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1 From the Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, and the 3 Department of Cell and Cancer Biology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
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Abstract |
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METHODS. Binding reactions were performed with 125I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with 125I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy.
RESULTS. Radioligand-binding assays showed that 125I-PEDF bound in a
specific and saturable fashion to one class of sites on retina
membranes (Kd = 2.5–6.5 nM; maximum
binding [Bmax] = 1–48 x
1010 sites/retina). A peptide of 44 amino acids (44-mer),
identified as the receptor-binding region of PEDF, competed efficiently
for 125I-PEDF binding to retina membranes with kinetics
similar to the full-length PEDF. Ligand blot analysis and
ligand–affinity chromatography revealed a specific and high-affinity
PEDF-binding protein of 
85 kDa in retina plasma membranes. Confocal
microscopy showed that fluorescein-conjugated PEDF stained exclusively
the inner segments of photoreceptors and cells of the ganglion cell
layer in retinal cryosections.
CONCLUSIONS. Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.
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Introduction |
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PEDF is a natural extracellular component of the retina.12 13 14 15 The localization of PEDF mRNA by in situ hybridization and Northern blot analysis reveals a distribution of high levels in the RPE and undetectable levels in the neural retina of bovine and mouse eyes.15 16 17 Immunoblot and immunohistochemistry analyses show that the PEDF protein localizes to the interphotoreceptor matrix in bovine, human, mouse, and rat eyes, and that cells of the RPE can secrete soluble PEDF into the media.1 12 14 18 19 These observations reveal that, in the native eye, the RPE is the main source of PEDF, which produces it as a diffusible factor toward the neural retina.
PEDF is a glycoprotein (50,000 molecular weight) that belongs to the
superfamily of serine protease inhibitors (serpin) by sequence
homology.16
18
20
21
However, it has no inhibitory effect
on serine proteases.22
23
24
25
Structure–function studies
have shown that a region toward the amino end of the polypeptide
(44-mer, amino acid positions 78-121 of the human PEDF) confers
neurotrophic activity on the PEDF polypeptide, whereas its
homologous serpin-reactive site, near the carboxy end, is not essential
for biologic activity.8
23
25
26
PEDF has high binding
affinity for cell-surface receptors in human retinoblastoma Y-79 cells
and rat cerebellar granule cell neurons
(Kd 
3 nM), identified as plasma
membrane proteins of approximately 80 kDa.26
In addition,
the binding of PEDF to these receptors was competed efficiently by the
44-mer peptide. Thus, the mechanism of action for the neurotrophic
activity of PEDF is independent of protease inhibition but mediated by
interactions between a region contained in the 44-mer and cell-surface
receptors expressed in target cells.
Given these biological activities of PEDF and its localization adjacent to the retina, investigation of the expression of PEDF receptors in the neural retina may yield important insights into the physiological relevance of PEDF–receptor interactions and distribution of PEDF’s activity among target cells in the retina. Because the binding of PEDF to its receptor is the first step mediating its biological effects, ligand-binding assays were developed for the bovine retina. We have used recombinant human PEDF24 labeled with 125I or fluorescein as the ligand for membrane extracts or cryosections of bovine retina, as well as unlabeled PEDF and the 44-mer peptide to further characterize the specificity of the binding. The present data provide evidence for spatially distributed neurotrophic receptors for PEDF in the neural retina.
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Methods |
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Membrane Extracts from Bovine Retina
All preparation procedures were performed at 4°C. Fresh adult
bovine eyes (J. W. Trueth & Sons, Baltimore, MD) were dissected
below the iris, the vitreous removed from the inner retinal surface,
and the neural retinas gently separated from the pigment epithelium
with forceps. Retinas were homogenized in a solution of cold 0.32 M
sucrose in Tris-buffered saline (TBS; 20 mM Tris/HCl [pH 7.5] and 150
mM NaCl) containing protease inhibitors (1 mM
aminoethyl-benzenesulfonyl fluoride hydrochloride [AEBSF], 5 µg/ml
aprotinin, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin) at 7.5 ml
per retina with a homogenizer (Polytron model 3000; Brinkman
Instruments, Westbury, NY) set at 10,000 rpm for 20 seconds. The
homogenized material was separated from tissue and cellular debris by
centrifugation at 1000g for 10 minutes and was subjected to
ultracentrifugation at 80,000g for 30 minutes. The pellets,
enriched in membranes were resuspended in cold 1% bovine serum albumin
(BSA) in PBS (1.5 ml per retina) and constituted the retinal membrane
extracts.
Solubilization of membrane proteins was performed as described previously.26 27 Briefly, membrane fractions were prepared as just described except that the homogenization buffer was 20 mM HEPES (pH 7), 100 mM KCl, 1 mM EDTA with protease inhibitors at 10 ml per retina. The membrane pellets were resuspended gently in buffer D (20 mM sodium phosphate [pH 6.5], 150 mM NaCl, 10% glycerol, 1 mM CaCl2, and 0.5% 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate [CHAPS]) at 0.4 ml per retina and centrifuged at 175,000g, 30 minutes at 4°C. The supernatant was transferred to a new tube and the pellet extracted a second time by the same procedure. The supernatants from both extractions were pooled (a final volume of approximately 0.8 ml per retina) and constituted the detergent-soluble membrane protein fraction, which was used immediately or stored at -20°C until further use.
125I-PEDF Binding Assays
The method of choice has been widely used and has a mechanism of
retention of receptors on polyethylenimine-treated glass fiber filters
based mainly on ionic interactions.27
Integral membrane
proteins tend to be acidic. Polyethylenimine binds strongly to glass,
which is negatively charged, and the resultant polycationic
polyethylenimine-coated glass should bind polyanions strongly. Because
binding of receptors to polyethylenimine filters is rather insensitive
to ionic strength, the ionic phenomenon is thought to be supplemented
by hydrophobic forces and hydrogen binding. Binding reactions were
performed by adding 125I-PEDF to membrane extract
suspension (0.15 ml) or detergent-soluble membrane fraction (0.08–0.15
ml) and incubations at 4°C. The specific
125I-PEDF binding to membrane extracts did not
change significantly with incubations between 1.5 and 16 hours. The
reaction was stopped by adding 5 ml cold 1% BSA-PBS and immediately
separating free and bound ligand by filtration under vacuum through
GF/C glass fiber filters (Whatman International Ltd., UK)
presoaked in 0.3% polyethylenimine. The filters were washed twice with
cold 1% BSA-PBS, allowed to dry, and placed in vials with 5 ml
scintillation cocktail. The radioactivity in the filters was determined
with a scintillation ß-counter (model LS3801; Beckman, Fullerton,
CA). Nonspecific binding was defined as bound radioactivity in the
presence of a 40-fold molar excess of rhPEDF over the radioligand
and specific binding as bound radioactivity minus nonspecific binding.
Each data point corresponds to the average of triplicate assays. Data
were analyzed (Prism, ver. 3; GraphPad, San Diego, CA) for nonlinear
regression with one-site binding and competition equations.
Ligand Blot
Detergent-soluble membrane proteins were resolved by SDS-PAGE
under nonreducing conditions and transferred to a 0.2-µm
nitrocellulose membrane. The membrane was first washed with 1% NP-40
in TBS for 15 minutes and then twice with TBS at 25°C for 10 minutes
each. The blot was incubated with blocking solution (1% BSA in TBST,
containing TBS with 0.05% Tween-20) at 25°C for 2 hours and then
with 2 nM 125I-PEDF in blocking solution at 4°C
for 16 hours. The blot was washed three times with TBST at 25°C for
15 minutes to remove the unbound ligand, air dried, and exposed to
x-ray film (BioMax ML; Eastman Kodak Co., Rochester, NY) to detect
bound radioligand by autoradiography.
Ligand-Affinity Column Chromatography
Fresh detergent-soluble membrane fractions from retina were
subjected to PEDF–affinity chromatography by a method described
previously.26
Briefly, rhPEDF was coupled to beads
of preactivated hydrophilic, cross-linked bis-acrylamide/azlactone
copolymers (3M Emphaze Ultralink; Pierce, Rockford, IL).
Detergent-soluble proteins obtained from 10 bovine eyes were passed
through a column of resin without ligand (1.8 ml). The unbound material
was mixed with PEDF-coupled resin (1.8 ml; 6 mg PEDF/ml resin) and
gently rotated at 4°C for 1 hour. The material was packed in a
column, washed with buffer D (20 column volumes or until absorbance at
280 nm was undetectable), followed by 1 M NaCl in buffer D (10 column
volumes). The bound material was eluted with 0.1 M glycine buffer (pH
11) 10% glycerol, 1 mM CaCl2, 0.15 NaCl, and
0.25% CHAPS (10 column volumes). Eluted proteins were concentrated to
100 µl by ultrafiltration with microconcentrators (Centricon-30;
Millipore). The microconcentrators were washed twice with 100 µl
buffer D.
Heparin-Affinity Column Chromatography
Heparin-affinity column chromatography was performed as
described previously.28
Briefly, rhPEDF or Fl-PEDF (30
µg) was incubated with heparin immobilized on acrylic beads at 4°C
for 30 minutes. Unbound material (flow-through) was removed by
extensive washes with binding buffer. Bound proteins were eluted with a
step gradient of 0.3 M and then 1 M NaCl in buffer H (20 mM sodium
phosphate [pH 6.5], 20 mM NaCl, and 10% glycerol). Fractions were
concentrated by ultrafiltration with microconcentrators.
Fl-PEDF Binding to Retinoblastoma Y-79 Cells
Human retinoblastoma Y-79 cells (6 x
105 cells/ml) were incubated in serum-free medium
at 37°C for 16 hours.22
Binding started by adding
Fl-PEDF (20 nM) and BSA (0.1%) to the culture medium and incubating at
4°C for 90 minutes. Unbound ligand was removed from the cells by
centrifugation and three washes with 1% BSA-PBS. The cells were
mounted in antifading solution (SlowFade; Molecular Probes) and scanned
with coherent light of 488 nm for fluorescein visualization under a
laser scanning microscope (model 510; Carl Zeiss, Oberkochen,
Germany).
Fl-PEDF Binding to Cryosections of Bovine Retina
Bovine eyes were sectioned below the iris, the vitreous was
removed, and a section of the posterior part of the eye encompassing
the retina, choroid, and sclera, was excised (0.5 x 2.5
cm2) and embedded in optimal temperature cutting
(OCT) compound (Sakura Finetek USA, Inc., Torrance, CA). Cryosections
(7 µm thick) were prepared on glass slides and used for in situ
Fl-PEDF binding. The frozen sections were prewashed in ice-cold 1 M
NaCl in 1% BSA-PBS and then in 1% BSA-PBS for 5 minutes each. The
prewash with 1 M NaCl did not affect the binding of Fl-PEDF and served
to preserve the cryosection from disintegrating through subsequent
incubation reactions. The cryosections were then incubated with Fl-PEDF
in 1% BSA-PBS plus 1 protease inhibitor cocktail tablet per 50 ml
(Complete; Roche, Indianapolis, IN), in a humid chamber at 4°C for 30
minutes, washed with ice cold 1% BSA-PBS (5 minutes) to remove unbound
ligand, and fixed with 10% formalin (3 minutes). Sections were
incubated with 1 µM 4',6-diamidino-2-phenylindole (DAPI; Molecular
Probes) at 25°C for 5 minutes, washed with PBS, and mounted with
antifading solution (SlowFade; Molecular Probes). Sections were scanned
with coherent light of 366 nm (for visualization of DAPI staining), 488
nm (fluorescein), and 546 nm (background in the red channel), using the
laser scanning microscope (Zeiss). Confocal images were obtained,
maintaining the same microscope settings for all samples to allow
comparisons between the treated sections and the control samples.
Western Blot Analysis
Western transfers and immunoreactions were performed as
described before.12
Briefly, immunoreactions with rabbit
polyclonal antiserum to human PEDF (Ab-rPEDF; diluted
1:1000)12
or
anti-Na+,K+-ATPase (0.5
µg/ml; Upstate Biotechnology, Lake Placid, NY) were followed by
sequential incubations with biotinylated anti-rabbit IgG (1:1000) and
ABC complex (Vector Laboratories, Inc., Burlingame, CA) and
immunostaining with 4-chloro-naphthol. Immunoreactions with a
monoclonal antibody for the human mitochondrial membrane protein of the
oxidative complex IV, cytochrome oxidase subunit I, was with anti-COX-I
(1D6-E1-A8; Molecular Probes) at 2 µg/ml in PBST (0.05% Tween-20 in
PBS) at 4°C for 1 hour. This was followed sequentially by washes with
PBST, incubation with horseradish peroxidase-conjugated goat anti-mouse
IgG (diluted 1:1000, Roche), washes with PBST, incubation in 10 ml of
chemiluminescent substrate solution (LumiLight; Roche), and exposure to
x-ray film (Biomax ML; Eastman Kodak, Co.) with development to
visualize the immunoreaction.
Other Assays
SDS-PAGE was performed with 10% to 20% or 4% to 12%
polyacrylamide gradient gels in SDS-tricine or SDS-Tris-glycine,
respectively (Novex, San Diego, CA). Protein concentration was
determined using a protein assay (Bio-Rad Laboratories, Hercules, CA).
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Results |
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85 kDa in retina plasma membranes consistent with the presence of
a cell-surface receptor protein in the bovine neural retina.
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Localization of PEDF Binding Sites in the Bovine Retina
Fl-PEDF was used to determine the distribution of PEDF binding
sites in cryosections of bovine retina. Confocal microscopy of
cryosections incubated with Fl-PEDF showed dense staining in the region
of the inner segments (IS) of photoreceptor cells (Figs. 5A
5C
5G) and in the ganglion cell layer (GCL; Figs. 5A
5J
). The
fluorescein signal decreased significantly when cryosections were
incubated with Fl-PEDF plus an excess of unmodified protein (Fig. 5H)
,
in the absence of ligand (Figs. 5D
5F)
or with anti-rabbit IgG labeled
with fluorescein (data not shown), as a negative control, indicating a
specific and competitive Fl-PEDF binding to the retina. The binding
pattern to the IS was found consistently among four different bovine
eyes. In three of the specimens, the staining was also observed in
large ganglion cells, but not all the cells were stained (Fig. 5L)
. The
RPE appeared to have intrinsic fluorescence, as illustrated by
comparing Figure 5I
with Figures 5G
and 5H
. Detection of Fl-PEDF on
cryosections by amplification of fluorescein signal by colorimetric
staining of anti-fluorescein and visualization by light microscopy
produced identical results.29
These results demonstrate a
discrete spatial distribution of specific Fl-PEDF–binding sites in the
photoreceptors and cells of the GCL of the bovine neural retina.
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These similarities suggest that PEDF interacts with a cell-surface protein in the bovine neural retina that is homologous to receptors in human retinoblastoma and rat cerebellar granule cells. The fact that the 44-mer peptide, the receptor-binding site of PEDF, blocked binding of PEDF to retina plasma membranes points to interactions with neurotrophic receptors present in the surface of retina cells. This conclusion is in agreement with in vitro survival effects of PEDF on retina cell cultures and in vivo protective effects on photoreceptor cells of the rd/rd and rds/rds mice.3
A spatial structural model for PEDF reveals that the 44-mer region, the receptor-binding site, is located in a distinct area opposite the glycosaminoglycan-binding region.28 The 44-mer has no amino acids with primary amines and is located in an area that has a negative electrostatic potential. In contrast, the glycosaminoglycan-binding region is densely populated with lysines that confer a basic electrostatic charge to the surface of the protein and are available to interact with the negatively charged glycosaminoglycans. Whereas 125I labeling modifies tyrosine groups, fluorescein labeling modifies primary amines, altering the basic electrostatic charge on the surface of the protein. We have shown that the chemical modification of PEDF’s primary amines with either fluorescein (Fig. 5D) or biotin28 abolishes the binding to glycosaminoglycans, but not to the PEDF receptor on the cell-surface of retinoblastoma Y-79 cells (Fig. 5A) . In addition, the Fl-PEDF is an active neurotrophic factor that shares the neuronal differentiating and survival activities in retinoblastoma and cerebellar granule cells with its unlabeled counterpart (personal observations, Vicente Notario and Joan P. Schwartz, 2000). Therefore, the use of Fl-PEDF allows the detection of interactions with the PEDF receptor while excluding those with glycosaminoglycan-rich areas of cells and tissue. These characteristics confer unique qualities on Fl-PEDF as a ligand for tissues and cells.
The present data also reveal the localization of PEDF binding sites in the neural retina. We found that the distribution of Fl-PEDF binding predominated in the IS of photoreceptor cells and also in cells of the GCL. Previous reports indicate that retinal pigment epithelial cells express the PEDF gene and secrete the mature PEDF protein (the ligand) into the interphotoreceptor matrix next to the neural retina.12 14 15 16 18 19 The Fl-PEDF–binding sites in the IS of the photoreceptor cells probably represent the cell-surface receptors available to interact with the extracellular ligand and agree with PEDF protective and morphogenetic effects on photoreceptors of the rd and rds mice,3 rat,4 and Xenopus laevis.5 Thus, PEDF may act directly on photoreceptor cells through a cell-surface receptor.
The effect of PEDF on ganglion cells is unknown, however, and the data
on Fl-PEDF–binding suggest for the first time that ganglion cells may
be targets for PEDF activity. Purified PEDF of 49,500 molecular weight
from bovine interphotoreceptor matrix has a retention time in a
TSK-300 gel column (TosaHaas, Japan) by HPLC that is slightly
behind than that of ovalbumin (a serpin of 43,000 molecular
weight).12
Because the Stokes radius of ovalbumin (hen
egg) is 3.05 nm (available at
http://itsa.ucsf.edu/hdeacon/Stokesradius.html), we estimate that
the one for PEDF is not larger than 3.05 nm. The size of PEDF suggests
a large protein and indicates a certain degree of difficulty in
diffusing through the outer limiting membrane (OLM). Although we have
not designed experiments to validate PEDF’s diffusion through the OLM
in the bovine retina, the following observations suggest that naturally
occurring PEDF molecules can cross the OLM in other species: (1)
Biotinylated-PEDF of 50 kDa injected in the vitreous of adult mice was
detected in the neural retina and RPE-choroid after injection (see Ref.
3
). (2) The intravitreally injected recombinant human
PEDF of 50 kDa had an effect on photoreceptors of rd and
rds mice throughout the retina.3
In addition,
immunohistochemistry of albino rat retina with polyclonal antiserum to
human PEDF shows some PEDF immunoreactivity distributed in cells of the
GCL,19
and as recently reported, PEDF
gene expression is present in cells of the GCL of the human
retina.30
Together, these observations point to
colocalization of PEDF receptors and ligand in cells of the GCL and
insinuate possible functional effects of PEDF on cells of the GCL.
Comparison with other neurotrophic factors indicates that ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) also have protective effects on photoreceptors.31 32 33 34 Localization of CNTF receptors by immunohistochemistry of the avian retina reveals a distribution in the outer segments of mature rods.35 In contrast, BDNF activates tyrosine kinase (Trk)-B receptors36 distributed in retinal pigment epithelial cells and cells of the inner retina,37 but not in photoreceptor cells of mouse38 or rat.39 Thus, the similarities between PEDF’s and CNTF’s protective effects and receptor distribution suggest that, as opposed to BDNF, both factors may act by interacting directly on photoreceptor cells.
In summary, using two PEDF ligands, we have demonstrated for the first time the presence of PEDF receptors in the neural retina. The demonstration of 125I-PEDF and Fl-PEDF binding in the bovine retina supports a role for this factor in the adult retina and provides an anatomic basis for investigations into the in vivo activity of PEDF in the neural retina. Further studies are necessary to confirm that the binding activity described herein is directed to functional receptors that, after interactions with their ligands, trigger the signal transduction events for neurotrophic activity on the neural retina. However, our data correlate with PEDF’s effects on the survival and morphogenesis of photoreceptor cells in vivo and retina cells in culture,2 3 4 5 suggesting that the PEDF binding sites on photoreceptor cells correspond to functional receptors. These observations imply that, in addition to binding in vitro, the PEDF–receptor interactions may serve to localize and direct PEDF activity in the neural retina.
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Acknowledgements |
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Footnotes |
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Submitted for publication February 16, 2001; revised July 2, 2001; accepted September 5, 2001.
Commercial relationships policy: P (SPB); N (all others).
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: S. Patricia Becerra, NIH-NEI-LRCMB, Building 6, Room 308, 6 Center Drive MSC 2740, Bethesda, MD 20892-2740. pbecerra{at}helix.nih.gov
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