Molecular Vision 2006; 12:892-901 <http://www.molvis.org/molvis/v12/a101/> Received 22 February 2006 | Accepted 4 August 2006 | Published 10 August 2006

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Retinoschisin forms a multimolecular complex with extracellular matrix and cytoplasmic proteins: interactions with β2 laminin and αB-crystallin

Marie-France Steiner-Champliaud,¹ José Sahel,¹ David Hicks^1,2
 
 

¹Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, INSERM U 592, Hópital St. Antoine, Paris, France; ²Laboratoire de Neurobiologie des Rythmes, CNRS UMR 7168/LC2, Strasbourg, France

Correspondence to: Dr. David Hicks, Laboratoire de Neurobiologie des Rythmes, CNRS UMR 7168/LC2, INCI, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France; Phone: 33 388 45 67 23; FAX: 33 388 45 66 54; email: photoreceptor67@hotmail.com

Abstract

Purpose: X-linked juvenile retinoschisis is a rare early-onset retinal degeneration characterized by the formation of cysts and loss of the electroretinogram "b" wave. The affected gene normally codes for retinoschisin (Rs1), a secreted protein containing a large discoidin homology domain. Rs1 seems to be principally synthesized in the photoreceptors, but its structure and spectrum of effects when mutated indicates association with other proteins. The present study searched for retinal proteins capable of interacting with Rs1.

Methods: Western blotting and RT-PCR of isolated outer nuclear (photoreceptors), inner nuclear and ganglion cell layers, and cell culture compartments were performed to verify sites of Rs1 synthesis and distribution. Potential Rs1 binding partners were searched for with affinity columns generated using specific Rs1- and β2 laminin-antisera. Following loading with total protein extracts from porcine retina, bound proteins were acid eluted and visualized by Coomassie blue staining of SDS-polyacrylamide gels, and selected bands were excised for tryptic peptide digestion and sequencing. Using single and double labeled immunohistochemistry, candidate binding partner distributions with that of Rs1.

Results: Whereas Rs1 mRNA was confined to the outer nuclear layer, Rs1 protein was found throughout the retina, including within the ganglion cell layer. One protein that was retained on Rs1 affinity columns was identified as αB crystallin, which showed partially overlapping distribution with Rs1 in the retina, mainly in the interphotoreceptor matrix and outer plexiform layer. Also, β2 laminin columns retained Rs1, and again shared partial distribution patterns. Finally, unidentified peanut agglutinin-binding proteins from the retina also bound to Rs1, αB crystallin and β2 laminin.

Conclusions: Taken together, these data demonstrate that Rs1 associates with different proteins during its synthesis and secretion, forming a multimolecular complex which presumably forms a stabilizing scaffold for retinal synapses, and possibly overall tissue integrity.

Introduction

X-linked juvenile retinoschisis is a recessively inherited, bilateral retinal degeneration affecting young males [1-4]. Affected individuals typically experience mild to severe loss in visual acuity in the first decade of life followed by progressive atrophy of the macula in mid to later years. About 50% of the patients also suffer from a decrease in visual field. Electroretinograms (ERGs) of most affected individuals exhibit normal a-waves, characteristic of normal photoreceptor function, but severely reduced b-waves, indicating a perturbation in synaptic transmission between photoreceptors and second order neurones (bipolar and horizontal cells) [5-7].

The gene (xlrs1) affected in X-linked juvenile retinoschisis has been identified by positional cloning [8], and consists of six exons encoding a 224 amino acid protein containing a 23 amino acid hydrophobic leader sequence with a consensus signal peptidase cleavage site. Retinoschisin protein (Rs1) consists mostly of a discoidin-like domain that is found in a heterogeneous family of proteins implicated in cell adhesion [9-11]. A recent studies [12] demonstrated it forms multimers through disulfide bonding. A spectrum of genetic mutations is found in individuals with X-linked retinoschisis, including missense and nonsense mutations, insertion and deletion mutations, intragenic deletions, and splice-site mutations [4,13,14]. These mutations occur chiefly within the discoidin-like domain, and result in protein misfolding, failure to insert into the retinoplasmic membrane and defective formation of multimers. Several studies indicate that missorting, protein retention and compromised subunit oligomerization as a consequence of xlrs1 mutations, are major pathogenic mechanisms [15-17]. In situ hybridization studies have shown that xlrs1 mRNA is uniquely expressed in photoreceptors [18-20]. Recent studies by the same group indicate that the protein is recaptured from the interphotoreceptor matrix by pinocytosis at the level of the apical microvillosities of the Müller glia, then transported downward into the interior of the tissue and subsequently re-released near its target sites [21]. Other groups have contested this model, and report in addition to expression in photoreceptors, protein localization in the inner nuclear and ganglion cell layers [22,23], consistent with the observed clinical manifestations of retinal splitting [4].

xlrs1 null mice display a retinal phenotype similar to that observed in humans, namely frequent cystoid spaces and splitting of inner retinal layers, together with a marked reduction in ERG b-wave amplitude [24]. Ultrastructural observation of these mice demonstrated structural abnormalities of synapses within the outer plexiform layer, including disorganized ribbons and large extracellular spaces. Viral-mediated gene therapy of xlrs1 null mice is able to rescue and even reverse the degenerative effects [25,26]. Since Rs1 is a secreted protein, its effects on synaptic organization would imply interaction with other molecules, both resident in the extracellular matrix (ECM) and within cells. We have generated affinity columns using anti-Rs1 polyclonal antibody, as well as candidate retinal ECM proteins, to examine potential molecular interactions. Both β2 laminin and αB crystallin emerge as candidate binding partners, as well as unidentified glycoproteins. In addition, separation of retinal layers by vibratome sectioning revealed xlrs1 mRNA only in the photoreceptor layer, while Rs1 protein was detected in abundance throughout the retina, including the ganglion cell layer.

Methods

Antibodies

Rabbit polyclonal anti-Rs1 antiserum was raised against the oligopeptide LSTEDEGEDPWYQKA (amino acid residues 23-37) conjugated to keyhole limpet hemocyanin and affinity purified on a HitrapTM Protein G HP (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Antibody specificity was verified by western blotting and immunohistochemistry. The D5 monoclonal antibody against laminin β2 chain was a generous gift of Dr. W. Brunken (Tufts University, Boston, MA) [27]. The anti-αB-crystallin monoclonal antibody was purchased from Stressgen Biotechnology (Victoria, BC, Canada). Biotinylated Peanut Agglutinin (PNA) was purchased from Sigma Life Science Technologies (Cergy Pontoise, France).

Elution of retinoschisin and interacting proteins

Eyes (about 25) from freshly killed adult domestic pigs were enucleated and transported from the slaughterhouse to the laboratory on crushed ice, where the retinas were dissected out as described previously [28]. They were then washed three times in 50 ml of cold 0.1 M phosphate buffered saline (PBS), pH 7.4, containing protease inhibitors (N-ethyl maleimide, 625 mg/l; phenylmethylsulfonyl fluoride, 150 mg/l). They were ground in a 50 ml that contained Tris HCl (50 mM, NaCl 0.5 M, Urea 2 M, EDTA 50 mM, pH 7.4, plus protease inhibitors using the same concentration as noted). The volume was adjusted to 100 ml and stirred overnight at 4 °C on a magnetic stirrer, after which the supernatant was collected and dialyzed against PBS (3 changes, 1 l each) at 4 °C. The extract was centrifuged at 45,000x g for 1 h, and Rs1 was purified by clearing the extract on gelatin-Sepharose, then passed over either a monoclonal (D5) antilaminin IgG/Sepharose affinity column or a polyclonal anti-Rs1 IgG/Sepharose column. Anti-Rs1 and antilaminin β2 antibody affinity columns were prepared by cyanogen bromide activation of Sepharose beads (1 g) previously published methods [29], and packed in mini-columns (length 5 cm, diameter 0.5 cm). Columns were connected to an automated fraction collector, with UV monitoring of 100 μl fractions at OD₂₈₀. The columns were washed repeatedly with PBS until the eluate returned to baseline values, and proteins eluted with 1 M acetic acid. In control experiments, protein extracts were loaded onto BSA-Sepharose columns and eluted in the same way.

Preparation of retinal layers

Different isolated strata of retina were prepared from 35-day-old C57 mice by planar vibratome sectioning as described before [30]. Briefly, neural retinas were dissected carefully to avoid mechanical disruption of layers, then cemented onto a gelatin block, photoreceptor face down. The vibratome blade was progressively lowered until just in contact with the inner limiting membrane, then successive cuts of 40 μm to remove the ganglion cell layer (GCL), and part of the inner plexiform layer (IPL), 60 μm to remove the inner nuclear layer (INL) and 120 μm to remove the outer nuclear layer (ONL) were performed. Light microscope monitoring of fractions was used to control purity of each fraction, based on morphological criteria (presence of abundant blood vessels and nerve fiber tracts in GCL; heterogeneous cell body sizes and blood vessels in INL; and regular cell body packing and outer segments in ONL). In addition, RT-PCR analysis of transcripts specific to INL (mGluR6, specific to rod bipolar cells [31]) and rhodopsin (specific to rods [32]; Table 1) were used to verify purity. Samples were collected in clean eppendorf tubes and immediately frozen in liquid nitrogen until use in western blotting and RT-PCR analyses.

Preparation of photoreceptor cultures

Highly enriched cultures of rod and cone photoreceptors were prepared from postmortem adult pig retinas using methods described previously [28]. Briefly, following dissection and digestion in papain, cells from the first two supernatants were collected and seeded at an initial density of 5x10⁶ in 35 mm tissue culture dishes in Neurobasal A medium supplemented with 2% fetal calf serum. After 2 days in vitro, medium was replaced with chemically defined medium, Neurobasal A containing B27 supplement. After a further 2 days in vitro, medium was replenished and left for a further 24 h. Medium (1 ml) was then collected and centrifuged at 10,000x g for 10 min, and soluble proteins were precipitated by TCA treatment, resuspended in 50 μl Laemmli buffer and analyzed by western blotting (see the fourth paragraph below).

Immunofluorescence analyses

Pig retinas were obtained from eyes collected at the slaughter house. Tissues were fixed in 4% paraformaldehyde in PBS for 2-6 h, then embedded in Tissue Tek (Sakura Finetek Europe BV, Zoeherwoude, Netherlands), frozen and sectioned by cryostat. The sections were blocked in PBS containing 0.1% bovine serum albumin (BSA), 0.1% Tween 20, 10% normal goat serum and 0.1% sodium azide (buffer A), then incubated overnight at 4 °C in the different primary antibodies (as indicated in the text and figure legends) or lectin diluted in the same buffer. Secondary antibody incubation was performed at room temperature for 60 min with Alexa (594 or 488) goat anti-rabbit or anti-mouse IgG conjugated antibodies (Molecular Probes Ltd., Eugene, OR). Sections were then washed thoroughly and mounted in PBS: glycerol for observation using either a Nikon Optiphot-2 fluorescence microscope equipped with differential interference contrast objectives, or by a confocal laser scanning microscope (Zeiss LSM 510 v2.5) scanning device mounted on a Zeiss Axiovert 100 inverted microscope.

Protein sequencing

Protein sequencing was performed with minor modifications of published methods [33]. Briefly, the eluate from the Rs1 column was resolved on a polyacrylamide gel in presence of 2-mercaptoethanol. The band of interest was excised and digested with trypsin. Analysis of the digest was performed at the Protein Sequencing Laboratory, IGBMC Strasbourg, with matrix-assisted laser desorption time-of-flight mass spectrometry performed on a Finnigan Lasermat 200 [34].

Reverse transcriptase polymerase chain reaction

RNA was purified from sheets obtained by vibratome sectioning of two adult normal (C57) mice retinas using the RNAeasy kit (Qiagen SA, Courtaboeuf France). RNA concentrations from pooled GCL, INL, and ONL fractions varied from 8.6-12.6 ng/μl. PCR amplifications using the different primer pairs listed in Table 1 were performed as follows: denaturation 2 min at 94 °C, 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, with a final extension period at 72 °C of 7 min. In addition, mRNA loading was checked with PCR primers for the housekeeping enzyme glycerol-6-phosphate dehydrogenase (data not shown). The experiments were repeated three times with identical results.

Western blotting

Total protein levels of single retinal layers were determined by Bradford assay, using an initial series of retinal preparations, as approximately 350-400 μg for ONL, 150-200 μg for INL, and 25-50 μg for GCL. Retinal layers were resuspended in 35 μl of Laemmli buffer, then run on 10% SDS-PAGE [35], the total contents of each separate fraction run on a single lane. Prestained low molecular weight range markers were run on a parallel lane. Proteins were transferred onto nitrocellulose membranes using a standard gel transfer apparatus, Tris 25 mM, glycine 0.192 M, methanol 20% for 1 h at 100 V [36]. The membrane was re-equilibrated in PBS-Tween 0.5% and incubated in PBS-5% fat-free milk for 1 h. After rinsing in PBS-Tween, the membrane was incubated in primary antibody overnight, rinsed three times in PBS-Tween and incubated in secondary antibody for 2 h, then rinsed three times in PBS-Tween. Immunoreactive bands were visualized using a Pierce Super Signal Substrate Kit (Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer's instructions. BioMax films were placed in contact with membranes for less than 25 s and developed according to the manufacturer's instructions.

Results

Distribution of retinoschisin

As previously published [22] for retinas from other mammalian species, immunostaining of adult pig retinal sections with anti-Rs1 antiserum showed intense labeling at the level of the interplexiform matrix (IPM) surrounding the photoreceptor inner segments. Additional label was seen surrounding some photoreceptor cell bodies and their processes within the ONL, principally lying along the scleral margin as well as within the outer plexiform layer (OPL) and in a subpopulation of INL neurones extending down their axons into the IPL (Figure 1). Immunohistochemical labeling was also detectable within the GCL and nerve fiber layer (NFL), although less intensely compared to other layers. Pre-adsorption of the Rs1 antibody with 100x excess immunizing peptide completely blocked all staining (data not shown).

These immunohistochemical data were complemented by western blotting and RT-PCR studies of retinal distributions of Rs1 protein and xlrs1 mRNA, respectively. Using a vibratome technique we separated adult mouse retina into three fractions; ONL (containing exclusively photoreceptor cell bodies and distal portions of Müller glia), INL (containing a mixture of bipolar, amacrine and Muller glial cell bodies), and GCL (containing retinal ganglion cells and their axons, as well as displaced amacrine cells and astrocytes). The protocol is shown schematically in Figure 2A. RT-PCR analyses were performed on mRNA prepared from single isolated samples. Quality and purity of the respective RNA preparations was confirmed by amplification of the cell-specific markers mGluR6 (bipolar cells) and rhodopsin (rods). Rhodopsin transcripts were detected in the ONL and INL (indicating contamination of the latter by rod mRNA), but not in the GCL. mGluR6 product was observed in the GCL and INL, but not in the ONL (Figure 2B). xlrs1 transcripts were only found in the ONL with a clear band observed at the expected product size (507 bp, Figure 2B). xlrs1 product transcripts were undetectable in the INL and GCL samples under the conditions used (Figure 2B). With respect to the western blotting analyses from similar fractions obtained from parallel samples, we observed an immunoreactive band of about 25 kDa in all three samples (Figure 2C). Finally, Rs1 protein was also detected in media conditioned by rod and cone photoreceptors during 24 h. It was also present in cell extracts prepared from the same cells, but was not found in medium that had not been incubated in the presence of cells (Figure 2D).

Interaction of retinoschisin with αB-crystallin

A crude total protein extract prepared from 25 pig retina was loaded onto the anti-Rs1 column, and after extensive washing with PBS the column was eluted with 1 M acetic acid. The eluate was resolved on SDS-Page and stained with Coomassie Blue. Many proteins eluted at molecular masses >60 kDa, but relatively few were visible at lower weights (Figure 3, left lane). The band at about 25 kDa represented Rs1 itself (as demonstrated by immunoblotting: data not shown). We chose to excise the band immediately below Rs1 (i.e., band at about 23 kDa) and process it for mass spectrometric sequence analysis after digestion with trypsin. Three peptides derived from this digestion were identified as corresponding to αB-crystallin, with an identical match to 28% of the protein. To confirm this result, we performed a western blot of the eluate using anti αB-crystallin antibody. In the right lane of Figure 3, a prominent band of about 23 kDa is present, in addition to another band at about 40 kDa which might be a dimer of αB-crystallin.

Double immunostaining of adult porcine retinal sections with monoclonal anti-αB-crystallin and polyclonal anti-Rs1 revealed partially overlapping distributions. αB-crystallin immunoreactivity was observed mainly within the outer IPM, although staining was also seen in the proximal IPM at the same level as Rs1. In addition, the two immunolabels were observed surrounding photoreceptor cell bodies in the ONL, and across the INL and IPL (Figure 4). αB-crystallin immunoreactivity was also strong in axon bundles within the NFL. Taken together with the affinity column data, these results suggest that Rs1 interacts with αB-crystallin.

Interaction of retinoschisin with laminin

The Rs1 antibody column retentate was also screened for the presence of β2 laminin by western blotting with a specific antibody. Western blot experiments showed the presence of a high molecular mass band about 180 kDa, corresponding to the β2 chain of laminin 14 and 15 (Figure 5A). To confirm a possible interaction between this extracellular matrix protein and Rs1, retinal protein extract was loaded onto an anti-β2 laminin antibody column and eluted as aforedescribed. Western blotting studies of the eluate with the Rs1 antibody clearly showed the presence of this protein (Figure 5B).

We also performed immunohistochemistry for β2 laminin and Rs1. Although we were unable to perform double labeling, as the D5 monoclonal only works on unfixed tissue, we again observed partially overlapping distribution patterns, though distinct from that seen for aB crystallin. β2 laminin immunostaining was observed within the IPM, surrounding cell bodies in the ONL and INL and outlining Müller cell extensions (Figure 6). Therefore, we conclude that Rs1 also has the potential to interact with laminins 14 and 15.

Interaction of retinoschisin and PNA-binding proteins

In a separate series of experiments, retinal extracts were loaded onto a PNA lectin affinity column and eluted with excess competing sugar (1 M D-galactose). The eluate was probed with antibodies against the three proteins: Rs1, αB-crystallin, and β2 laminin. All three proteins were detected at their approximate expected molecular masses under denaturing conditions (Figure 7). Labeling of adult pig retinal sections with Rs1 and PNA revealed overlapping patterns at the level of the cone matrix sheaths within the IPM, as well as surrounding cell bodies within the ONL (Figure 8). Although there was general correspondence between the two labels within the OPL, PNA was more concentrated on presynaptic terminals while Rs1 localized to postsynaptic structures (Figure 8E-G). Distributions of PNA lectin histochemical staining, αB-crystallin and β-laminin immunoreactivity also shared common domains, all three surrounding cell bodies in the ONL (compare Figure 4C, Figure 6C, and Figure 8C).

Discussion

These studies were aimed at extending the knowledge of Rs1 protein distribution in the retina, and beginning to identify proteins capable of interacting with it and participating in the formation of multimolecular scaffolds that provide the structural basis for the role of Rs1 in stabilizing synaptic organization. The principal findings were as follows western blotting suggests a wider tissue distribution than seen by immunohistochemistry; xlrs1 mRNA could only be detected in photoreceptor samples; and least two proteins can be implicated in Rs1 binding interactions.

There are major differences in xlrs1 mRNA and Rs1 protein distribution patterns reported by different laboratories. The original studies coming from the laboratory of Farber demonstrated that xlrs1 mRNA could only be detected in photoreceptors, highly concentrated at the level of the inner segments [18,19]. Subsequent studies by the same group showed that Rs1 protein was more widespread, and that this was accomplished by directed transport involving the radial Müller glial cells [20,21]. In contrast, we observed that xlrs1 mRNA persisted in mutant mice lacking rods and cones, and Rs1 protein could be seen on bipolar cells in vitro [22]. Furthermore, the laboratory of Sieving, using more sophisticated staining techniques, was able to detect both mRNA and protein outside the photoreceptors, at the level of the ganglion cells and inner retina [23]. Using a vibratome-based technique developed in our laboratory in order to isolate photoreceptor layers for transplantation [37] or tissue culture [30], we prepared mRNA from fractions of GCL, INL, and ONL. Relative purity of each fraction was assessed by RT-PCR analysis of specific cell markers contained within distinct layers. Hence mGluR6 is expressed uniquely by rod bipolar cells and constitutes a biomarker for INL [31]. Product for this gene was present in INL fractions but not in ONL, showing that the latter were not contaminated by INL cells. However, mGluR6 was also amplified in GCL fractions, which presumably arose from mRNA within cells or processes in the IPL. Similarly, as expected rhodopsin primers were unable to detect message within the GCL, but did show transcripts in the ONL and INL. This latter shows that some cellular material from the ONL was included in the INL preparations. Nevertheless, RT-PCR studies using xlrs1 primers only revealed mRNA within the ONL, not in the INL or GCL, in accordance with the original findings of Reid and co-workers [18-20]. It is possible we did not detect low levels of expression that would require further rounds of amplification.

Immunohistochemical analyses by different laboratories using different antisera have revealed Rs1 staining to be concentrated within the IPM, ONL, INL, and GCL [18,20,22,23]. Using a newly generated polyclonal antibody (but directed against the same epitope used previously [22]), we also observed abundant labeling of IPM and INL. However western blotting of separated retinal layers using the same antibody exhibited intense staining of GCL fractions. Although part of this staining is accounted for by inclusion of IPL membranes in the GCL fraction, considering the total protein concentrations of GCL preparations were far lower than those used for the other layers, the data indicate a pool of Rs1 residing at this level which is not readily recognized in sections. It is possible that this pool differs in its conformation, partly masking the epitope recognised by the amino terminal antibody, but which is revealed under denaturing gel electrophoresis conditions. Recent studies using highly sensitive immunodetection techniques demonstrated that Rs1 is expressed at the level of the GCL, appearing early in development [23]. This wider distribution is consistent with the inner retinal splitting observed in affected patients and transgenic mice [4,6,24]. We also examined the presence of Rs1 in conditioned medium prepared from highly enriched cultures of adult photoreceptors [28]. Western blotting revealed the presence of a faint band at about 25 kDa after 24 h in culture, indicating that the protein was effectively secreted into the medium. Much higher levels of Rs1 protein were observed in cell lysates prepared from the same cultures, representing either intracellular pools or protein bound to cell surfaces.

Given the secretory nature of Rs1, and the observed effects on synaptic disorganization within the plexiform layers, we reasoned that the protein likely interacts with both other extracellular proteins and transmembrane and intracellular proteins to constitute a complex scaffold involved in stabilizing cellular structure. Importantly, the discoidin domain has been shown to interact with collagen [38]. Also, such multimolecular complexes have been observed in several cases, notably in the interactions of the dystrophin and dystrophin-associated family members with membrane-bound neurotransmitter receptors and the cytoskeleton [39-41], including within the retina [42]. In addition, both dystrophin animal mutants and afflicted humans exhibit features associated with loss of the ERG b wave, as seen in xlrs1 mutants [43,44]. As techniques such as yeast double hybrid trapping are not particularly suitable for membrane proteins, we chose two complementary approaches to begin searching for candidate Rs1-interacting proteins: screening against potentially relevant ECM proteins, and affinity isolation of binding proteins. Laminins 14 and 15, of chain composition α4β2γ3 and α5β2γ3, are known to be present in the IPM [45], and knockout mice exhibit a retinal phenotype with some resemblances to xlrs1 -/- mice (ERG b-wave abnormalities), and it was therefore logical to investigate if these laminins could bind to Rs1. Three lines of evidence suggest this may be the case: (1) Rs1 is detected in retentates on laminin antibody columns; (2) laminin is detected in retentates of Rs1 antibody columns; and (3) the two proteins exhibit partial overlap in their retinal distributions.

As revealed by Coumassie blue staining, acid elution of proteins retained on an affinity column prepared using anti-Rs1 antibody produced a wide range of molecular mass species. Sequencing of a selected band running just underneath that of Rs1 itself identified aB crystallin as a binding protein. Several previous reports have shown this member of the crystallin family to be present in the neural retina, notably within photoreceptors and Müller glia [46,47]. It associates tightly with post-Golgi transport vesicles in the photoreceptor cytoplasm [48], has been shown to possess chaperone-like activities [49,50], and is involved in prevention of apoptosis [51]. It is seen to be upregulated in retina subjected to various insults such as light-induced stress or inherited degeneration [52,53]. Thus its association with Rs1 seems plausible, enabling it to be involved in transport and release of Rs1-containing vesicles within the photoreceptor cytosol.

In addition to this investigation, previous studies on Rs1 expression have observed large amounts of protein at the level of the photoreceptor inner segments if not within the subretinal space [19-24]. This localization does not correspond with any apparent function, since Rs1 mutations do not appear to display major defects in photoreceptors themselves [1-7]. Nevertheless, given the prominence of Rs1 immunostaining within this compartment, especially at the level of the cone matrix sheath, it also seemed reasonable to speculate that it may interact with galactose-containing glycoproteins enriched in this space [54]. PNA staining is also seen within the OPL, another site where Rs1 is abundant, although close observation of their binding indicates they do not coincide. It is of interest that not only Rs1 itself, but also β2 laminin and αB crystallin were present in PNA column retentates. However, it is important to point out that laminins are glycoproteins containing several consensus glycosylation motifs [55], and that galactose itself has been reported to constitute part of the carbohydrate moiety [56]. Furthermore, a large body of data demonstrates that crystallins accept sugar residues through nonenzymatic reactions, termed glycation [50], which occur as a consequence of disease or aging. Thus these proteins could also be pulled down directly by PNA binding, and may not interact through Rs1 or PNA binding proteins.

In conclusion, these studies indicate that Rs1 forms multimolecular complexes with a minimum of two candidate partners, β2 laminin and αB crystallin, and possibly with unidentified glycoconjugates. Although these findings represent only a first step, they provide original data on the molecular components to which Rs1 may bind. Interactions with αB crystallin are most likely to be intracellular, while the association with β2 laminin probably occurs at the level of the extracellular scaffold involved in stabilizing synapses. Use of interphotoreceptor matrix preparations [57] or photoreceptor extracts instead of total proteins, should refine protein-protein interactions. It will be of especial interest to delimit the binding domains involved in these interactions, and to examine whether known mutations in Rs1 affect its ability to bind. For example, since some mutations compromise Rs1 transport and secretion [15,17], αB crystallin association may be diminished.

Acknowledgements

MFSC was supported by a grant from the Fondation de France; other sources of financial support included a core grant from INSERM.

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