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1 From the Department of Biochemistry and Molecular Biology and 4 Center for Macular Research, Department of Ophthalmology, University of British Columbia, Vancouver, British Columbia, Canada; 2 Hopitaux Universitaires de Strasbourg-ULP, France; and 3 Institut für Humangenetik, Biozentrum, Am Hubland, Würzburg, Germany.
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Abstract |
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METHODS. Rs1h mRNA expression was measured from the eyes of wild-type and rd/rd mice by Northern blot analysis and reverse transcription–polymerase chain reaction (RT-PCR). Specific antibodies raised against the N terminus of RS1 were used as probes to examine the properties and distribution of RS1 in retina, retinal cell cultures, and transfected COS-1 cells by Western blot analysis and immunofluorescence microscopy.
RESULTS. Rs1h mRNA expression was detected in the retina of postnatal day (P)11 and adult CD1 mice, but not homozygous rd/rd mice by Northern blot analysis. However, Rs1h expression was detected in rd/rd mice by RT-PCR. RS1 migrated as a single 24-kDa polypeptide under disulfide-reducing conditions and a larger complex (>95 kDa) under nonreducing conditions in the membrane fraction of retinal tissue homogenates and transfected COS-1 cells. RS1 antibodies specifically stained rod and cone photoreceptors and most bipolar cells, but not Müller cells, ganglion cells, or the inner limiting membrane of adult and developing retina as revealed in double-labeling studies. RS1 antibodies also labeled retinal bipolar cells of photoreceptorless mice and retinal bipolar cells grown in cell culture.
CONCLUSIONS. RS1 is expressed and assembled in photoreceptors of the outer retina and bipolar cells of the inner retina as a disulfide-linked oligomeric protein complex. The secreted complex associates with the surface of these cells, where it may function as a cell adhesion protein to maintain the integrity of the central and peripheral retina.
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Introduction |
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A characteristic feature of X-linked retinoschisis is the presence of streaks radiating outward from the parafoveal region of the retina. This spoke-like pattern results from cystic cavities that split the inner retina at the level of the nerve fiber and ganglion cell layers.6 7 8 Bilateral schisis is also found in the peripheral retina in approximately half of the affected persons. The extracellular space adjacent to the schisis cavity has been reported to contain amorphous material and filaments that merge with Müller cell membranes.8 Degeneration of photoreceptors and underlying retinal pigment epithelium is also evident in the macula and affected peripheral regions of the retina.
The gene (RS1, formerly XLRS1) responsible for X-linked juvenile retinoschisis has been identified by positional cloning.9 It consists of six exons and encodes a 224-amino-acid protein containing a hydrophobic leader sequence with a consensus signal peptidase cleavage site. RS1, also called retinoschisin, consists of a discoidin-like domain that is found in a family of proteins implicated in cell adhesion.9 10 11 12 13 14 A spectrum of genetic mutations is found in persons with X-linked retinoschisis. These include missense and nonsense mutations, insertion and deletion mutations, intragenic deletions, and splice-site mutations.9 15 16
RS1 expression is restricted to retinal tissue.9 In situ hybridization studies have further shown that the murine orthologue Rs1h is abundantly expressed in photoreceptors.14
To begin to define the role of RS1 in retinal cell biology and X-linked juvenile retinoschisis, we have examined the biochemical properties, cellular expression, and localization of RS1 in mammalian retina and cell cultures. We report here that RS1 is assembled as a disulfide-linked oligomeric protein complex that is expressed and secreted from bipolar cells as well as rod and cone photoreceptors. It interacts with the surface of these cells where it may function as a cell adhesion protein to stabilize the organization of the retina.
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Methods |
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RNA Isolation, Northern Blot Analysis, and cDNA Probes
Total RNA was isolated from whole eyes of adult,
rd/+ and rd/rd
mice (
8 weeks old) by the guanidinium thiocyanate
method.17
For Northern blot analysis, 12 µg total RNA
was run on each lane of a 1.2% agarose gel in the presence of
formaldehyde and blotted onto a nylon membrane. A 436-bp cDNA probe
(3F6R) representing coding exons 3 through 6 of the murine
Rs1h gene was amplified using primers rsm3F
(5'-TACCTCCTTAGACTGTATTCC-3') and rsm6R
(5'-GATGAAGCGGGAAATGATGG-3').13
The murine
Crx cDNA probe was generated by polymerase chain
reaction (PCR) using primers Crx-F (5'-GTCCCCCACCTCCTTGTCAG-3') and
Crx-R (5'-CCTCAAGTTCCCAGCAATCC-3').18
A 289-bp murine
ß-actin probe was generated by PCR using primers XAHR20
(5'-ACCCACACTGTGCCCATCTA-3') and XAHR17 (5'-CGGAACCGCTCATTGCC-3';
GenBank accession number X03765). The probes were radiolabeled by
random priming and consecutively hybridized to the same filter at
65°C in 0.5 mM sodium phosphate buffer (pH 7.2), 7% sodium dodecyl
sulfate (SDS), and 1 mM EDTA.19
Reverse transcription–polymerase chain reaction (RT-PCR) reactions were performed in a volume of 25 µl. Each reaction contained 1 µl of first-strand cDNA (from whole-eye mouse RNA) as template and 15 picomole primers. PCR conditions were: 94°C, 5 minutes; 94°C, 30 seconds; TA of each primer pair, 30 seconds; 72°C, 30 seconds; 72°C, 5 minutes. Each PCR was performed for 30 cycles. The primer sequences and reaction conditions were as follows:
Generation of the RS1 Antibody
A 17-amino-acid peptide (LSSTEDEGEDPWYQKAC) corresponding to
amino acids 22-39 of the human RS1 precursor protein9
was
conjugated to keyhole limpet hemocyanin and used to immunize a rabbit.
To confirm the specificity of immunolabeling, the RS1 antibody was
affinity purified from the antiserum, as previously
described.20
Another RS1 antibody was raised in a mouse
immunized with a glutathione-S-transferase fusion
protein containing the same N-terminal peptide.
Isolation of Retina Tissue and COS-1 Cell Extracts
Retina tissue from mouse or rat eyes was incubated in 400 µl
hypotonic buffer (5 mM Tris-HCl [ pH 7.4] containing 1 mM
Pefabloc SC protease inhibitor; Boehringer-Mannheim, Germany)
for 1 hour at 4°C and homogenized in a microfuge tube. The homogenate
was layered on 10% sucrose and centrifuged at 26,000 rpm for 20
minutes in a swinging-bucket rotor (model TLS-55; Beckman, Berkeley,
CA). The fraction collected above the 10% sucrose solution was defined
as the soluble fraction. The pellet resuspended in 100 µl of 10%
sucrose was defined as the retinal membrane fraction. Protein
concentration was determined by BCA assay (Pierce, Rockford, IL).
Monkey kidney COS-1 cells were transfected with the full-length human RS1 cDNA in pcDNA3 (Invitrogen, San Diego, CA), as previously described.21 Cells were homogenized in 10 mM MOPS buffer (3-[N-morpholino] propanesulfonic acid; pH 7.5) and separated into a soluble and membrane fraction by centrifugation on a gradient consisting of 40% (wt/wt) and 60% (wt/wt) sucrose.
SDS-PAGE and Western Blot Analysis
For sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE), samples (3 mg/ml protein) were added to an equal volume
of SDS cocktail (125 mM Tris-HCl [pH 6.8], 4% SDS, 40% sucrose,
0.02% bromophenol blue in the presence or absence of 8%
ß-mercaptoethanol) and run on a 12% polyacrylamide gel. Proteins
were transferred onto membranes (Immobilon; Amersham Pharmacia Biotech,
Arlington Heights, IL) and Western blots were labeled with the RS1
antiserum diluted 1:3000 in phosphate buffered saline-0.1% Tween 20
(PBS-T) or affinity-purified antibody diluted 1:500 in PBS-T in the
presence or absence of competing RS1 peptide, as previously
described.20
The blots were developed by enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech, Baie d’Urfé,
Quebec).
Immunofluorescence Labeling
Retina tissue was fixed with 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 1 to 2 hours and rinsed in 0.1 M
phosphate buffer (pH 7.4) containing 10% sucrose. Cryosections were
blocked with PBS containing 0.2% Triton X-100 and 10% goat serum for
20 minutes and labeled overnight with the RS1 antiserum or affinity
purified antibody diluted 1:2000 in PBS-T containing 2% goat serum.
Samples were rinsed in PBS-T and labeled for 1 hour with Alexi
594–conjugated goat anti-rabbit immunoglobulin (Molecular Probes,
Eugene, OR). In control samples, sections were labeled with the RS1
antibody in the presence of 0.1 mg/ml RS1 peptide. For double-labeling
studies, sections were also labeled with cell-specific monoclonal
antibodies (mAbs) and Alexi 488–conjugated goat anti-mouse
immunoglobulin. mAbs were: protein kinase C (PKC
) and vimentin
(Sigma, St. Louis, MO); PKCß (Seikagaku America, Falmouth, MA);
monoclonal antibody (MAb) 115A1022
against rat olfactory
bulb (ROB; kindly provided by Shinobu C. Fujita, Mitsubishi Kasei
Institute of Life Sciences, Tokyo, Japan). Polyclonal antibody
to cellular retinal binding protein (CRABLP) was a generous gift from
Jack Saari (University of Washington, Seattle, WA). Labeled
retinal sections were examined under a fluorescence microscope
(Axioplan2; Zeiss, Munich, Germany) equipped with a digital image
analysis system (Eclipse; Zeiss).
Retinal Cell Cultures
Retinal cell cultures were established as previously
described.23
Briefly, retinas from adult pig eyes were
digested with 0.2% papain in PBS for 20 minutes at 37°C, triturated
by passage through a Pasteur pipette and seeded at 5 x
105 cells/cm2 onto laminin-treated coverslips
in Dulbecco’s modified Eagle’s medium (DMEM)-Ham’s F12
supplemented with 2% fetal calf serum. The medium was refreshed every
week. For immunofluorescence microscopy, the cells were fixed in 4%
paraformaldehyde, blocked in PBS-T, and incubated overnight with
anti-RS1 antiserum diluted 1:3000. After a rinsing in PBS, the cells
were permeabilized in 0.1% Triton X-100 and relabeled with
retinal-cell–specific antibodies and fluorescence-labeled secondary
antibodies, as described.
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Analysis of RS1 in Retinal and Transfected COS-1 Cell Membranes
Antibodies raised against a peptide corresponding to the N
terminus of mature RS1 were used to examine the biochemical properties
of RS1 in mouse retinal extracts by SDS-PAGE and Western blot analysis.
RS1 migrated in the membrane fraction of retinal homogenates as single
24-kDa polypeptide under disulfide-reducing conditions and a large
complex (>95 kDa) near the top of the gel under nonreducing conditions
(Fig. 2A
). No signal was detected in the soluble fraction. In control studies,
labeling was abolished when excess competing RS1 peptide was included
during the primary-antibody–labeling step (data not shown).
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Distribution of RS1 in Mammalian Retina
The distribution of RS1 in mammalian retina was examined by
immunofluorescence microscopy. The pattern of outer and inner retina
labeling was similar for bovine (Fig. 3)
, human (Fig. 4
and 5)
, mouse (Fig. 6) , and rat (Fig. 7)
and when RS1 antibodies raised in either a rabbit or mouse were used
for immunolabeling. Intense staining was observed in the photoreceptor
inner segment layer, particularly in the ellipsoid region immediately
adjacent to the outer segments. Below the outer limiting membrane, more
moderate staining of the photoreceptor cell body and outer plexiform
layers was observed. In bovine (Fig. 3)
and human retina (Figs. 4
5)
,
cone inner segment and cell-body staining was outlined against the more
abundant rod photoreceptors, a labeling pattern that is consistent with
the distribution of RS1 along the surface membrane of cone cells. Weak,
diffuse labeling of the photoreceptor outer segment layer was observed
in sections containing intact retinal pigment epithelial (RPE) cells
(Fig. 3)
. This labeling most likely represented weak staining of the
interphotoreceptor matrix surrounding the photoreceptor outer segments.
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(Fig. 3)
. As previously
reported,24
the anti-CRALBP antibody labeled the RPE layer
and Müller cells that extend from the outer limiting membrane
down through the INL and GCL to the inner limiting membrane. RS1
staining did not resemble CRALBP staining. PKC antibody stained cell
bodies and neurites of rod bipolar cells.25
26
Some PKC
immunoreactive bipolar cells were double labeled by the RS1 antibody,
although the majority of RS1-immunopositive cells were PKC
immunonegative (see also Figs. 5
and 7
). The specificity of RS1 immunolabeling of retinal sections was examined in a series of control studies. The pattern of labeling was the same for antiserum or affinity-purified RS1 antibody and for antibodies raised in a rabbit or mouse. Moreover, addition of excess RS1 peptide during labeling abolished the staining of the photoreceptors and inner retinal cells (see Fig. 6 ).
RS1 Staining in Human Macula and Peripheral Retina
Because X-linked juvenile retinoschisis affects
primarily central vision, we examined the distribution of RS1 within
the macula and foveal regions of human retina. The RS1 antibody labeled
the inner segments of foveal and macular cone photoreceptors (Figs. 4A 4C)
. The distal tips of cone inner segments were intensely labeled.
There was also moderate staining of photoreceptor cell bodies and a few
inner retinal cells, presumably bipolar cells (discussed later) that
were distributed close to the inner limiting membrane. Double labeling
of foveal sections with Müller-cell–specific vimentin
antibody,27
clearly demonstrated that RS1 immunostaining
was distinct from Müller cell staining (Figs. 4A
4B)
. In the
macula, the pattern of RS1 staining was similar to that observed for
peripheral retina (Fig. 4C)
: Very prominent labeling of rod and cone
inner segments, strong labeling of cone cell bodies, and moderate
labeling of Henle fibers and many cells within the INL. RS1 labeling
was absent from the INL margins and entire GCL, and distinct from the
vimentin and CRABLP labeling of Müller cells (Fig. 4D)
.
To more precisely identify inner retinal cells that were immunopositive
for RS1, human macula sections were double labeled with RS1 and
antibody markers for bipolar cell subclasses.26
These
included PKC specific for rod bipolar cells, PKCß specific for
cone off-bipolar cells and ROB MAb 115A10 specific for a subclass of
cone on-bipolar cells and rod bipolar cells.22
26
Figure 5
shows that RS1 immunoreactivity was associated with the majority of
bipolar cell types labeled with PKC and ROB antibodies (staining with
RS1 and a cocktail of the three mAbs produced coincident labeling in
>90% of the cases, data not shown).
RS1 Staining of Photoreceptorless Mice
The retina of wild-type and rd/rd
mice were labeled with the RS1 antibody to determine whether inner
retina cell labeling persisted in the absence of a
photoreceptor cell layer (Figs. 6A 6B
6C
6D
6E
6F)
. As in the case of
wild-type retina, the RS1 antibody specifically stained the cell bodies
and processes of bipolar cells in rd/rd mice 2
and 12 months old. Irregular immu-nostaining below the GCL was
observed in rd/rd mice that was not observed in
wild-type mice. Because rd/rd mice are known to
contain a small number of surviving cone photoreceptors, we labeled the
retina of an rd/rd cl mouse, which is without
both types of photoreceptors.28
As shown in Figure 6H
, the
RS1 antibody specifically stained bipolar cells of the inner retina
similar to that observed for wild-type and rd/rd
mouse.
Developmental Expression of RS1
The temporal expression and distribution of RS1 during development
of the rat retina was also examined by immunofluorescence microscopy
(Fig. 7) . At P6, weak RS1 staining was seen along the scleral margin of
the neuroblastic zone, and by P8, labeling of the newly forming
photoreceptor outer nuclear layer was evident. The intensity of outer
retinal staining increased over time with the emergence of intense
staining of the newly formed inner segment layer at P10. Weak RS1
labeling of inner retinal cells just below the photoreceptor cell layer
was also first observed at this time. By P12, the adult pattern of
expression was present. Staining continued to intensify with age, and
as in the case of human retina, many RS1-immunopositive cells in the
adult rat INL were also stained with the bipolar-specific ROB MAb
115A10 antibody (Figs. 7J
7K)
.
Expression of RS1 in Cultured Retinal Bipolar Cells
RS1 expression in retinal cell cultures derived from enzymatically
dissociated retinal tissue was examined by immunofluorescence
microscopy. No RS1 immunolabeling was detected for retinal cells
maintained in culture for 1 week (data not shown). However, after 4
weeks, a significant number of cells were labeled with the RS1 antibody
(Fig. 8A ). Diffuse labeling extended to the periphery of the cells, a pattern
characteristic of cell surface labeling of nonpermeabilized
cells.21
A number of RS1 positive cells were also PKC
positive indicating that they were rod bipolar cells (Fig. 8B)
. PKC
labeling, however, was primarily localized around the nucleus, a
pattern characteristic of intracellular labeling. A large number of
RS1-positive cells also labeled with MAb 115A10 indicating that RS1 was
expressed in a subset of cultured on-bipolar and rod bipolar cells.
Horizontal and ganglion cells in these cultures were not labeled with
the RS1 antibody, as revealed in double-labeling studies using
cell-specific antibodies.23
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In the present study, we have confirmed that RS1 is abundantly expressed in rod and cone photoreceptor cells. However, our results differ from the earlier studies cited in two important respects. First, our studies indicate that RS1 is expressed in bipolar cells of the inner retina as well as photoreceptors, although at lower levels. Second, double-labeling experiments using a variety of cell surface markers reveal that RS1 is specifically associated with rod and cone photoreceptors of the outer retina and bipolar cells of the inner retina, but not Müller cells, ganglion cells, or the inner limiting membrane.
Several lines of evidence indicate that RS1 is expressed in retinal bipolar cells. By immunofluorescence microscopy, we detected RS1 on bipolar cells of all mammalian retinas examined including 1-year-old rd/rd mice without rods and most cones, and rd/rd cl mice devoid of both rod and cone photoreceptors. We also observed RS1 expression on bipolar cells dissociated from retinal tissue and maintained in culture for 4 weeks. Because RS1 was not detected in these cells after 1 week in culture, we conclude that RS1 expression in bipolar cells maintained in culture for 4 weeks arises from newly synthesized protein. Finally, although we did not detect Rs1h mRNA expression in rd/rd mouse retina by Northern blot analysis, in agreement with the results of Reid et al.,14 we detected Rs1 expression using the more sensitive technique of RT-PCR. Together, these results provide strong evidence that RS1 is expressed in bipolar cells as well as in photoreceptors.
It is becoming increasingly clear that in situ hybridization and Northern blot analysis are useful techniques to detect abundantly expressed mRNA but often do not have the sensitivity or reliability to measure low-level mRNA expression. For example, the transcription factor Crx was initially thought to be expressed only in photoreceptors of the retina on the basis of a strong in situ hybridization signal in the photoreceptor layer and no signal18 31 or an extremely weak signal32 in the inner retina. In the present study, we also were unable to detect Crx expression in rd/rd mice by Northern blot analysis. However, recent immunocytochemical labeling studies have established that Crx is also expressed at low levels in retinal bipolar cells.33 The photoreceptor ABC transporter ABCA4 (formerly ABCR) mRNA expression has been observed in rod, but not cone, cells by in situ hybridization.34 Recently, however, ABCA4 has been detected in human cone as well as rod cells by immunofluorescence microscopy and Western blot analysis techniques, a result that has important implications in the pathogenesis of Stargardt disease.35 These and other studies indicate that caution must be exercised in interpreting cell-specific expression when relatively insensitive techniques are used. The inability to detect RS1 expression in nonphotoreceptor cells by conventional in situ hybridization and Northern blot analysis is most likely due to the limited sensitivity of these techniques.
Immunofluorescence labeling studies using highly specific RS1 antibodies and a variety of cell-specific markers identified the RS1-immunopositive inner retinal neurons as rod and cone bipolar cells in all mammalian species examined. RS1 labeling was not detected on Müller glial cells, ganglion cells, or along the inner limiting membrane of either peripheral or central retina. These results are in marked contrast to previous reports showing more widespread retinal tissue labeling.29 30 It is unclear why the antibodies used in our study showed selective staining of photoreceptors and bipolar cells, whereas an N-terminal antibody used by another group of investigators29 30 labeled photoreceptors and most other cells of the inner retina. However, it is interesting to note that the antibody used in later studies labeled multiple bands in Western blot analysis of retinal extracts, leading one to question the specificity of this antibody.
The RS1 gene codes for a polypeptide that has primary structural features characteristic of an extracellular cell adhesion protein.9 It has an N-terminal 23-amino-acid hydrophobic signal sequence with a signal peptidase cleavage site, a signature characteristic of proteins destined for secretion from cells. It also contains a discoidin domain found in discoidin I and II, hemocytin, neuropilin, and other proteins implicated in cell adhesion processes.9 10 11 12 13 14 36 37
Biochemical and immunocytochemical experiments performed in this study support the view that RS1 is a secreted protein complex that interacts with cell surfaces. RS1 from retinal and COS-1 cell membranes was found to migrate as a single 24-kDa polypeptide under reducing conditions and a large (>95-kDa) complex under nonreducing conditions. This indicates that RS1 subunits assemble into a large, disulfide-linked complex within the oxidizing environment of the endoplasmic reticulum lumen. The intense peripheral RS1 staining of bipolar cells and cone inner segments and RS1 labeling of nonpermeabilized cultured bipolar cells is consistent with the localization of a significant fraction of RS1 on the surface of these cells. Together, these results support the model in which RS1 is synthesized in photoreceptor and bipolar cells as a disulfide-linked oligomeric complex. This secreted complex associates with the surface of photoreceptor and bipolar cells. The molecular composition of the RS1 complex and identity of the interacting cell surface components are currently under investigation.
Many missense mutations associated with X-linked juvenile retinoschisis involve the introduction or substitution of cysteine residues.9 15 It is likely that these cysteine mutations cause misfolding and defective subunit assembly of RS1, as recently reported for the photoreceptor-specific protein, peripherin/rds.38
The role of RS1 in the pathogenesis of X-linked juvenile retinoschisis remains to be determined at a molecular level. Earlier histopathologic and electrophysiological studies led to the conclusion that defective Müller cells were directly responsible for the disease state.5 8 However, expression and localization of RS1 on photoreceptors and bipolar cells suggest that these cells are directly involved in the pathogenesis of X-linked juvenile retinoschisis. The discoidin-like domain of RS1 probably participates in cellular adhesion, possibly by stabilizing the association of the extracellular matrix to the surface of photoreceptors and bipolar cells. Mutations in RS1 that compromise the putative role of RS1 as an adhesion protein could destabilize the retinal tissue. The foveal region may be especially compromised because of its unique cellular organization, constituting a weak spot. This fragility is seen in another human retinal disorder, that of the macular hole in which vitreal shrinkage pulling at the retina leads to detachment at the level of the macula.39 It is unclear why microcysts form between the GCL and the inner limiting membrane, a region that appears to contain little, if any, RS1. Perhaps, the extracellular matrix required to maintain the integrity of the retinal tissue between the GCL and the inner limiting membrane extends into the INL where it is anchored to retinal bipolar cell surfaces through the RS1 complex. Loss of the RS1 adhesion function in persons with X-linked retinoschisis could lead to destabilization of the matrix and splitting of the inner retina cell layer.
Most persons with X-linked retinoschisis produce ERGs with normal a-waves originating from photoreceptors but reduced or absent b-waves.3 4 Although the b-wave was initially thought to be derived from Müller cell depolarization, there is growing evidence to suggest that the b-wave originates directly from depolarizing bipolar cells.3 40 41 Immunostaining showing that RS1 is associated with bipolar cells is consistent with the direct involvement of bipolar cells in b-wave activity and in X-linked juvenile retinoschisis.
In summary, the results of this study indicate that RS1 is expressed in both photoreceptors and bipolar cells as a secreted, disulfide-linked oligomeric protein. On export to the extraretinal space this complex binds to the surface of photoreceptors and bipolar cells where it may function as a cellular adhesion protein to maintain the integrity of the retina tissue.
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Footnotes |
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Supported by the Medical Research Council of Canada, the National Eye Institute (EY 02422), the Deutsche Forschungsgemeinschaft (We 1259/12-1) and a North Atlantic Treaty Organization Travel Fellowship (960236).
Submitted for publication September 6, 2000; revised November 9, 2000; accepted November 15, 2000.
Commercial relationships policy: N.
Corresponding author: Robert S. Molday, Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. molday{at}interchange.ubc.ca
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