solution phase and
plastic-absorbed proteins. In addition, the LRP-2 antagonist RAP was
shown to competitively inhibit 125I-SVS-II binding to
LRP-2-coated wells (Fig. 3C), and an inhibition constant
(Ki) of 9.0 ± 2.5 nM
(n = 4) was determined. The findings indicate that
LRP-2 can bind to SVS-II with high affinity and that the interaction
can be blocked by RAP.

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Fig. 3.
Homologous and heterologous ligand
displacement assays demonstrate the ability of solution-phase and
immobilized LRP-2 to bind SVS-II. Panel A shows
125I-LRP-2 binding to purified SVS-II immobilized on
microtiter well plastic. The binding to SVS-II could be inhibited by
incubation with unlabeled LRP-2. A dissociation constant
(Kd) of 10.4 ± 4.9 nM
(n = 6) was determined after fitting the data using a
single class of binding sites model and the computer program Ligand.
Panel B shows 125I-SVS-II binding to wells
coated with LRP-2, and a Kd of 5.8 ± 2.5 nM (n = 5) was determined for the binding
interaction. In panel C, the LRP-2 antagonist RAP was used
to competitively inhibit 125I-SVS-II binding to
LRP-2-coated wells, and an inhibition constant (Ki)
of 9.0 ± 2.5 nM (n = 4) was
determined.
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Given that LRP-2 shares a number of ligands in common with LRP-1, we
were interested in evaluating the ability of LRP-1 to bind to SVS-II.
As shown in Fig. 4A,
125I-LRP-1 bound to purified SVS-II immobilized on
microtiter well plastic, and the binding could be inhibited by
incubation with solution phase SVS-II. Additionally,
125I-SVS-II was found to bind to wells coated with LRP-1
but not to ovalbumin-coated wells (Fig. 4B). A
Kd of 1.4 ± 1.0 nM
(n = 3) was determined for the binding of SVS-II to
immobilized LRP-1. RAP was found to also inhibit the binding of SVS-II
to immobilized LRP-1 (Ki = 4.4 ± 0.6 nM) (Fig. 4C). A Ki of
16.5 ± 3.5 nM (n = 2) was determined
for SVS-II inhibition of binding of radiolabeled LRP-1 to immobilized
SVS-II. The results indicate that LRP-1, like LRP-2 can bind to SVS-II
in vitro; however, given that LRP-1 was not immunologically
detected in the seminal vesicle (data not shown), the significance of
this interaction remains uncertain.

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Fig. 4.
Homologous and heterologous ligand
displacement assays demonstrate the ability of solution phase and
immobilized LRP-1 to bind SVS-II. Panel A shows
125I-LRP-1 binding to purified SVS-II immobilized on
microtiter-well plastic. The binding of LRP-1 to SVS-II could be
inhibited by incubation with unlabeled SVS-II. An inhibition constant
(Ki) of 16.5 ± 3.5 nM
(n = 2) was determined after fitting the data using a
single class of binding sites model and the computer program Ligand.
Panel B shows 125I-SVS-II binding to wells
coated with LRP-1, and a dissociation constant (Kd)
of 1.4 ± 1.0 nM (n = 3) was
determined for the binding interaction. In panel C, the
LRP-1 antagonist RAP was used to competitively inhibit
125I-SVS-II binding to LRP-1-coated wells, and an
inhibition constant (Ki) of 4.4 ± 0.6 nM (n = 2) was determined.
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LRP-2 Binds to a Site within the 13-Residue Repeat Region of
SVS-II--
To determine the binding site within the SVS-II protein, a
series of fragments of SVS-II were generated recombinantly and evaluated for their ability to promote LRP-2 binding in gel-blot overlay assays. The recombinant fragments corresponding to the 85-residue N-terminal domain, the 103-residue C-terminal domain, or the
204-residue 13-residue repeat domain of SVS-II (Fig.
5) were expressed as fusions with GST. As
shown in Fig. 6, LRP-2 bound the
204-residue 13-residue-repeat domain-containing fragment but not the N-
and C-terminal domain fragments. As controls, LRP-2 bound
filter-immobilized SVS-II and RAP, but not GST. LRP-2 binding to
SVS-II, the 204-residue 13-residue-repeat domain-containing fragment
and RAP was inhibited by RAP (Fig. 6, C).

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Fig. 5.
Schematic diagram depicting the domain
organization of rat SVS-II. Sizes of individual domains in amino
acid residues are indicated in parentheses. The locations of
oligonucleotide primers described in Table I for PCR amplification of
cDNAs encoding regions of SVS-II are indicated. Asterisk
indicates the position of the cysteine residue presumably involved in
disulfide bond-stabilized dimer formation.
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Fig. 6.
LRP-2 binds to a central 13-residue-repeat
domain within SVS-II. Shown in panel A is a
Coomassie-stained 4-12% polyacrylamide gradient gel containing
molecular weight standards, (lane 1), GST (1 µg;
lane 2), SVS-II N-terminal domain-GST fusion protein (1 µg; lane 3), SVS-II C-terminal domain-GST fusion protein
(1 µg; lane 4), SVS-II 13-residue-repeat domain-GST fusion
protein (1 µg; lane 5), rat SVS-II protein (1 µg;
lane 6), and RAP (1 µg; lane 8)
electrophoretically separated in the absence of reducing agent.
Panels B and C show autoradiographs of a membrane
containing proteins from duplicate gels that were transferred to PVDF
membranes and probed with LRP-2 (1 nM; panel B)
or LRP-2 plus RAP (1 µM) and detected with monoclonal
anti-LRP-2 antibody. Indicated on the left are molecular
mass values of the marker proteins in kDa.
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LRP-2 Mediates Cellular Uptake and Lysosomal Degradation of
SVS-II--
To determine whether LRP-2 functions to mediate
endocytosis leading to lysosomal degradation of SVS-II, we evaluated
the ability of LRP-2-expressing cells to internalize and degrade
radioiodinated SVS-II. As shown in Fig.
7, cultured F9 cells, treated to augment expression of LRP-2 (24, 27) mediated internalization and degradation
of 125I-SVS-II. Both processes could be inhibited by
polyclonal LRP-2 antibodies that have been previously shown to block
LRP-2 function (24, 27, 33). Control IgGs had no effect on
125I-SVS-II internalization and degradation. The magnitude
of the inhibition by LRP-2 antibodies (200 µg/ml) was nearly as great as that obtained using either 1000-fold molar excess of unlabeled SVS-II or RAP as competitors. The degradation of radiolabeled SVS-II
was inhibited by chloroquine (Fig. 7A), a drug that blocks lysosomal proteinase activity through its ability to increase the pH of
lysosomal vesicles. As expected, the chloroquine treatment resulted in
an intracellular accumulation of 125I-SVS-II (Fig.
7B). The findings indicate that LRP-2 can function to
mediate endocytosis and lysosomal degradation of
125I-SVS-II.

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Fig. 7.
LRP-2 antibodies and RAP inhibit endocytosis
and lysosomal degradation of 125I-SVS-II by
LRP-2-expressing cells. RA/Bt2cAMP-differentiated F9
cells were incubated with 125I-SVS-II (1 nM) in
the presence of unlabeled SVS-II (1 µM), RAP (1 µM), affinity purified anti-LRP-2 IgG (200 µg/ml),
control rabbit IgG (200 µg/ml), or chloroquine (0.1 mM)
for 5 h at 37 °C, 5% CO2. Shown are the amounts of
125I-SVS-II internalized (B) and degraded
(A) by the cells. Plotted values are means ± S.D. of
triplicate values and are representative of duplicate
experiments.
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DISCUSSION |
Evidence is presented in this study to indicate that LRP-2, a
multi-ligand endocytic receptor and a member of the LDL receptor family, is expressed by seminal vesicle epithelial cells and is potentially a component of seminal vesicle-secretory vesicles. In
addition, it was discovered that LRP-2 binds with high affinity (Kd = 5.6 nM) to SVS-II, a prominent
seminal vesicle-derived coagulatory protein. The LRP-2 binding site was
mapped to a 204-amino acid region of the protein containing a series of
13-residue repeats. As evidence that LRP-2 is also capable of mediating
cellular clearance of SVS-II, LRP-2-expressing cells were shown to
endocytose and degrade SVS-II. Both processes can be inhibited by
anti-LRP-2 IgG or RAP, a known antagonist of LRP-2 function (23, 24, 33, 34).
The physiological significance of the LRP-2 interaction with SVS-II is
not yet known. LRP-2-mediated endocytosis of SVS-II may function
in vivo to clear multimerized and/or
transglutaminase-cross-linked SVS-II, thereby preventing coagulation
within the male reproductive tract and the urinary tract which might
lead to an obstruction. Proteinaceous plugs have been reported in the
accessory sex glands and urinary bladders of male rats (35, 36). The
expression of LRP-2 in epithelial cells proximal to the ductal region
of the gland might be important to clear SVS-II that has become
cross-linked by retrograde flow of transglutaminase-containing fluids
derived from the coagulatory and prostate glands. LRP-2 that is
expressed by epithelial cells lining the female reproductive tract (1, 4, 37) might also mediate clearance of proteolytic fragments of SVS-II
generated during dissolution of the coagulatory plug. Indeed, following
copulation, seminal vesicle antigens have been detected within uterine
epithelial cells, indicative of endocytosis as a mechanism of their
elimination (38).
Immunohistological staining of the seminal vesicle not only showed
LRP-2 to be localized within the epithelial cells lining the ductal
region of the gland, but possibly in membrane bound vesicular bodies
found in the lumen of the gland. While LRP-2-expressing epithelial
cells lacking apparent vesicular budding were observed, numerous
regions of epithelium in the ductal area appeared to be active in
secretion of LRP-2-containing vesicular bodies. The pronounced LRP-2
staining of the budding vesicles in these areas may indicate that LRP-2
serves a role in vesicle secretion. Vesicular bodies have been observed
by electron microscopy in the lumen of the seminal vesicle and other
reproductive organs including the epididymis, vas deferens and ampulla
(39). In addition, such vesicular bodies have been isolated from
seminal vesicle (39-41), epididymal (42, 43), and prostatic fluids
(44, 45). Following our previous finding of LRP-2 expression by
epididymal epithelial cells (2), we isolated vesicular bodies from rat cauda epididymal fluid and found that they contained LRP-2 (unpublished observations, Ranganathan and Argraves). Vesicular bodies in seminal and epididymal fluids have been speculated to play a role in sperm maturation, perhaps involving proteolytic and glycolytic modification of spermatozoa membrane glycoproteins (46-48). It remains to be determined what role is served by LRP-2 associated with seminal and
epididymal vesicular bodies.