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J. Biol. Chem., Vol. 277, Issue 47, 45400-45407, November 22, 2002
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From the Laboratory of Retinal Cell and Molecular Biology, NEI,
National Institutes of Health, Bethesda, Maryland 20892-2740
Received for publication, August 14, 2002, and in revised form, September 10, 2002
Pigment epithelium-derived factor (PEDF), a
neurotrophic and antiangiogenic serpin, is identified in tissues rich
in collagen, e.g. cornea, vitreous, bone, and cartilage. We
show that recombinant human PEDF formed complexes with collagens from
the bovine cornea and vitreous. We have examined the direct binding of
PEDF to collagen I and found that interactions were ionic in nature and
occurred when PEDF and collagen I were both in solution, when either
one was immobilized, or even when collagen I was denatured under
reducing conditions. 125I-PEDF bound to immobilized
collagen I in a saturable fashion (KD = 123 nM). Compared with neurotrophic PEDF-derived peptides,
ovalbumin and angiogenic inhibitors, only full-length PEDF competed
efficiently with 125I-PEDF for the binding to immobilized
collagen I (EC50 = 3 µg/ml). The collagen-binding region
was analyzed using controlled proteolysis and chemically modified PEDF.
Cleavage of the serpin exposed loop did not prevent binding to collagen
I. Conjugation of lysines with fluorescein increased the collagen
binding affinity. However, treatment of PEDF with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide abolished it, implicating
the PEDF aspartic and/or glutamic acid residues in its interaction with
collagen I. A negatively charged region on the surface of the PEDF
molecule is rich in acidic residues (Glu41,
Glu42, Glu43, Asp44,
Asp64, Asp256, Asp258,
Glu290, Glu291, Glu296,
Asp300, Glu304) available to interact directly
with positively charged areas of collagen. This represents the first
collagen-binding site described for a serpin, which in PEDF, is
distinct from its heparin-binding region, neurotrophic active site, and
its serpin exposed loop. The collagen-binding property of PEDF may play
a role in surface localization and modulation of its antiangiogenic
effects in the eye and bone.
Pigment epithelium-derived factor
(PEDF)1 is an extracellular
protein that has neurotrophic and antiangiogenic activities expressed mainly in compartments of the eye. It acts in neuronal survival and
differentiation on photoreceptor cells and on neuronal cells of the
retina and central nervous system (1-5). Several reports show that
PEDF is a major inhibitor of neovascularization and is responsible for
excluding vessels from invading the cornea, vitreous, and retina
(6-8). These biological activities are of great importance for the
development, morphology, and vision process in the eye.
PEDF is highly secreted by a variety of cells and associates intimately
with extracellular matrix (5, 9-12). In particular, cells of the
retinal pigment epithelium secrete PEDF protein, which associates with
the interphotoreceptor matrix consistent with its affinity for binding
glycosaminoglycans, e.g. heparin and heparan sulfate (13,
14). Other areas in the eye that contain PEDF include the vitreous gel
and the cornea. In the bovine vitreous, PEDF accumulates at 20 nM accounting for <1% of the total protein of cell-free
extracts (15). In the human cornea, PEDF immunolabeling has been
detected in the epithelium and endothelium (16). Outside of the eye,
PEDF has been detected in teeth, bone, and cartilage matrix (10). It is
worth noting that in addition to containing PEDF and being rich in
collagen, the adult vitreous, cornea, and bone and cartilage matrixes
are vessel-free.
Collagens comprise a family of 19 proteins with a broad range of
structural and physiological functions, which are strictly located in
the extracellular space. They are composed of three chains that fold to
form at least one triple helical domain. All collagens are present in
tissues as homotrimeric and/or heterotrimeric assemblies and have
specific tissue distributions and functions, e.g. they are
involved in hemostasis, wound healing, cell adhesion, and migration. In
the cornea and vitreous they are known to give transparency, strength,
and elasticity. Collagen type I is the major protein of the cornea
(94% of the total collagen in the bovine cornea) and coassembles in
heterotypic fibrils with collagen type V. Collagen type II is the main
protein of the vitreous (70-80%) followed by collagens type V/XI and
IX (17).
Studies on structure-function relationships have revealed that
PEDF is a glycoprotein of 50 kDa that folds like members of the
superfamily of serine protease inhibitors (serpins) (13, 18, 19), but
its neurotrophic and antiangiogenic activities are mediated by pathways
independent of its serine protease inhibition potential (1, 4, 7, 19).
The molecular mechanisms of action are better understood for its
neurotrophic activities than for its antiangiogenic properties. Upon
interactions with receptors on the surface of cells, PEDF can activate
the necessary signal transduction events for neurotrophic activities
(20-22). The binding sites for receptors are distinct and
non-overlapping from those for glycosaminoglycans, with affinities for
PEDF >1000-fold different (KD ~3 nM
for the receptor; KD ~4 µM for
heparin) and locations in the folded PEDF protein molecule separated
about 180° (12, 14, 20, 21). The receptors interact with a
neurotrophic region spanning amino acid positions 78-121 in the human
PEDF (termed 44-mer), while heparin binds to a region rich in basic amino acids (lysines and arginines) located opposite to the 44-mer. Both of these binding sites are distinct and away from the homologous serpin reactive loop.
Given the antiangiogenic activities of PEDF and colocalization
with collagen in extracellular matrix, it was of interest to study the
binding between these two proteins for structure-function studies. We
have examined the interactions of PEDF with collagens present in bovine
eyes and developed assays for PEDF-collagen binding. Using recombinant
human PEDF polypeptides, synthetic PEDF peptides, and three
different chemically modified PEDF versions, we have analyzed the
binding site of PEDF to collagen I. We discuss how these data may aid
in further understanding the antiangiogenic functions of PEDF.
Materials and Reagents--
PEDF was purified from the culturing
media of baby hamster kidney cells containing an expression vector
for human PEDF as described (12). 125I-PEDF and Fl-PEDF
were prepared as described previously (14, 21). Collagen type I
purified from rat tail, collagen type III from human placenta, and
24-well plates coated with collagen I were all purchased from BD
Biosciences. Collagen type II purified from chicken sternal
cartilage, monoclonal antibodies to bovine skin collagen type I (mouse
IgG1 isotype, clone COL-1), pig collagen type II (mouse IgM isotype,
COL-2) and human collagen type III (mouse IgG1 isotype, FH-7A), and
heparin immobilized on acrylic beads were obtained from Sigma. The
monoclonal antibody to PEDF, anti-PEDF, was obtained from Chemicon.
Rabbit polyclonal antiserum to fluorescein (IgG fraction), anti-Fl, was
from Molecular Probes. Protease inhibitor tablets and
Lumi-LightPLUS Western blotting kit were from Roche
Molecular Biochemicals. Vectastin ABC elite kit for immunoreactions was
obtained from Vector Laboratories. Centricon-100 and centricon-30
devices were obtained from Millipore. PBS was prepared from 10×
phosphate-buffered saline, pH 7.4, from Invitrogen (catalog
number 70011-044). 1-Ethyl-3-(3-dimethylaminopropyl)carbomiide HCl
(EDC) and BupH MES-buffered saline were from Pierce. Ethylenediamine was from Acros, and ethanolamine was from J. T. Baker Inc.
Preparation of Cornea and Vitreous Extracts--
Bovine eyes
were purchased from J. W. Trueth & Sons, Baltimore, MD. After
dissection of the eye, the corneas were removed, suspended in a
solution of cold 20 mM HEPES, pH 7, 100 mM KCl, 1 mM EDTA containing protease inhibitors at 10 ml per cornea, and homogenized with a Polytron (Brinkmann PT
3000) set at 10,000 rpm for 20 s. The homogenized material was
separated from tissue and cellular debris by centrifugation
at 1000 × g for 10 min at 4 °C and then subjected
to ultracentrifugation at 80,000 × g for 30 min at
4 °C. The supernatant termed corneal extract was stored at
Solution Binding Assays--
These assays were based on the
separation of complexes formed between PEDF and other proteins by
ultrafiltration in which PEDF complexes >100-kDa are retained by a
membrane of Mr 100,000 exclusion limit, while
free PEDF molecules of 50 kDa are filtered through (14). PEDF proteins
(100 µg/ml, unless indicated) were mixed with either ocular extracts
or collagen (100 µg/ml, unless indicated) in PBS, pH 7.4, containing
10% glycerol and incubated at 4 °C for 1 h. Because stock
solutions of collagen II and III contained 10 mM acetic
acid, NaOH was added to neutralize the pH of the reaction mixtures.
PEDF complexes were separated from free PEDF with Centricon-100 devices
centrifuged at 5000 rpm for 40 min followed by three washes with 10%
glycerol/PBS. The retained material was resolved by SDS-PAGE and
visualized by Coommassie Blue staining or immunostaining as described
below. To quantify the binding, the intensity of PEDF bands was
quantified using NIH Image. The binding data were analyzed using Prism
GraphPad software.
Ligand Affinity Chromatography--
PEDF affinity resin was
prepared as described previously (20). A total of 50 µl of PEDF
affinity beads and 100 µg/ml protein from each of collagen I,
corneal, and vitreal extracts was mixed in an 1.5-ml tube with gentle
rotation at 4 °C for 1 h. Bound and unbound material was
separated by centrifugation at 1600 × g for 10 min at
4 °C. The beads were washed three times with 300 µl of 10%
glycerol/PBS. SDS-PAGE sample buffer (50 µl) was added to the beads,
mixed, heated at 100 °C, centrifuged, and the supernatant was
resolved by SDS-PAGE.
Ligand Blotting--
Protein samples were resolved by SDS-PAGE
and transferred to nitrocellulose membranes. The membranes were washed
with 1% Nonidet P-40 in Tris-buffered saline (TBS: 20 mM
Tris/HCl, pH 7.5, 150 mM NaCl) for 15 min at room
temperature with gentle rocking and washed twice with TBS for 10 min.
The membranes were blocked with Blocking Solution
(Lumi-LightPLUS kit) for 1 h at room temperature with
gentle rocking before incubation with 2 nM Fl-PEDF in
Blocking Solution for 2 h at room temperature with gentle
rocking. The membranes were rinsed and washed four times for 2 min each
with TBST (TBS with 0.05% Tween 20). Bound Fl-PEDF was detected by
immunostaining with anti-fluorescein as described below for Western
blot with Lumi-lightPLUS.
Solid-phase Binding Assays--
Binding reactions were performed
with 125I-PEDF (2 nM, unless indicated) in
0.1% BSA/PBS, pH 7.4, to collagen I immobilized on plastic of 24-well
plates. After incubations at 4 °C for 90 min with gentle rocking,
the binding solution was removed, and the wells were washed three times
with 0.1% BSA/PBS. Then 1 N NaOH was added to the wells,
incubated at room temperature for 30 min, and transferred to
scintillation vials to determine the amount of radioactivity using a
Surface Plasmon Resonance Assays--
Assays for PEDF-collagen I
interactions were performed immobilizing either 4 ng of collagen I or
PEDF on a CM5 sensor chip, by N-hydroxysuccinimide (NHS)/EDC
activation, followed by covalent amine coupling of the proteins to the
surface using Biacore 3000. In particular we treated the
carboxymethyl-dextran surface of the sensor chip with NHS/EDC to
activate it in preparation for amine coupling. The NHS/EDC creates
reactive ester groups where the carboxyl groups were on the dextran
(only about 40% of the COO EDC Treatment of PEDF--
Purified PEDF (2.25 mg/ml; 45 µM) was mixed with 6 mM ethylenediamine or 12 mM ethanolamine and increasing concentrations of EDC (as
indicated) in 0.1 MES, 0.9% NaCl, pH 4.7. The mixtures were incubated
at room temperature for 2 h. To purify the conjugates, 2 ml of PBS
were added to the mixtures and then subjected to ultrafiltration through centricon-30 (Mr cut-off = 30,000). This was repeated four times. The protein concentration in the
final samples ranged between 0.5-0.8 mg/ml.
Heparin Affinity Column Chromatography--
EDC-treated PEDF was
subjected to heparin affinity column chromatography (14). EDC-treated
PEDF (20 µg) was diluted in a 0.6-ml total volume of buffer S (20 mM phosphate buffer, pH 6.5, 20 mM NaCl) and
loaded onto a column of heparin immobilized on acrylic beads (0.5 ml
bed volume). The flow-through was collected and the column washed with
10 column volumes of buffer S. The bound protein was eluted with a step
gradient of NaCl in buffer S (0.6 ml per fraction). The flow-through
and eluted fractions were concentrated using Centricon-30 before
SDS-PAGE.
Controlled Proteolysis--
Limited proteolysis of PEDF and
Fl-PEDF was with chymotrypsin at a protease:substrate ratio of 1:100
(w:w) as described previously (19). Cleavage of the serpin exposed loop
was confirmed by SDS-PAGE.
PAGE--
PAGE was performed using premade 10-20%
polyacrylamide gels (Invitrogen) with Tricine-SDS running buffer
following the manufacturer's instructions followed by either Coomassie
Blue staining or Western analysis.
Western Blot--
Proteins in gels were transferred to
nitrocellulose membranes (pore size of 20 µm). Immunostaining of PEDF
and fluorescein was by ECL using Lumi-LightPLUS following
the manufacturer's instructions. Briefly, the membranes were
sequentially incubated in a 1:100,000 dilution of mouse anti-PEDF or
1:200,000 dilution of rabbit anti-Fl in Blocking Solution, then
in secondary antibody (anti-mouse IgG-POD (1:1,000) or anti-rabbit IgG-POD (1:20,000)) and finally in Lumi-LightPLUS substrate
solution and then exposed to Kodak Biomax ML film. For colorimetric
immunostaining, the membranes were sequentially incubated in a 1:750
dilution of anti-collagen I or anti-collagen III, then in biotinylated
anti-mouse IgG (H+L) (1:1000) and followed by ABC solution (Vectastin
ABC elite kit). Color was developed with horseradish peroxidase
(HRP) color development reagent (Bio-Rad).
PEDF Binds to Collagens of Cornea and Vitreous--
Because PEDF
is a natural component of cornea and vitreous, we evaluated its ability
to interact with proteins from these ocular components. Binding
reactions were performed at 4 °C to allow interactions to occur with
minimal proteolytic degradation. Using a method based on size-exclusion
ultrafiltration, a PEDF of 50 kDa was retained by the membrane
(exclusion limit of Mr 100,000) only when
incubated with the ocular extracts (Fig.
1A). The amount of bound PEDF
increased with increasing amounts of corneal and vitreal protein in the
reactions. Next, we used ligand affinity chromatography to identify
PEDF-binding proteins in the cornea and vitreous. Several proteins of
the cornea and vitreous had binding affinity for immobilized PEDF,
including proteins that comigrated with collagen I and immunoreacted
with antibodies to collagens (Fig. 1B). Note that the
commercial collagen I also bound to the PEDF affinity resin. Ligand
blotting of collagen I showed that PEDF bound to immobilized denatured
collagen I and comigrating proteins from vitreous and cornea after
SDS-PAGE under reducing conditions (Fig. 1C). These data
demonstrated that PEDF can bind to bovine corneal and vitreal collagens
in solution and when immobilized.
Binding of PEDF to Collagens in Solution--
Then we assayed the
binding of PEDF to purified collagen types I and II in solution. Fig.
2 shows that PEDF bound to each type of
collagen, unlike the non-inhibitory serpin ovalbumin. The binding was
collagen concentration-dependent with an apparent efficiency greater for collagen type I than the other types (Fig. 2B). Non-linear regression analyses of PEDF binding with
increasing concentrations of collagen I and II showed a best fitted
equation for a sigmoidal dose response (variable slope) with
half-maximal concentration binding EC50 = 11.9 µg/ml collagen I, Hill coefficient = 4.342 and EC50 = 117.5 µg/ml collagen II, Hill coefficient = 2.423, implying a
cooperative behavior for the PEDF-collagen interactions. The binding
between PEDF and collagens occurred at physiological conditions of 150 mM NaCl and pH 7.4 and at a similar extent with incubations
between 0 and 25 °C.2
However, decreasing the pH of the reaction from 8.2 to 5.0 had an
inhibitory effect on the binding with optimum binding at pH 7.4-8.2
(Fig. 2C). A similar pH effect was observed for collagen III
binding (data not shown). Thus PEDF formed specific complexes with
collagens in solution, binding most efficiently to type I.
Binding of 125I-Labeled PEDF to Collagen
I--
Because PEDF had higher affinity for collagen type I compared
with the other types, we analyzed these interactions in more detail.
PEDF radiolabeled with 125I was used as a ligand for
collagen I binding. Solution assays showed that 43% of the 200 nM 125I-PEDF was retained by
ultrafiltration in reactions with collagen I (20 µg/ml), in contrast
to 5% without collagen I, which decreased with an excess of
unlabeled PEDF over the radioligand (data not shown). Thus, as with
unmodified PEDF, the 125I-PEDF modified on tyrosines by
iodination formed specific complexes with collagen I in solution.
To determine the binding parameters of the PEDF-collagen I
interactions, solid-phase binding assays were performed with soluble radioligand and immobilized collagen I on plastic. The binding reactions were under the same conditions as the solution assays above;
however, one advantage was that they could be performed with lower
concentrations of radioligand. 125I-PEDF at 2 nM bound to the immobilized collagen I and the unlabeled ligand competed efficiently for the binding with a half-maximal competition concentration of EC50 = 1.69 µg/ml (34 nM PEDF) (Fig. 3A). A binding saturation
curve was calculated (Fig. 3B), and the binding parameters
revealed an affinity of KD = 124 nM for
the PEDF-collagen I interactions.
Radioligand competion assays were performed with PEDF fragments. BH is
a bacterially derived recombinant human PEDF fragment spanning amino
acid positions 44-418 (23), and the 34-mer and 44-mer peptides are
synthetic peptides designed from the human PEDF sequence between
positions 44-77 and 78-121, respectively. The 44-mer peptide contains
the receptor-binding region of PEDF and has neurotrophic activity on
retinoblastoma cells (20). Fig. 3C shows that additions of
an excess of BH inhibited the 125I-PEDF binding, comparable
with additions of unlabeled PEDF. The presence of 75 mM
urea in the binding reactions decreased the 125I-PEDF
binding to 60%, probably due to partial unfolding of the ligand.
However the 34-mer and 44-mer peptides did not have a major effect on
the binding of PEDF to collagen I. Similarly, increasing concentrations
of angiostatin or endostatin, established antiangiogenic inhibitors,
did not compete with 125I-PEDF for binding to collagen
I.3 When collagen I in
solution was used to test for displacement of bound
125I-PEDF from the immobilized collagen I, additions of
increasing concentrations of collagen I resulted in a gradual loss in
radioligand binding, reaching ~40% inhibition with 100 µg/ml
collagen I (Fig. 3D).
Surface Plasmon Resonance Assays--
Biacore kinetic and
affinity analyses were performed on the interaction between PEDF
and collagen I (Table I), and
little or no nonspecific binding was observed with this method. Binding was reproducible and yielded data suitable for analysis. When the assay
was performed in an orientation where the PEDF monomer protein was in
solution (following a simple 1:1 interaction mechanism), the resultant
affinities from both steady-state analysis and kinetically derived
KD matched those obtained from solid-phase assays with radioisotopes in solution. When the orientation was reversed, and
the trimeric collagen was in solution as the analyte, the kinetics
became somewhat complicated probably due to avidity effects of
multivalency (e.g. more than one PEDF-binding site on a
collagen molecule). This observation was consistent with the
cooperative binding found above with collagen in solution (see Fig. 2).
Even though the measured affinities fell within a 10-fold range
(i.e. 100-800 nM) for the bivalent steady-state
model proposed for collagen binding to immobilized PEDF, and were
roughly consistent with affinities measured in the opposite orientation
(i.e. collagen I surface), the results from the latter
orientation (i.e. collagen I surface) and the simple 1:1
model are to be preferred.
Effect of NaCl on the PEDF-Collagen I Interactions--
To
elucidate the type of interactions between PEDF and collagen I, we
examined the effect of increasing the ionic strength in the reactions
using NaCl. Fig. 4 shows that the binding
of both unmodified PEDF and 125I-PEDF to collagen I was
lost with an increase in NaCl concentration. The affinity of both PEDF
versions for collagen I decreased significantly with 400 mM
NaCl and 600 mM NaCl and was completely lost with Modifications of PEDF at Lysines with Fluorescein Increase Its
Binding Affinity for Collagen I--
Given the ionic nature of the
PEDF-collagen I interactions, we investigated the role that charges of
the protein play on collagen I binding. The three-dimensional structure
of PEDF has revealed two distinct areas with opposite ionic potentials
on its surface (14, 24). In previous studies, we demonstrated that
modification at the positively charged lysine residues of PEDF
abolished its binding affinity for polyanions, such as
glycosaminoglycans (14, 21). To determine whether the basic region of
PEDF is required for collagen binding, we used PEDF chemically modified
with an activated N-hydroxysuccinimide ester form of
fluorescein, which reacts with primary amines such as those on basic
lysine residues. Binding of Fl-PEDF to increasing concentrations of
collagen I was tested in solution assays. Fig.
5 shows that about 50 ng of Fl-PEDF were
retained in assays with 2.5 µg/ml collagen I, which increased more
than 5-fold with 10 µg/ml collagen I. In contrast, the bound
unmodified PEDF was not detected with 2.5 µg/ml collagen I and barely
reached 50 ng with 10 µg/ml collagen I. Fig. 5B shows a
comparison of the affinities for collagen I of unmodified PEDF and
Fl-PEDF in the presence of NaCl. The modified Fl-PEDF required a higher
ionic strength (1350 mM NaCl) to release from collagen I
than the unmodified PEDF (300 mM NaCl). These results
showed that the modifications to PEDF did not prevent binding to
collagen I, but rather increased it. In addition to excluding the basic region of the PEDF protein as site for interaction with collagen I,
these data show that neutralization of its positive charged lysines
increased its binding affinity for collagen I. Furthermore, the
homologous serpin reactive loop of PEDF has a chymotryptic site (19).
PEDF and Fl-PEDF were cleaved by controlled proteolysis with
chymotrypsin and tested for collagen I binding. Fig. 5C
shows that the binding ability was not abolished upon cleavage of the homologous serpin reactive loop of either PEDF or Fl-PEDF, indicating that this region is also dispensable for the collagen I
interactions.
Modified PEDF at Aspartic Acid and Glutamic Acid Loses Its Binding
to Collagen I--
To determine whether the negatively charged area of
PEDF is directly involved in binding to collagen I, we treated PEDF
with EDC. EDC modifies carboxyl groups, such as the ones in aspartic acid and glutamic acid residues. Treatments were with increasing concentrations of EDC in the presence of a molar excess of amine containing reagents such as ethylenediamine or ethanolamine to prevent
protein-protein cross-linking. The resulting PEDF proteins migrated as
50-kDa monomers, with minimal formation of polymers detected only with
the highest concentration of EDC (Fig.
6A). To test whether the
treatments had affected the basic region of PEDF, the EDC-treated PEDF
proteins were subjected to heparin affinity column chromatography. Fig.
6B shows that the treated proteins had retained their
heparin-binding site with a higher heparin affinity as the EDC
concentration increased, implying that their folded conformation had
been maintained. Note that those treated in the presence of excess of
ethylenediamine had slightly higher heparin affinity than ethanolamine
(compare samples 3 and 6), probably due to
differences in charge-to-protein ratio. The modified PEDF proteins were
then tested for collagen I binding in solution. Treatments with
increasing concentrations of EDC resulted in a gradual loss of affinity
for binding collagen I (Fig. 6C). These results demonstrated
that the aspartic and glutamic acid residues of PEDF were required for
its binding to collagen I and implied that they are directly involved
in interactions with collagen I.
Effect of Polyanions on the PEDF-Collagen I Interactions--
The
requirement of negative charges/acidic region of PEDF for the binding
to collagen I suggests interactions at positively charged regions in
collagen I. Heparin was used as a polyanionic competitor for the
binding of the acidic region of PEDF to collagen I. Solution binding
assays were under ionic strength conditions in which the PEDF-heparin
interactions do not occur (see Ref. 14). Fig. 8 shows that
increasing concentrations of heparin competed with unmodified PEDF
molecules for the binding to soluble trimeric collagen I. These results
imply that PEDF shares a positively charged binding region in collagen
I with heparin and further verify that the binding site for collagen I
in PEDF is formed by a negatively charged region on the surface of the protein.
In this study we have characterized and analyzed the interactions
between PEDF and collagen and provided evidence for a collagen I-binding site on PEDF. Our results show that the extracellular neurotrophic and antiangiogenic serpin PEDF binds to sites on collagens
I-III under physiologic conditions. Interactions can occur when PEDF
and collagen I are both in solution, when one is immobilized, or even
when collagen I is denatured under reducing conditions. We present
evidence for ionic interactions between a negatively charged area in
the intact and correctly folded PEDF protein rich in glutamic acid and
aspartic acid residues and positively charged area(s) in collagen I
molecules. These interactions may occur in vivo and may play
important roles in regulating the local availability of PEDF and/or in
modulating its biological activities, e.g. antiangiogenic
activities in the cornea and vitreous.
PEDF has the highest affinity for collagen I among collagens I-IV
immobilized (10) and in solution, as shown here. When collagens are in
solution, the PEDF-collagen interactions have a cooperative binding
nature as demonstrated by the preferred binding equation for sigmoidal
dose response. When collagen is immobilized, both the radioligand
binding and Biacore analyses resulted in the same affinity measurement
for the PEDF-collagen I interactions (Table I), confirming and
validating each other as a means for measuring affinity. In addition,
real-time surface plasmon resonance analysis revealed that this
interaction has rapid kinetics and is transient. In other words, when
the local concentration of reactants falls below the
KD of the interaction, the complex is likely to
rapidly dissociate. This is likely to provide additional insight into
the binding mechanism in vivo. For example, transient
interactions in the range of 10 Our data also provide evidence for a collagen I-binding site on PEDF.
The affinity of PEDF for immobilized collagen I
(KD = ~130 nM) is distinct from its
affinity for neurotrophic receptors (KD = ~3
nM) and glycosaminoglycans (KD = ~4 µM for PEDF-heparin) (12, 14, 20), implying a distinct
and separate binding site for collagen I on the PEDF protein molecule. In this regard, the lack of competition by the 44-mer peptide containing the PEDF receptor-binding site (20, 21) is in agreement with
separate and non-overlapping binding sites for collagen I and the
neurotrophic receptor. The fact that modifications of lysines of PEDF,
e.g. Fl-PEDF, abolishes the binding affinity for heparin
(14, 21) but not for collagen I, also points to distinct and
non-overlapping binding sites for glycosaminoglycans and collagens.
Moreover, these three binding sites are independent of the homologous
serpin reacting loop in PEDF.
The loss of the affinity of PEDF for collagen I with an increase in
ionic strength, a lower pH, or modifications of carboxylic groups of
PEDF, e.g. EDC-treated PEDF, is consistent with an
acidic/negatively charged region of PEDF as a binding site for collagen
I. The three-dimensional structure of PEDF has two distinct regions
with opposite ionic potentials located almost 180° apart from
each other (14, 24). The positively charged region is rich in basic
amino acid residues, lysines and arginines, with side chains containing
NH Our data also provide evidence for PEDF-binding sites in the collagen I
molecule. The competition between heparin and PEDF for collagen I
binding points to overlapping binding sites for these two ligands in
collagen I. The binding affinity of heparin for collagen I
(KD = 150 nM) (30) is similar to that of
PEDF-collagen I, suggesting that heparin and PEDF can be
interchangeable ligands for collagen I. Mapping of ligand-binding sites
in collagen I has shown that collagen I has positively charged areas
that interact with glycosaminoglycans (31). Several sites on collagen I
have been proposed to mediate heparin/heparan sulfate binding to type I
collagen. One of them at position 87-90 of Comparison with serpins known to bind to collagen I such as HSP47 and
maspin shows that the PEDF-collagen I interactions have binding
parameters more similar to those for HSP47 but biological activities
resembling those of maspin. For example, using surface plasmon
resonance with immobilized collagen I and maspin as analyte, Blacque
and Worral (29) found a KD of 630 nM
with an association rate constant (ka) of 3.2 × 103 M The collagen binding property of PEDF suggests that PEDF
regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. At the molecular level,
PEDF has binding sites for collagens and glycosaminoglycans, which are
characteristic features of cell adhesion molecules. The actual affinity
of PEDF for extracellular matrix may be even higher than that for
individual extracellular matrix molecules, as its affinity for
collagen increases when the glycosaminoglycan-binding site of PEDF is
neutralized and vice versa and could even change with the pH of the
extracellular matrix. Therefore, qualitative and quantitative changes
of extracellular matrix molecules, e.g. collagen,
glycosaminoglycans, as well as pH changes of extracellular matrix,
which occur through development, with aging and in certain pathologic
conditions, such as corneal dystrophies, diabetic retinopathy, and
wound healing, (17, 35-42), may alter molecular assembly and the
location of the antiangiogenic activities of PEDF.
We thank Evan Behre for technical support
with the Biacore 3000 and insightful discussions, Bijan Ahvazi for
protein modeling, and Shurid Rahman for technical assistance with
ligand blotting.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M208339200
2
C. Meyer, personal observations.
3
S. P. Becerra, unpublished observations.
The abbreviations used are:
PEDF, pigment
epithelium-derived factor;
Fl, fluorescein;
PBS, phosphate-buffered
saline;
EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbomiide HCl;
MES, 4-morpholineethanesulfonic acid;
BSA, bovine serum albumin;
NHS, N-hydroxysuccinimide;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
Mapping the Type I Collagen-binding Site on Pigment
Epithelium-derived Factor
IMPLICATIONS FOR ITS ANTIANGIOGENIC ACTIVITY*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until use and contained ~1.7 mg/ml protein. Vitreous extracts were obtained as described before (15). Briefly, after dissection of the anterior of the eye, the vitreous gel was transferred to a tube, homogenized with a Polytron, and subjected to centrifugation at 1300 × g for 15 min at 4 °C. The supernatant
termed vitreal extract was stored at 80 °C until use and contained
~0.4 mg/ml protein.
-scintillation counter. Nonspecific binding was determined from
fractions with >100-fold molar excess of unlabeled PEDF over
radioligand. Binding data were analyzed by non-linear regression using
Prism GraphPad software.
groups are derivitized). The
protein designed to be bound to the activated CM5 chip is exposed to
the dextran with the reactive esters. Primary amines on the protein
(e.g. N-terminal and possibly lysine groups) then attach to
the dextran by nucleophilic substitution of the reactive ester. The
remaining free surface (about 60%) was then blocked with 0.1 M Tris, pH 8.0, and the matrix washed with 0.5 M NaCl solution and then re-equilibrated with binding buffer (PBS, 10% glycerol). Eight different dilutions of PEDF or
collagen I were prepared in binding buffer with concentrations ranging
from 0 to 1.0 µM and injected from low to high
concentration, and then the series was repeated, to study the
interaction of both free PEDF on a collagen I matrix and the inverse
orientation. Each injection was followed by a 0.5 M NaCl
regeneration step. The data were then fitted to several binding models
for a kinetic analysis. The best fittings were obtained with a simple
1:1 Langmuir model for the collagen surface binding assay and with a
bivalent analyte model for the opposite orientation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (55K):
[in a new window]
Fig. 1.
PEDF binds to collagens of the cornea and
vitreous. A, complex formation between PEDF (5 µg)
and proteins from cornea and vitreous in solution (100 µl final
volume). Control reactions were with BSA. A Western blot immunoreacted
with anti-PEDF is shown. Lane 1, 0.25 µg of PEDF.
Lanes 2-11 correspond to assays of reactions with no
extracts (lane 2), corneal protein (10, 34, and 67 µg in
lanes 3-5, respectively), vitreal protein (10, 34, and 67 µg in lanes 6-8, respectively) and BSA (10, 34, and 67 µg in lanes 9-11, respectively). B, PEDF
affinity chromatography of proteins from cornea, vitreous, and collagen
I (lanes 1, 2, and 3, respectively).
SDS-PAGE followed by Coomassie Blue staining and immunostaining with a
mixture of anti-collagen I and III are shown for load and eluted
samples, as indicated. C, ligand blot. Corneal (lane
1) and vitreal (lane 2) extracts, and collagen I
(lane 3), were resolved by SDS-PAGE under reducing
conditions. The blots with transferred proteins were incubated with
Fl-PEDF and the bound ligand detected by immunostaining with
anti-fluorescein.
View larger version (40K):
[in a new window]
Fig. 2.
Complex formation between PEDF and
collagens. A, binding reactions were conducted with
PEDF or ovalbumin and collagen I or II (200 µg/ml) followed by
ultrafiltration. Lane 1, PEDF and collagen I; lane
2, PEDF and collagen II; lane 3, ovalbumin and collagen
I; lane 4, ovalbumin and collagen II; lane 5,
PEDF; lanes 6 and 7, correspond to 1.2 µg of
PEDF and ovalbumin, respectively, not subjected to ultrafiltration.
B, bar graphs of complexed PEDF (intensity of each PEDF band
shown above each lane) with increasing concentrations of collagen types
I and II are shown. C, effect of pH on the binding of PEDF
to collagen I. The pH in reactions were as indicated. Polyacrylamide
gels shown in all panels were stained with Coomassie Blue.
View larger version (39K):
[in a new window]
Fig. 3.
Binding of 125I-labeled PEDF to
collagen I. Recombinant human PEDF labeled with 125I
was used as ligand for collagen I in solid-phase assays. A,
binding to immobilized collagen I was with 100 ng/ml (2 nM)
125I-PEDF and increasing concentrations of unlabeled PEDF.
The bound-to-total ratio without unlabeled PEDF was 3.3%. Amounts of
radioactivity of 125I-PEDF bound per well ± S.D. are
shown. Each point was the average of triplicate assays. B,
binding isotherm of PEDF to immobilized collagen I. Binding data of
A was transformed and subjected to nonlinear regression
using GraphPad PrismTM. Equations for one- and two-site
binding (hyperbola) were fitted and compared with an F test. The best
fitted equation was for one-site binding with best-fit values of
Bmax = 0.59 ± 0.02 pmol per well and
Kd = 123.5 ± 18.12 nM. Specific
binding ± S.E. is shown. The inset displays the
Scatchard transformation of the binding data. C, radioligand
competition. Bar graph of 125I-PEDF per well with PEDF
fragments in molar excess over the radioligand, as indicated. Binding
was with 2.8 nM radioligand, and each point was the average
of triplicate assays ± S.D. Human PEDF fragments were BH (amino
acid positions 44-418), 33-mer (positions 44-77), and 44-mer
(78-121, containing the neurotrophic active site of PEDF). Reactions
for BH (0 and 50×) contained 75 mM urea. D,
inhibition of 125I-PEDF binding by collagen I in solution.
Plot of 125I-PEDF bound per well versus
increasing concentrations of collagen I. Each point was the average of
duplicate assays ± S.D.
Comparison of methods to measure affinity and kinetics of the
PEDF-collagen I interactions
1
M NaCl. These data showed that the binding of PEDF to
collagen was sensitive to increasing ionic strength, implying an ionic nature for their interactions, and that the modification on tyrosines by conjugation with 125I did not affect this binding
characteristic of PEDF.
View larger version (23K):
[in a new window]
Fig. 4.
Effect of increasing the ionic strength on
the PEDF-collagen I interactions. A, solution assays.
Binding reactions of PEDF and collagen I and washes of the bound
proteins were with increasing concentrations of NaCl. Bound PEDF was
resolved by SDS-PAGE followed by Coomassie blue staining. Binding
reactions were without collagen I (lane 2), in 0.15 M NaCl with 0.15 M NaCl washes (lane
3), in 0.15 M NaCl with 0.6 M NaCl washes
(lane 4), and in 1 M NaCl with 1 M
NaCl washes (lane 5). Lane 1, PEDF (2 µg) not
subjected to ultrafiltration. B, solid-phase binding of
125I-PEDF (2 nM) to immobilized collagen I with
increasing concentrations of NaCl. The amount of radioactivity of bound
125I-PEDF per well is shown, with each data point being the
average of triplicates ± S.D.
View larger version (36K):
[in a new window]
Fig. 5.
Binding of fluoresceinated PEDF to collagen
I. Solution binding assays with 25 µg/ml PEDF or Fl-PEDF to
collagen I. A, binding reactions were with increasing
concentrations of collagen I, as indicated. Lanes on the
right were with PEDF or Fl-PEDF not subjected to
ultrafiltration. B, binding reactions were with 10 µg/ml
collagen I and increasing concentrations of NaCl, as indicated. Western
analysis using anti-PEDF is shown for A and B. C, PEDF and Fl-PEDF were cleaved with chymotrypsin at the
serpin exposed loop and assayed for collagen I binding in solution. A
Coomassie Blue-stained gel of bound ligands is shown. U and
C correspond to uncleaved and cleaved PEDF species,
respectively. Arrows point to migration positions of cleaved
and uncleaved ligands.
View larger version (36K):
[in a new window]
Fig. 6.
Binding of EDC-treated PEDF to collagen
I. PEDF protein samples were treated with increasing
concentrations of EDC in the presence of ethylenediamine
(samples 1-3) or ethanolamine (samples 4-6).
A, SDS-PAGE of each purified EDC-treated PEDF protein (2 µg protein/lane). Reactions were with 0 mM EDC
(lanes 1 and 4), 1.3 mM EDC
(lanes 2 and 5), and 13 mM EDC
(lanes 3 and 6). B, EDC-treated PEDF
samples were subjected to heparin affinity column chromatography.
Flow-through and eluted fractions with a step gradient of NaCl were
resolved by SDS-PAGE. Numbers to the left
correspond to protein loaded to each column and were as lanes in
A. C, the EDC-PEDF was assayed for binding to
collagen I in solution. Bound PEDF was resolved by SDS-PAGE. Numbers to
the top correspond to protein ligand in each reaction and were as lanes
in A. All gels shown were stained with Coomassie Blue.
*, monomers; **, dimmers; ***,
trimers.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 to
105 M are typical with molecules
involved in cell adhesion (25-29).
groups exposed to the surface
of the protein (Fig. 8). As the pH is lowered the carboxyl groups
become protonated, and the attracting force between the acidic region
of PEDF and collagen I decreases (Fig. 2D). At the same
time, however, in regions that were electroneutral at pH 7, as the
carboxyl groups become protonated, the number of positively charged
amino groups that are no longer neutralized increases, and the
attracting force for polyanions increases. When the basic region is
blocked, e.g. Fl-PEDF, the number of positive charged amino
groups decreases causing a reduction in attracting the negatively
charged heparin (21) and an increase in attraction for collagen I (Fig.
6). Conversely, when the acidic region of PEDF is blocked,
e.g. EDC-treated PEDF, the number of negatively charged
carboxyl groups diminishes, causing a reduction in the attracting force
for positively charged areas in collagen I and an increase in
attraction for heparin (Fig. 7). Together our data demonstrate that all or part of the aspartic and glutamic acids forming the acidic region of the PEDF protein are available to
interact directly with positively charged areas in collagen I. In the
three-dimensional structure of PEDF, this acidic region, corresponding
to the collagen I-binding site, is distinct and non-overlapping from
both of the binding sites for the neurotrophic receptor and heparin and
all away from the serpin exposed loop (see Fig.
8).
View larger version (17K):
[in a new window]
Fig. 7.
Effect of heparin on the binding of PEDF to
collagen I. Solution assays of PEDF-collagen I binding were in the
presence of increasing concentrations of heparin, as indicated. A
Coomassie Blue-stained gel of bound PEDF is shown. Lane to the
left corresponds to 2 µg of PEDF as standard
protein.
View larger version (36K):
[in a new window]
Fig. 8.
Three-dimensional structure of human PEDF
(from Protein Data Bank ID 1IMV) to illustrate the location of the
proposed collagen I-binding site. The two structures
are rotated about 180 °C from each other. Highlighted in
green are positions 78-121 (receptor binding area) (20), in
blue a positively charged area on the protein surface
(glycosaminoglycan binding area) (14), and in red a
negatively charged area on the protein surface (collagen binding area),
and P2 corresponds to the residue next to the homologous serpin
reactive site, P1.
1(I) overlaps with one
of the integrin 11-binding sites and is a
near neighbor of the integrin 21-binding
sites. These observations suggest that the binding of heparin/heparin
sulfate proteoglycans and/or PEDF to collagen I could modulate
integrin-collagen interactions, which play a role in cell adhesion an
important event in angiogenesis. In addition, triple helical peptides
of the basic N-terminal sequence of this area were found to be
inhibitory in the collagen I-mediated endothelial tube formation
in vitro, an assay for angiogenesis (30). In the same
fashion, in corneal neovascularization assays or other angiogenesis
assays requiring the presence of collagen, PEDF could regulate
endothelial cell adhesion and migration on collagen.
1
s1, and a dissociation rate constant
(KD) of 0.002 s1, wherease
MacDonald and Balcinger (32), studying the binding of mouse and chicken
HSP47 to native bovine collagen I by fluorescence quenching and
cooperative binding, observed a collagen concentration at
half-saturation (Khalf) of 110-140
nM and a Hill coefficient of 3.2-4.3. However, the
collagen binding in maspin is localized to amino acids 84-112, which
aligns with the collagen-binding region of HSP47 amino acids 104-169
(29, 33) but is not related to the collagen-binding site for PEDF. In
contrast to PEDF, HSP47 is an intracellular serpin and plays a role in
chaperoning collagen during its synthesis, while maspin is involved in
inhibition of angiogenesis and cell adhesion (27, 34).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: NEI, NIH, Bldg. 6, Rm.
308, 6 Center Dr., MSC 2740, Bethesda, MD 20892-2740. Tel.: 301-496-6514; Fax: 301-451-5420; E-mail:
pbecerra@helix.nih.gov.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Becerra, S. P.
(1997)
Adv. Exp. Med. Biol.
425,
223-237[Medline]
[Order article via Infotrieve]
2.
Cayouette, M.,
Smith, S. B.,
Becerra, S. P.,
and Gravel, C.
(1999)
Neurobiol. Dis.
6,
523-532[CrossRef][Medline]
[Order article via Infotrieve]
3.
Cao, W.,
Tombran-Tink, J.,
Elias, R.,
Sezate, S.,
Mrazek, D.,
and McGinnis, J. F.
(2001)
Investig. Ophthalmol. Vis. Sci.
42,
1646-1652 4.
Houenou, L. J.,
D'Costa, A. P., Li, L.,
Turgeon, V. L.,
Enyadike, C.,
Alberdi, E.,
and Becerra, S. P.
(1999)
J. Comp. Neurol.
412,
506-514[CrossRef][Medline]
[Order article via Infotrieve]
5.
Crawford, S. E.,
Stellmach, V.,
Ranalli, M.,
Huang, X.,
Huang, L.,
Volpert, O., De,
Vries, G. H.,
Abramson, L. P.,
and Bouck, N.
(2001)
J. Cell Sci.
114,
4421-4428 6.
Bouck, N.
(2002)
Trends Mol. Med.
8,
330-334[CrossRef][Medline]
[Order article via Infotrieve]
7.
Dawson, D. W.,
Volpert, O. V.,
Gillis, P.,
Crawford, S. E., Xu, H.,
Benedict, W.,
and Bouck, N. P.
(1999)
Science
285,
245-248 8.
Stellmach, V.,
Crawford, S. E.,
Zhou, W.,
and Bouck, N.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2593-2597 9.
Ortego, J.,
Escribano, J.,
Becerra, S. P.,
and Coca-Prados, M.
(1996)
Investig. Ophthalmol. Vis. Sci.
37,
2759-2767 10.
Kozaki, K.,
Miyaishi, O.,
Koiwai, O.,
Yasui, Y.,
Kashiwai, A.,
Nishikawa, Y.,
Shimizu, S.,
and Saga, S.
(1998)
J. Biol. Chem.
273,
15125-15130 11.
Perez-Mediavilla, L. A.,
Chew, C.,
Campochiaro, P. A.,
Nickells, R. W.,
Notario, V.,
Zack, D. J.,
and Becerra, S. P.
(1998)
Biochim. Biophys. Acta
1398,
203-214[Medline]
[Order article via Infotrieve]
12.
Stratikos, E.,
Alberdi, E.,
Gettins, P. G.,
and Becerra, S. P.
(1996)
Protein Sci.
5,
2575-2582[Medline]
[Order article via Infotrieve]
13.
Wu, Y. Q.,
Notario, V.,
Chader, G. J.,
and Becerra, S. P.
(1995)
Protein Expression Purif.
6,
447-456[CrossRef][Medline]
[Order article via Infotrieve]
14.
Alberdi, E.,
Hyde, C. C.,
and Becerra, S. P.
(1998)
Biochemistry
37,
10643-10652[CrossRef][Medline]
[Order article via Infotrieve]
15.
Wu, Y. Q.,
and Becerra, S. P.
(1996)
Investig. Ophthalmol. Vis. Sci.
37,
1984-1993 16.
Karakousis, P. C.,
John, S. K.,
Behling, K. C.,
Surace, E. M.,
Smith, J. E.,
Hendrickson, A.,
Tang, W. X.,
Bennett, J.,
and Milam, A. H.
(2001)
Mol. Vis.
7,
154-163[Medline]
[Order article via Infotrieve]
17.
Bishop, P. N.
(2000)
Prog. Retin. Eye Res.
19,
323-344[CrossRef][Medline]
[Order article via Infotrieve]
18.
Steele, F. R.,
Chader, G. J.,
Johnson, L. V.,
and Tombran-Tink, J.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
1526-1530 19.
Becerra, S. P.,
Sagasti, A.,
Spinella, P.,
and Notario, V.
(1995)
J. Biol. Chem.
270,
25992-25999 20.
Alberdi, E.,
Aymerich, M. S.,
and Becerra, S. P.
(1999)
J. Biol. Chem.
274,
31605-31612 21.
Aymerich, M. S.,
Alberdi, E. M.,
Martinez, A.,
and Becerra, S. P.
(2001)
Investig. Ophthalmol. Vis. Sci.
42,
3287-3293 22.
Yabe, T.,
Wilson, D.,
and Schwartz, J. P.
(2001)
J. Biol. Chem.
276,
43313-43319 23.
Becerra, S. P.,
Palmer, I.,
Kumar, A.,
Steele, F.,
Shiloach, J.,
Notario, V.,
and Chader, G. J.
(1993)
J. Biol. Chem.
268,
23148-23156 24.
Simonovic, M.,
Gettins, P. G.,
and Volz, K.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
11131-11135 25.
Wild, M. K.,
Huang, M. C.,
Schulze-Horsel, U.,
van der Merwe, P. A.,
and Vestweber, D.
(2001)
J. Biol. Chem.
276,
31602-31612 26.
Mehta, P.,
Cummings, R. D.,
and McEver, R. P.
(1998)
J. Biol. Chem.
273,
32506-32513 27.
Ngamkitidechakul, C.,
Burke, J. M.,
O'Brien, W. J.,
and Twining, S. S.
(2001)
Investig. Ophthalmol. Vis. Sci.
42,
3135-3141 28.
Nicholson, M. W.,
Barclay, A. N.,
Singer, M. S.,
Rosen, S. D.,
and van der Merwe, P. A.
(1998)
J. Biol. Chem.
273,
763-770 29.
Blacque, O. E.,
and Worrall, D. M.
(2002)
J. Biol. Chem.
277,
10783-10788 30.
Sweeney, S. M.,
Guy, C. A.,
Fields, G. B.,
and San Antonio, J. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7275-7280 31.
Di Lullo, G. A.,
Sweeney, S. M.,
Korkko, J.,
Ala-Kokko, L.,
and San Antonio, J. D.
(2002)
J. Biol. Chem.
277,
4223-4231 32.
Macdonald, J. R.,
and Bachinger, H. P.
(2001)
J. Biol. Chem.
276,
25399-25403 33.
Ball, E. H.,
Jain, N.,
and Sanwal, B. D.
(1997)
Adv. Exp. Med. Biol.
425,
239-245[Medline]
[Order article via Infotrieve]
34.
Zhang, M.,
Volpert, O.,
Shi, Y. H.,
and Bouck, N.
(2000)
Nat. Med.
6,
196-199[CrossRef][Medline]
[Order article via Infotrieve]
35.
Robert, L.,
Legeais, J. M.,
Robert, A. M.,
and Renard, G.
(2001)
Pathol. Biol. (Paris)
49,
353-363[Medline]
[Order article via Infotrieve]
36.
Kern, P.,
Sebert, B.,
and Robert, L.
(1986)
Clin. Physiol. Biochem.
4,
113-119[Medline]
[Order article via Infotrieve]
37.
Labat-Robert, J.,
Kern, P.,
and Robert, L.
(1992)
Ann. N. Y. Acad. Sci.
673,
16-22[CrossRef][Medline]
[Order article via Infotrieve]
38.
Lu, D. W.,
Chang, C. J.,
and Wu, J. N.
(2001)
J. Ocul. Pharmacol. Ther.
17,
343-350[CrossRef][Medline]
[Order article via Infotrieve]
39.
Holmes, J. M.,
Zhang, S.,
Leske, D. A.,
and Lanier, W. L.
(1999)
Investig. Ophthalmol. Vis. Sci.
40,
804-809 40.
Nishikawa, T.,
Giardino, I.,
Edelstein, D.,
and Brownlee, M.
(2000)
Curr. Eye Res
21,
581-587[CrossRef][Medline]
[Order article via Infotrieve]
41.
Reiser, K. M.,
Amigable, M. A.,
and Last, J. A.
(1992)
J. Biol. Chem.
267,
24207-24216 42.
Hadley, J. C.,
Meek, K. M.,
and Malik, N. S.
(1998)
Glycoconj J
15,
835-840[CrossRef][Medline]
[Order article via Infotrieve]
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 H. Baba, Y. Yonemitsu, T. Nakano, M. Onimaru, M. Miyazaki, Y. Ikeda, S. Sumiyoshi, Y. Ueda, M. Hasegawa, I. Yoshino, et al. Cytoplasmic Expression and Extracellular Deposition of an Antiangiogenic Factor, Pigment Epithelium-Derived Factor, in Human Atherosclerotic Plaques Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1938 - 1944. [Abstract] [Full Text] [PDF]
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L. Notari, A. Miller, A. Martinez, J. Amaral, M. Ju, G. Robinson, L. E. H. Smith, and S. P. Becerra Pigment Epithelium-Derived Factor Is a Substrate for Matrix Metalloproteinase Type 2 and Type 9: Implications for Downregulation in Hypoxia Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2736 - 2747. [Abstract] [Full Text] [PDF]
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 S. Filleur, K. Volz, T. Nelius, Y. Mirochnik, H. Huang, T. A. Zaichuk, M. S. Aymerich, S. P. Becerra, R. Yap, D. Veliceasa, et al. Two Functional Epitopes of Pigment Epithelial-Derived Factor Block Angiogenesis and Induce Differentiation in Prostate Cancer Cancer Res., June 15, 2005; 65(12): 5144 - 5152. [Abstract] [Full Text] [PDF]
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R. H. P. Law, J. A. Irving, A. M. Buckle, K. Ruzyla, M. Buzza, T. A. Bashtannyk-Puhalovich, T. C. Beddoe, K. Nguyen, D. M. Worrall, S. P. Bottomley, et al. The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix J. Biol. Chem., June 10, 2005; 280(23): 22356 - 22364. [Abstract] [Full Text] [PDF]
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 T. A. Rege, C. Y. Fears, and C. L. Gladson Endogenous inhibitors of angiogenesis in malignant gliomas: Nature's antiangiogenic therapy Neuro-oncol, April 1, 2005; 7(2): 106 - 121. [Abstract] [PDF]
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 S. Kanda, Y. Mochizuki, T. Nakamura, Y. Miyata, T. Matsuyama, and H. Kanetake Pigment epithelium-derived factor inhibits fibroblast-growth-factor-2-induced capillary morphogenesis of endothelial cells through Fyn J. Cell Sci., March 1, 2005; 118(5): 961 - 970. [Abstract] [Full Text] [PDF]
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M. Al-Ayyoubi, P. G. W. Gettins, and K. Volz Crystal Structure of Human Maspin, a Serpin with Antitumor Properties: REACTIVE CENTER LOOP OF MASPIN IS EXPOSED BUT CONSTRAINED J. Biol. Chem., December 31, 2004; 279(53): 55540 - 55544. [Abstract] [Full Text] [PDF]
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M. Garcia, N. I. Fernandez-Garcia, V. Rivas, M. Carretero, M. J. Escamez, A. Gonzalez-Martin, E. E. Medrano, O. Volpert, J. L. Jorcano, B. Jimenez, et al. Inhibition of Xenografted Human Melanoma Growth and Prevention of Metastasis Development by Dual Antiangiogenic/Antitumor Activities of Pigment Epithelium-Derived Factor Cancer Res., August 15, 2004; 64(16): 5632 - 5642. [Abstract] [Full Text] [PDF]
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S. Halin, P. Wikstrom, S. H. Rudolfsson, P. Stattin, J. A. Doll, S. E. Crawford, and A. Bergh Decreased Pigment Epithelium-Derived Factor Is Associated with Metastatic Phenotype in Human and Rat Prostate Tumors Cancer Res., August 15, 2004; 64(16): 5664 - 5671. [Abstract] [Full Text] [PDF]
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 G. P. Cosgrove, K. K. Brown, W. P. Schiemann, A. E. Serls, J. E. Parr, M. W. Geraci, M. I. Schwarz, C. D. Cool, and G. S. Worthen Pigment Epithelium-derived Factor in Idiopathic Pulmonary Fibrosis: A Role in Aberrant Angiogenesis Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 242 - 251. [Abstract] [Full Text] [PDF]
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R. Abdulhussein, C. McFadden, P. Fuentes-Prior, and W. F. Vogel Exploring the Collagen-binding Site of the DDR1 Tyrosine Kinase Receptor J. Biol. Chem., July 23, 2004; 279(30): 31462 - 31470. [Abstract] [Full Text] [PDF]
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 H. Liu, J.-G. Ren, W. L. Cooper, C. E. Hawkins, M. R. Cowan, and P. Y. Tong Identification of the antivasopermeability effect of pigment epithelium-derived factor and its active site PNAS, April 27, 2004; 101(17): 6605 - 6610. [Abstract] [Full Text] [PDF]
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S. V. Petersen, T. D. Oury, L. Ostergaard, Z. Valnickova, J. Wegrzyn, I. B. Thogersen, C. Jacobsen, R. P. Bowler, C. L. Fattman, J. D. Crapo, et al. Extracellular Superoxide Dismutase (EC-SOD) Binds to Type I Collagen and Protects Against Oxidative Fragmentation J. Biol. Chem., April 2, 2004; 279(14): 13705 - 13710. [Abstract] [Full Text] [PDF]
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