RS1, a Discoidin Domain-containing Retinal Cell Adhesion Protein Associated with X-linked Retinoschisis, Exists as a Novel Disulfide-linked Octamer*

RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys59-Cys223 intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys40-Cys40 bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys63-Cys219 and Cys110-Cys142) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys83) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein.

individuals show a significant loss in central and in some cases peripheral vision, a splitting of the inner layers of the retina, and a loss in the b-wave of the electroretinogram. The gene responsible for XLRS was identified by positional cloning and shown to encode a retinal-specific 224-amino acid protein, known as RS1 or retinoschisin, containing a discoidin domain (4).
RS1 is expressed and secreted from photoreceptor cells of the outer retina and bipolar cells of the inner retina as a multisubunit protein (5)(6)(7)(8). The secreted protein associates with the surface of rod and cone photoreceptors at the level of the inner segment, outer nuclear, and outer plexiform layers and the surface of bipolar cells within the inner nuclear and inner plexiform layers of the retina. Biochemical studies further show that RS1 is tightly associated with the membrane fraction of retinal cell homogenates (6). RS1 is generally believed to function as a retinal cell adhesion protein, because mice deficient in RS1 have a highly disorganized retina with displacement of bipolar cells into the outer retinal layer, gaps between bipolar cells within the inner retina, disruption of the photoreceptor-bipolar synapse, and progressive degeneration of rod and cone photoreceptors (9).
The dominant structural feature of the RS1 polypeptide is the 157-amino acid discoidin domain, also known as an F5/8 type C domain, which comprises over 75% of the processed polypeptide chain (4). Discoidin domains are present in a wide range of membrane and extracellular proteins where they mediate a variety of cell adhesion and cell signaling processes (10,11). Some proteins that contain discoidin domains are Factors V and VIII involved in blood coagulation, neuropilins 1 and 2, which mediate nervous system regeneration and degeneration, discoidin domain receptors implicated in cancer metastasis, and discoidin I involved in cellular adhesion during slime mold differentiation and development (10 -13). Still other members of the discoidin family play a role in key physiological processes ranging from heart development, milk lactation, and spermegg adhesion in vertebrates, to immunity and metamorphosis in the silkworm, and to post-fertilization events in the sea urchin zygote (14 -17). Bioinformatic studies have also identified a number of discoidin domain-containing proteins of unknown function (11).
The high resolution structures of the C2 discoidin domain of Factors V and VIII and the b1 discoidin domain of neuropilin 1 * This work was supported in part by National Institutes of Health Grant EY 02422, by the Canadian Institutes for Health Research (Grant MT5822), and by the Macular Vision Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by scholarships from the Natural Sciences and Engineering Research Council (NSERC, Canada) and the Michael Smith Foundation for Health Research. have been determined (13,18,19). These domains consist of eight antiparallel ␤-strands arranged in a barrel-like structure with two or three loops, or "spikes," projecting from one end of the core structure. In the case of Factors V and VIII, the spikes are involved in the attachment of these proteins to the phosphatidylserine-rich surface of platelets as a key step in the blood coagulation process (12). The discoidin domain of RS1 has been modeled from the C2 discoidin domain of Factors V and VIII (20,21), and, like these domains, the structure consists of eight core beta strands and three spike regions.
In addition to its discoidin domain, RS1 contains a 23-amino acid leader or signal sequence, followed by a 39-amino acid segment known as the Rs1 domain. The leader sequence plays an essential role in the insertion of the nascent RS1 polypeptide chain into the ER membrane. It is subsequently cleaved in the lumen of the ER by a signal peptidase as a key step in the secretion of RS1 from cells (4,20). The Rs1 domain contains four cysteine residues. One of these residues, Cys 59 , forms an intermolecular disulfide bond with Cys 223 of another subunit to form a disulfide-linked homo-oligomeric complex (20). The redox state or function of the three other cysteines in the Rs1 domain has not been determined.
To date more than 133 mutations in the RS1 gene have been associated with XLRS (available at www.dmd.nl/rs). HEK 293 or COS-7 cells expressing RS1 mutants have been used to understand how selected mutations cause this disease (20,22). Most disease-linked missense mutations in the RS1 discoidin domain result in severe misfolding of the protein and retention in the ER. Mutations in the leader sequence prevent insertion of RS1 into the ER membrane resulting in mislocalization of these mutant polypeptides to the cytoplasm and rapid proteolytic degradation (22). Finally, two disease-linked cysteine mutations (C59S and C223R) outside the discoidin domain do not significantly affect protein folding or secretion from cells (20). However, these mutants fail to assemble into a normal multisubunit complex suggesting that oligomerization is crucial for the functioning of RS1 as an extracellular adhesion protein.
Although previous studies have shown that RS1 exists as a disulfide-linked oligomeric protein, the number and arrangement of these subunits in the native complex and the role of intramolecular and intermolecular disulfide bonds and noncovalent interactions in the assembly and stabilization of the RS1 complex have not been investigated. In this study we have used site-directed mutagenesis and expression together with velocity sedimentation and peptide mapping by mass spectrometry to gain insight into the oligomeric structure of retinal and recombinant RS1 under native and denaturing conditions. Here, we show that native RS1 has an unusual oligomeric structure consisting of disulfide-linked dimers within a disulfide-linked homo-octameric complex. Cysteines involved in the formation of key intramolecular and intermolecular disulfide bridges are identified.
Cell Transfections and Protein Preparation-A version of HEK 293 cells known as HEK EBNA 293 (H293) cells (American Type Culture Collection) were transfected with WT and mutant RS1 cDNA as previously described (20). The cellular fraction of the protein was obtained by washing the cells twice in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ) by low speed centrifugation. The final pellet was resuspended in 200 l of PBS containing 20 mM NEM (Calbiochem) and added to an equal volume of PBS containing 2% Triton X-100, 20 mM NEM, and Complete Protease Inhibitor (Roche Applied Science, Basel, Switzerland). After stirring for 20 min at 4°C, the solution was centrifuged at 100,000 ϫ g in a TLA-100.4 rotor (Beckman, Fullerton, CA) for 20 min, and the supernatant was retained for SDS gel electrophoresis. The secreted fraction of the protein was obtained by centrifuging 10 ml of the media at 300,000 ϫ g in a TLA-75 rotor (Beckman) at 4°C for 20 min to remove any insoluble membrane material. The samples were then incubated for 2 h with the Rs1 3R10-Sepharose 2B affinity matrix. After washing with column buffer (20 mM Tris, 0.2% Triton X-100, and 20 mM NEM), bound protein was eluted with 4% SDS in column buffer.
Gel Electrophoresis and Western Blotting-Proteins were denatured in a denaturing solution (10 mM Tris, pH 6.8, 1% SDS, 10% glycerol) in the presence or absence of 4% ␤-mercaptoethanol and separated on 6.5% or 10% polyacrylamide SDS gels or gradient polyacrylamide SDS gels as indicated. For 6.5% or 10% polyacrylamide SDS gels, a denaturing mixture of 10 mM Tris, pH 6.8, 1% SDS, and 10% glycerol was used. For gradient gels, the above mixture was used except 1% SDS was replaced with 2% lithium dodecyl sulfate, and 2 mM EDTA was also included. Gels were transferred to Immobilon P membranes for 20 min in transfer buffer (25 mM Tris, 192 mM glycine) containing 20% methanol. For analysis of the C38S/C40S/C42S/C83S mutant, a 6.5% nonreducing gel was used, and the protein was transferred for 20 min in transfer buffer containing 7% methanol. Blots were blocked with 0.5% skim milk in PBS and labeled with the Rs1 3R10 antibody containing 0.5% skim milk in PBS and 0.1% Tween 20 for 1 h. The blots were then washed and labeled with goat-anti-mouse Ig conjugated to CW800 dye (Licor, Lincoln, NE) containing 0.5% skim milk in PBS and 0.2% SDS for 30 min for detection by the Odyssey Infrared Imaging System (Licor).
For octamer analysis, the samples were run on a 4 -12% NuPAGE gradient gel (Invitrogen) using 50 mM MOPS, 50 mM Tris, 0.1% SDS, and 1 mM EDTA as the running buffer. NuPAGE gels were transferred using transfer buffer containing 25 mM bicine, 25 mM Bis-Tris, 1 mM EDTA, and 10% methanol. The nonreduced form of bovine retinal RS1 and the nonreduced form of the C59S/C223S RS1 dimer were analyzed on 3-8% NuPAGE gradient gels under denaturing conditions. Ammonium Sulfate Precipitation of RS1 from Cell Culture Media-Human WT RS1 and the C59S/C223S mutant were transfected as described above in T150 flasks. The media containing 10% fetal calf serum was replaced the following day with fresh media. One week later, it was replaced with media containing 2% fetal calf serum. The media containing 2% fetal calf serum was harvested once a week for 2 or 3 weeks. The secreted RS1 protein was precipitated with 40% ammonium sulfate (final concentration) at 4°C and resuspended in 1/40th the volume of the cell culture media. The protein solution was dialyzed overnight against 20 mM Tris, pH 7.4, 0.1 M NaCl at 4°C.
Extraction of RS1 from Bovine Retina-Bovine retinas were washed twice in low salt containing 10 mM Tris, pH 7.4, by centrifugation at 14,000 ϫ g in an SS-34 Sorvall rotor (Kendro Laboratory Products, Asheville, NC). The cells were then disrupted as follows: the pellet was resuspended in lysis buffer consisting of 10 mM Tris, pH 7.4, 10 mM NEM, and Complete Protease Inhibitor and incubated for 10 min on ice, followed by several passages through a 26-gauge needle attached to a syringe. To obtain the membrane fraction, the lysed cells were applied to a 60% sucrose gradient and centrifuged for 30 min at 38,000 ϫ g in a TLS 55 rotor (Beckman). The membranes were collected at the top of the sucrose solution and washed with 10 mM Tris, pH 7.4, by centrifugation at 100,000 ϫ g in a TLA 100.4 rotor. The membranes were then incubated with 2 M urea and centrifuged at 100,000 ϫ g for 10 min. The washing procedure with 2 M urea was repeated, and retinal RS1 was subsequently extracted with buffer containing 6 M urea, 10 mM CHAPS, and 0.5 M NaCl. The extract was spun down at 100,000 ϫ g and the supernatant was dialyzed overnight against 20 mM Tris, pH 7.4, and 0.1 M NaCl to remove the urea and CHAPS.
Immunoprecipitation of RS1 from Bovine Retina-RS1 was immunoprecipitated from bovine retina on an Rs1-3R10-Sepharose column as follows: Five frozen bovine retinas were thawed and treated with lysis buffer as described above. The cells were then solubilized in buffer containing 18 mM CHAPS, 0.15 M NaCl, 10 mM NEM, Complete Protease Inhibitor, and 20 mM Tris, pH 7.4, for 20 min and subsequently centrifuged at 100,000 ϫ g for 10 min. The supernatant was incubated with 100 l of the Rs1-3R10-Sepharose beads for 1 h and washed in column buffer consisting of 0.15 M NaCl, 20 mM Tris, pH 7.4, and 2 mM CHAPS. After elution with column buffer containing 4% SDS, the sample was denatured in SDS solution and analyzed by SDS gel electrophoresis.
Velocity Sedimentation of Native RS1-Bovine retinal RS1, recombinant WT RS1, and RS1 containing the C59S/C223S mutation were sedimented through a 5-20% (w/w) linear sucrose gradient containing PBS at 4°C for 17 h and 72,449 ϫ g in a Beckman TLS-55 rotor. Bovine retinal RS1 was obtained by urea and CHAPS extraction as described above. The secreted fraction of transfected WT and C59S/C223S RS1 was concentrated by ammonium sulfate precipitation as described above before subjecting to velocity sedimentation. Mouse IgG having a molecular mass of 160 kDa was run as a standard. All samples were spun for 20 min at 100,000 ϫ g in a Beckman TLA 100.4 rotor prior to loading onto the sucrose gradient to remove any insoluble material. Three-drop fractions were collected, run on reducing gels, and blotted with the Rs1 3R10 antibody. The intensity of the labeled bands was quantified with the Odyssey Infrared Imaging System. Sucrose concentration in each fraction was determined by refractometry to plot the percentage of each protein species versus the percentage of sucrose.
In-gel Proteolytic Digestion of RS1 for Mass Spectrometry-In-gel digestion was performed by punching out the Coomassie Blue-stained RS1 bands with a glass Pasteur pipette. The gel pieces were washed with water several times to remove acetic acid. They were then destained in a 1:1 mixture of 100 mM ammonium bicarbonate and 100% acetonitrile several times. After shrinking and drying the gels with 100% acetonitrile, the gels were incubated with either 50 mM NEM for nonreducing samples, or 10 mM DTT (Sigma) followed by 50 mM IAM (Sigma) for reducing samples, washed with ammonium bicarbonate, and dried from 100% acetonitrile. The gel pieces containing the samples were then incubated with the proteases at 12 ng/l for 30 min on ice. The protease solution was removed, and the gel pieces were overlaid with 50 mM ammonium bicarbonate and 5 mM calcium chloride. Nonreduced samples also contained 0.6 M urea. The samples were digested for 18 h at 37°C. The solution was collected in a tube and the gel pieces were re-extracted with 50 mM ammonium bicarbonate/66% acetonitrile (basic extraction) and with 5% formic acid/66% acetonitrile (acidic extraction). The samples were pooled, dried in a SpeedVac, and resuspended in 10 l of 50 mM ammonium bicarbonate. For MALDI-TOF mass spectrometry, protease-digested RS1 (2 l) was applied to an H4 chip (Ciphergen, Fremont, CA). The sample was dried and washed with two quick rinses of water before applying 20% ␣-cyanohydroxycinnamic acid matrix in a solvent with 50% acetonitrile and 0.1% trifluoroacetic acid. Samples were analyzed on a surface-enhanced laser desorption ionization-time of flight (Ciphergen). Masses obtained were average masses. A Qstar XL LC/MS/MS (Applied Biosystems, Foster City, CA) was used for MS/MS sequencing of the RS1 peptides. Trypsinized samples were obtained as above and lyophilized and reconstituted in formic acid. A PepMap C18 column, with a 3-mm particle size and 100-Å pore size column from LC Packings (Amsterdam, Netherlands) was used for peptide separation. Solvents B and A contained 20% acetonitrile in water and 5% acetonitrile in water, respectively. LC conditions started at 2% solvent B, with a gradient to 60% B over 60 min, to 95% B at 93 min, and held for 3 min before returning to 2% B. Masses obtained were monoisotopic masses. The enzymes used were trypsin (Promega, Mad-FIG. 1. Analysis of disulfide-linked RS1 oligomers on nonreducing SDS gels. WT and C38S/C40S/C42S RS1 were expressed in HEK 293 cells, purified from the secreted fraction and analyzed on a nonreducing SDS 4 -12% polyacrylamide gradient gel. The ladder of oligomeric species was detected on Western blots labeled with the Rs1 3R10 monoclonal antibody. A, the C38S/C40S/C42S mutant (lanes 1 and 2) and WT (lanes 3 and 4) at two different concentrations. Positions of the oligomeric species are indicated. B, serial dilutions of WT RS1 were run on nonreducing gels to determine the migration distance of the various oligomers. C, data from B was used to plot the logarithm of the molecular weight of the six lower bands versus logarithm of the percentage acrylamide. The line-of-best-fit equation was determined using SigmaPlot. The number of subunits for the 185-kDa WT RS1 protein (top prominent band) was 7.7.
ison WI), endo-LysC (Roche Applied Science), and endo-AspN (Roche Applied Science). Background control enzyme digestions of blank gel pieces were also performed to subtract out peaks due to autoproteolysis and other contaminating peptides.

RS1
Assembles as a Disulfide-linked Homo-octamer-In previous studies, RS1 was shown to migrate on SDS gels as a 24-kDa monomer under disulfide-reducing conditions and a high molecular weight oligomer under nonreducing conditions (20). To determine the size of the RS1 oligomer, a C38S/C40S/ C42S triple mutant displaying partial disruption of the oligomeric complex was expressed, secreted from HEK 293 cells, and analyzed on nonreducing SDS-polyacrylamide gradient gels. As shown in Fig. 1A, the secreted mutant showed a ladder of eight distinct bands ranging from the 24-kDa monomer to the prominent 185-kDa octameric protein.
The secreted fraction of HEK 293 cells expressing WT RS1 was also analyzed by SDS gel electrophoresis under nonreducing conditions. As shown in Fig. 1B, a ladder of bands was also evident for WT protein when Western blots were exposed for extended times, with the 185-kDa complex being the most prominent species. Unlike the C38S/C40S/C42S mutant, however, only seven bands were resolved for WT RS1, presumably due to the masking of the heptamer by the dominant octameric complex. This was confirmed by plotting the logarithm of the apparent molecular mass for the six lower bands against the logarithm of the acrylamide percentage at which these species migrated (23). From the linear relation (Fig. 1C), the number of subunits in the prominent top band of the WT complex was calculated to be 7.7. This suggests that WT RS1, like the C38S/C40S/C42S mutant, exists as a disulfide-linked octamer.
Analysis of Cysteine Residues Involved in Disulfide-linked Dimer Assembly-Previously, we showed that the Cys 59 and Cys 223 residues of RS1 are responsible for disulfide-linked oligomer (octamer) assembly (20). Substitution of these cysteine residues with serine resulted in a loss of the disulfide-linked oligomer and the appearance of a disulfide-linked dimer when analyzed on nonreducing SDS gels. To determine which of the one or more cysteine residues in RS1 are responsible for disulfide-mediated dimer formation, we individually replaced each of four cysteine residues (Cys 38 , Cys 40 , Cys 42 , and Cys 83 ) with a serine in a C59S/C223S octamer-defective mutant, and analyzed the cellular and secreted protein fractions from transfected HEK 293 cells. These four cysteines were selected because previous studies had suggested that the remaining cysteines (Cys 63 , Cys 110 , Cys 142 , and Cys 219 ) are involved in intramolecular disulfide bonds within the discoidin domain (20). Under disulfide-reducing conditions, all four mutants exhibited similar levels of cellular expression and secretion on SDS gels and migrated as 24-kDa monomers (Fig. 2). Under nonreducing conditions, the parent C59S/C223S mutant from the secreted fraction ran solely as a 47-kDa dimer, whereas the three C59S/C223S mutants containing an additional C38S, C42S, or C83S mutation migrated as a mixture of dimers and monomers. Only the C59S/C223S mutant containing a C40S mutation ran as the monomer under nonreducing conditions in both the cellular and secreted fractions without detectable dimer. These studies suggest that Cys 40 residues of individual subunits are directly involved in dimer formation. Partial loss in dimer formation present in the C38S and C42S mutants, and to a lesser extent the C83S mutant, most likely result from structural perturbations, which partially restrict the formation of Cys 40 -mediated intermolecular disulfide bonds.
All of these cysteine mutants exhibit an additional protein band just above the 24-kDa monomer in the cellular fraction of nonreduced gels (Fig. 2, lower left). This upper band, not found in the secreted fraction, most likely corresponds to a misfolded monomer that migrates anomalously on nonreducing SDS due to the presence of abnormal intramolecular disulfide bonds. This species is recognized by the quality control system of the ER as a misfolded protein and therefore is not secreted from the cell.

RS1 Contains Disulfide-linked Dimers within the Disulfidelinked Octamer-
The finding that intermolecular disulfide bonds are formed been Cys 59 and Cys 223 , and between individual Cys 40 residues of different subunits, suggests two models. In one model (Fig. 3A, left), the Cys 59 -Cys 223 disulfide bonds are solely responsible for octamer formation, and additional Cys 40 disulfide bonds form between two adjacent or opposing subunits within the octamer. In another model (Fig. 3A, right) the Cys 59 -Cys 223 intermolecular disulfide bonds result in the formation of tetramers. Two tetramers further link together in a head-to-head arrangement through Cys 40 disulfide bonds to form the octamer. For the first model, one predicts that RS1 containing a C40S dimer-defective mutation should still form an octamer, whereas for the second model, this mutation should result in tetramers. To distinguish between these models, we expressed a C38S/C40S/C42S/C83S mutant (termed ⌬4Cys), which is defective in disulfide-linked dimer formation, for analysis on reducing and nonreducing SDS gels.  shows that the secreted C38S/C40S/C42S/C83S mutant, like WT RS1, migrated as a 24-kDa monomer under reducing conditions and a 185-kDa octamer under nonreducing conditions. Similar results were obtained for the C40S single mutant (data not shown). These results support the first model in which an octamer is formed via Cys 59 -Cys 223 disulfide bonds even in the absence of Cys 40 mediated dimer formation.
RS1 from Retina Tissue Is Heterogeneous-To determine if RS1 from retina tissue exhibits a similar disulfide-linked octameric structure as recombinant protein, we compared the migration behavior of these proteins, along with the C59S/ C223S mutant, on SDS-polyacrylamide gradients gels under reducing and nonreducing conditions. As shown in Fig. 4, a fraction of RS1 from the retina migrated as a 24-kDa monomer under reducing conditions and a 185-kDa octamer under nonreducing conditions similar to recombinant WT RS1. However, a significant fraction of retinal RS1 migrated more slowly. Under reducing conditions a portion of RS1 migrated as a 47-kDa dimer and another fraction ran as a diffuse band between the monomer and dimer. Heating at 60 or 95°C for 5 min in the presence of reducing agent (␤-mercaptoethanol) and SDS did not abolish the slow migrating bands in the retinal RS1 sample. Under nonreducing conditions, a diffuse band was observed above the 185-kDa octamer. A similar diffuse RS1labeled band was reported by Reid et al. (7) for mouse retinal extracts under nonreducing conditions. These additional species were not observed for the recombinant WT or C59S/C223S mutant protein expressed in HEK 293 cells (Fig. 4), nor were they seen in RS1 derived from Weri RB1 retinoblastoma cells (data not shown). The banding pattern of RS1 from frozen bovine retina was also observed for freshly dissected bovine and mouse retina tissue (data not shown) indicating that the diffuse bands observed in the retina sample are not due to artifacts generated from aged tissue or from the use of bovine tissue. These data suggest that a portion of RS1 expressed in photoreceptor and/or bipolar cells undergoes a heterogeneous type of posttranslational modification.
Velocity Sedimentation of RS1 under Nondenaturing Conditions-Velocity sedimentation studies were carried out to determine whether RS1 exists as an octamer or a higher ordered oligomer under nondenaturing conditions. Fig. 5 shows sedimentation profiles for retinal and recombinant RS1 generated from Western blots of fractions collected from a 5-20% sucrose gradient. The WT recombinant protein (rWT) sedimented just ahead of retinal RS1 and mouse IgG (ϳ160 kDa) used as a standard. This indicates that both retinal and WT RS1 exist as a disulfide-linked octamer under nondenaturing conditions, as well as under denaturing (nonreducing) conditions, and does not form higher ordered oligomers through noncovalent interactions. Heterogeneous post-translational modification may account for the small decrease in sedimentation rate and broader peak profile observed for retinal RS1 relative to the recombinant WT protein (Fig. 5). The octamer-defective C59S/C223S mutant migrated much more slowly, consistent with its smaller size, presumably a dimer as observed under nonreducing, denaturing conditions. This suggests that noncovalent interactions do not play a significant role in octamer formation or stabilization.
Peptide  Bovine retinal RS1 was solubilized with CHAPS in the presence of NEM and purified on an Rs1 3R10 immunoaffinity column. Samples were run on polyacrylamide gels. For electrospray LC/MS/MS, bands were excised, reduced with DTT, alkylated with IAM, and digested with trypsin. For MALDI-TOF MS, samples were reduced with DTT and alkylated with IAM after digestion. Control digestions without trypsin were performed for both electrospray and MALDI-TOF MS. For cysteine-containing peptides, the mass noted is the mass after alkylation with IAM. See Fig. 6 for the locations of the peptides.  Table I and illustrated in Figs. 6 and 7, 14 tryptic peptides were identified from various regions of RS1. Peptide A (m/z of 1592.6 for bovine RS1 and m/z of 1583.6 for human RS1 (not shown)) corresponds to the N-terminal tryptic peptide of the processed RS1 polypeptide, confirming that signal peptidase cleavage had occurred at Ser 23 as part of the protein secretion pathway (Fig. 7). The remaining peptides were derived from the RS1 discoidin domain (Table I). In addition a broad peak corresponding to a peptide mass of ϳ7 kDa was observed (data not shown). This peptide was in agreement with the mass of the large tryptic fragment extending from position 40 to 100. The broad nature of the peak may reflect, in part, the heterogeneous nature of retinal RS1.

MS. As listed in
The mass spectrum of tryptic peptides from nonreduced RS1 is shown in Fig. 7. In addition to peptides identified in reduced RS1, a peak at m/z of 2548.8 was observed. This peak corresponded to a tryptic peptide (1410.6Da) containing Cys 110 disulfide bonded to a tryptic peptide (1140.3 Da) containing Cys 142 . (To calculate the theoretical mass of the nonreduced peptide, a mass of 2 Da is subtracted from the sum of the masses of these two cysteine-containing peptides due to removal of two hydrogens upon disulfide bond formation.) Another peak at m/z of 2564.7 most likely corresponds to the same disulfide-linked peptide, but with the single methionine (located on the Cys 142 peptide) in an oxidized state. To confirm that these peptides indeed contain a disulfide bond, we reduced this sample with DTT and alkylated the free cysteines with IAM. Under these conditions, the peaks at m/z of 2548.8 and 2564.7 disappeared and a new peak at 1467.6 appeared (Fig. 7). The mass of this new peptide is consistent with the tryptic peptide containing an IAM-modified Cys 110 residue. The tryptic peptide containing the alkylated Cys 142 residue was not observed by MALDI-TOF mass spectrometry, but was detected by LC/MS/MS (Table I).
Cys 83 of RS1 Exists in Its Reduced State-To determine the redox state of Cys 83 , nonreduced RS1 was alkylated with NEM and double-digested with trypsin/LysC, or trypsin/AspN. The trypsin/LysC sample contained a peptide with an m/z of 3819, and the trypsin/AspN sample contained a peptide with an m/z of 2705 ( Fig. 8 and Table I). These masses correspond to peptides containing Cys 83 that have been alkylated with NEM. This indicates that the Cys 83 within the discoidin domain of RS1 exists in its reduced state. Additional peaks that have slightly higher masses appear to correspond to the same peptide containing one or more oxidized amino acids. DISCUSSION In this study, we have analyzed the oligomeric structure of RS1 as an important step in understanding how this extracellular protein functions in retinal cell adhesion and how mutations in RS1 cause XLRS. RS1 is composed of eight identical subunits that are linked together through intermolecular disulfide bonds between Cys 59 and Cys 223 residues on adjacent subunits. These disulfide bonds are required for octamer assembly, because substitution of Cys 59 and Cys 223 with serine abolishes octamerization of RS1 under nonreducing denaturing conditions as observed by SDS-gel electrophoresis and native conditions as analyzed by velocity sedimentation. In addition to the Cys 59 -Cys 223 intermolecular disulfide bond, RS1 contains a Cys 40 -Cys 40 disulfide bond responsible for dimer formation (Fig. 9A). Dimerization is independent of octamerization, and octamerization is independent of dimerization, because each multimeric species forms in the absence of the other (Fig. 9B). Accordingly, RS1 is composed of four disulfide-linked dimers within a disulfide-linked octameric structure. The exact arrangement of the subunits, however, awaits determination of the high resolution structure of RS1.
The cysteine residues involved in these intermolecular disulfide bonds are located in segments that flank the discoidin domain, i.e. the 39-amino acid Rs1 domain upstream and a 5-amino acid segment downstream of the discoidin domain (Fig. 9A). Therefore, a principal function of these flanking regions is to assemble RS1 into a disulfide-linked octamer. Octamerization appears to be essential for the function of RS1 in retinal cell adhesion, because the C59S and C223R mutations, which do not significantly affect the folding and secretion of dimeric RS1, are known to cause XLRS (20,24,25). However, it is unclear if Cys 40 -mediated dimerization is critical for RS1 function, because substitution of Cys 40 with serine does not affect folding, secretion, or octamerization of RS1 (20), and to date no disease-linked missense mutations at position 40 have been found. It is possible that Cys 40 -linked dimerization may simply contribute to the stability of the octameric complex but not be critical for its function. A biochemical assay for retinal cell adhesion would help to resolve this issue.
In addition to cysteine at position 40, the RS1 domain contains two additional cysteine residues at positions 38 and 42. Substitution of these residues with serine does not significantly affect octamerization (20) and only partially affects dimerization, as shown here. We were not able to determine the redox state of Cys 38 and Cys 42 by mass spectrometry. It is possible that these residues form an intramolecular disulfide bond or additional intermolecular disulfide bonds that facilitate dimer formation. Alternatively, the Cys 38 and Cys 42 cysteine residues may exist in their reduced state. Interestingly, this -CKCDC- FIG. 8. Mass spectrum of the C83-containing peptide from nonreduced and alkylated RS1. Detergent-solubilized bovine retinal RS1 was treated with NEM and purified on an Rs1 3R10 immunoaffinity column. After elution with SDS, nonreduced RS1 was separated by SDS-gel electrophoresis. Bands were excised, washed, and doubly digested with trypsin/LysC or trypsin/AspN. Samples were analyzed by MALDI-TOF mass spectrometry in the nonreduced form and subsequently in the reduced form after reduction with DTT and alkylation with IAM. Peaks corresponding to NEM-modified Cys 83 -containing peptides are indicated. Additional peaks differing by ϳ16 Da are likely oxidized forms of the peptide. motif is highly conserved in all vertebrate RS1 proteins that have been sequenced to date.
The discoidin domain is the main functional part of the RS1 protein. Like the discoidin domains of other proteins, RS1 contains conserved cysteine residues at the beginning (Cys 63 ) and the end (Cys 219 ) of its discoidin domain. High resolution structures of the discoidin domains of Factor V, Factor VIII, and neuropilin 1 indicate that these cysteines marking the beginning and end of the discoidin domain form an intramolecular disulfide bond. Because Cys 63 and Cys 219 have been shown to come in close proximity to each other in modeling studies of RS1 and are required for proper protein folding (20), it is likely that these cysteines form an intramolecular disulfide bond in the RS1 polypeptide. In addition to Cys 63 and Cys 219 , the RS1 discoidin domain contains 3 additional cysteine residues. The Cys 110 and Cys 142 cysteines, located in spike 2 and spike 3, respectively, had been suggested to form another intramolecular disulfide bond based on molecular modeling and site-directed mutagenesis (20). This has been confirmed directly in the present study through the mass spectrometric identification of a Cys 110 -Cys 142 containing proteolytic fragment generated from nonreduced RS1. The function of the spike regions in the RS1 discoidin domain has yet to be determined. However, analogous spikes in the C2 discoidin domain of Factors V and VIII are known to insert into the lipid bilayer of phosphatidylserine-rich platelet membranes as part of the blood coagulation process. Partial sequence conservation of RS1 with the C2 domain of Factors V and VIII in the vicinity of the spike regions (12), together with molecular dynamic simulation, have led to the suggestion that RS1 may interact with membranes in a similar fashion (21). On this basis, the Cys 110 -Cys 142 disulfide bond may be important in stabilizing the spike regions of RS1 for insertion into the lipid bilayer.
An additional cysteine residue, not found in the discoidin domain of other proteins, is present at position 83 of the RS1 discoidin domain. Mass spectrometric analysis of nonreduced and alkylated proteolytic fragments indicates that this cysteine is in its reduced state. This is consistent with molecular mod- eling studies, which indicates that this residue is largely buried within the protein structure (18). Substitution of Cys 83 with serine has little effect on the folding, secretion, or oligomeric assembly of RS1, suggesting that this cysteine residue may not be essential for the structure or function of RS1. Furthermore, to date no disease-linked missense mutations at position 83 have been reported. A model depicting the role of various cysteines in disulfide bonding is shown in Fig. 9.
Our results suggest a sequence of events leading to the secretion of RS1 from cells (Fig. 10). As in the case of many secreted proteins, the leader sequence initiates the insertion and translocation of the nascent RS1 polypeptide chain across the ER membrane. Signal peptidase in the ER lumen cleaves the leader peptide at Ser 23 to produce the processed RS1 polypeptide. The folding of the discoidin domain occurs with the formation of the Cys 63 -Cys 219 and Cys 110 -Cys 142 intramolecular disulfide bonds. Subsequently, intermolecular disulfide bonds Cys 40 -Cys 40 and Cys 59 -Cys 223 within the segments flanking the discoidin domain form to produce the final RS1 octamer for secretion from cells. Our studies suggest that a portion of RS1 in retina tissue undergoes an additional post-translational modification, the nature of which remains to be determined. Mutational studies indicate that the monomeric and dimeric forms of RS1 can pass through the ER quality control system for secretion, suggesting that the proper folding of the RS1 discoidin domain occurs independently of disulfide-linked subunit assembly. However, the function of RS1 as a cell adhesion protein requires proper octamerization. The multivalent nature of the RS1 homo-octamer may facilitate cell-cell adhesion via its insertion into the surface membranes of adjacent cells. Alternatively, the multisubunit nature of RS1 may enhance its binding to the surface of a cell by providing multiple points of attachment. In this case, RS1 may interact with other extracellular proteins or carbohydrates to promote cell adhesion and stabilize the cellular architecture of the surrounding retina tissue.