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Originally published In Press as doi:10.1074/jbc.M201279200 on May 3, 2002

J. Biol. Chem., Vol. 277, Issue 28, 25592-25600, July 12, 2002
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Molecular Cloning and Characterization of Chick Sialoprotein Associated with Cones and Rods, a Developmentally Regulated Glycoprotein of Interphotoreceptor Matrix*

Masahiro ZakoDagger §, Masayoshi IwakiDagger , Masahiko Yoneda, Osamu Miyaishi||, Jinsong ZhaoDagger , Yasuhiko Suzuki**, Makoto TakeuchiDagger , Goichiro MiyakeDagger , Hiroshi IkagawaDagger , and Koji KimataDagger Dagger

From the Departments of Dagger  Ophthalmology and || Pathology, the Dagger Dagger  Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, the  Aichi Prefectural College of Nursing and Health Nagoya, Aichi 463-8502, and the ** Tosei Municipal Hospital, Oiwake, Seto, Aichi 489-0803, Japan

Received for publication, February 7, 2002, and in revised form, May 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylated N- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15-16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MY-174, a monoclonal antibody, has been shown to recognize chick PG-M/versican (1), a member of the hyaluronan-binding chondroitin sulfate proteoglycan family (2-4). In the eye, MY-174 specifically stains the photoreceptor layer (1). However, recently we demonstrated an absence of PG-M/versican at the chick photoreceptor layer using a polyclonal antibody that recognizes all alternatively spliced forms of PG-M/versican (5). These inconsistent results suggest that MY-174 does not recognize PG-M/versican but another molecule in the photoreceptor layer.

The IPM (interphotoreceptor matrix),1 resides in an extracellular compartment between the outer limiting membrane of the neural retina and apical surface of the retinal pigment epithelium and is composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans (6, 7). A variety of important reactions relating to vision, including visual pigment chromophore exchange, metabolite trafficking, photoreceptor alignment, and membrane turnover, is thought to be mediated by the IPM (8). However, one of the most essential functions of IPM is that of a biological glue for retinal adhesion through its viscous adhesive properties (9, 10). Retinal adhesiveness can be weakened by treatments with enzymes such as neuraminidase, hyaluronidase, and chondroitinase ABC (11, 12). Intracellular blocking of glycosylation with xyloside also prevents the secretion of proteoglycans and results in retinal detachment (13). These results suggest that the proteoglycans, glycosaminoglycans, or glycoproteins of the IPM are involved in retinal adhesion. However, the specific IPM molecule that mediates adhesion has not been identified.

SPACR (sialoprotein associated with cones and rods) is a hyaluronan-binding glycoprotein newly identified in adult human PBS (phosphate-buffered saline)-insoluble IPM (14-17). This 147- to 150-kDa glycoprotein was purified by wheat germ agglutinin-affinity chromatography and characterized (17). It has been shown that (a) SPACR is heavily sialylated, (b) both N- and O-linked glycoconjugates are present in the molecule, and (c) glycoconjugates account for ~30% of the molecular mass (17). Recently, mouse SPACR was also cloned and characterized (18). Both human and mouse SPACRs have a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain near the C-terminal, and several potential hyaluronan-binding motifs in common. Interestingly, biochemical studies showed that SPACR is a glycoprotein in human and a proteoglycan in mouse. Except for the ability of SPACR to bind hyaluronan (17), other properties or functional roles of SPACR remain unknown.

In the present study, we first examined the localizations of the retinal antigen that binds to MY-174 in the photoreceptor layer. We then determined a full-length cDNA of MY-174 antigen at photoreceptor layer and showed that the antigen corresponded to chick SPACR. Finally, to investigate the biological significance of SPACR, we compared the expressions of chick SPACR and the occurrence of adhesion between neural retina and retinal pigment epithelium during development.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Fertilized eggs from white leghorns were maintained in a humidified incubator at 37 °C, and embryos at the different developmental stages were obtained according to the standards used by Hamburger and Hamilton (19) and their retinas used for study. Retinas from adult white leghorns and Sprague-Dawley rats were also used. All experimental procedures in this study conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Mouse monoclonal antibody, MY-174 (1), was purified by E-Z-SEP (Amersham Biosciences, Uppsala, Sweden). Rabbit antibody against rhodopsin was purchased from LSL Co., Tokyo, Japan. Affinity-purified fluorescein isothiocyanate-conjugated goat antibody against mouse IgM was from TAGO, Burlingame, CA. Peroxidase-conjugated goat IgG fractions to mouse immunoglobulins (IgG, IgA, and IgM) were from Organon Teknika Corp., Durham, NC. IgM fractions from nonimmunized mouse serum were from Zymed Laboratories, South San Francisco, CA, and used as a control for immunohistochemical study.

Chondroitinase ABC (protease-free), hyaluronidase, and Endo-alpha -N-acetylgalactosaminidase (O-glycanase) were purchased from Seikagaku Corp., Tokyo, Japan. Recombinant N-glycanase and neuraminidase (sialidase) were from Genzyme Corp., Cambridge, MA. Protease inhibitors were from Roche Molecular Biochemicals, Tokyo, Japan. A newborn chick retinal cDNA library was established using pTriplEx vector by CLONTECH (Palo Alto, CA) from neural retinas prepared from 20 newborn chicks.

Immunofluorescent Microscopy-- Chick eyes were cut into two pieces and then fixed in 3.7% (w/v) formaldehyde solution neutralized with calcium carbonate in 0.1 M sodium phosphate buffer, pH 6.8, for 1 h at room temperature with gentle shaking. Fixed retinas were rinsed in PBS containing 0.1% (w/v) glycine at 4 °C overnight and then embedded in OCT compound (Miles Scientific, Naperville, IL) on petroleum ether-dry ice and dissected. Cryostat sections (6-8 µm) were incubated with MY-174 in 10% (w/v) normal swine serum for 30 min at room temperature. After rinsing three times with PBS for 3 min for each rinse, the sections were incubated with goat antibody against mouse IgM (TAGO, Burlingame, CA) and then with fluorescein isothiocyanate-conjugated swine antibody against goat IgG (TAGO) in PBS containing 10% (w/v) normal swine serum. Finally, the sections were rinsed in PBS and mounted in mounting media (Shandon Lipshaw, Detroit, MI). Immunolabeled tissue sections were observed using a fluorescence microscope (Olympus, Tokyo, Japan). Photographs were taken using Kodak Tri-X pan film (Eastman Kodak Co., Rochester, NY). Immunohistochemical staining was performed on 20 different eyes, and reproducible results were achieved. A control for nonspecific staining omitted the primary antibody and replaced it with mouse nonimmune serum at the same protein concentration. No staining was observed in control sections.

In some cases, enzymatic treatments were performed prior to immunostaining. Acylneuraminyl hydrolase and O-glycosidase digestions were performed on microscope slides with neuraminidase in 0.5 M Tris-HCl, pH 6.5, for 1 h at 37 °C and with O-glycanase (endo-alpha -N-acetylgalactosaminidase) in 0.2 M citrate buffer, pH 4.5, for 1 h at 37 °C, respectively.

Immunoelectron Microscopy-- Chick eyes were excised into small pieces and fixed in 3.7% formaldehyde, 0.01% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 6.8, for 30 min with gentle shaking at 4 °C. The fixed tissues were rinsed overnight at 4 °C in PBS containing 1% glycine. Then the tissues were treated with 10% (w/v) sucrose in PBS followed with 20% (w/v) sucrose in PBS for 2 h at 4 °C. The frozen tissues embedded in OCT compound were cut into sections 10-15 µm thick. After treatment with 5% (w/v) bovine serum albumin for 1 h, the sections were immunostained using MY-174 antibody. Then the sections were fixed again with 1% glutaraldehyde in PBS for 30 min on ice, and postfixed with 2% OsO4 for 1 h on ice. After sequential dehydration with ethanol, the sections were embedded in Epon resin and observed using an electron microscope.

Western Blot Analyses-- PBS-soluble and -insoluble IPM samples from retinas were prepared according to reported procedures (16, 17). PBS-insoluble IPM samples were also prepared from human and rat retinas. Each sample (10 µg of protein) was digested with enzymes in the presence of protease inhibitors. N-Glycosidase digestion was performed with recombinant N-glycanase in 0.5% SDS, 50 mM beta -mercaptoethanol, and 0.2 M Tris-HCl, pH 8.0, for 3 h at 37 °C. Acylneuraminyl hydrolase digestion was performed with neuraminidase (sialidase) in 0.5 M Tris-HCl, pH 6.5, for 1 h at 37 °C. O-Glycosidase digestion was performed with O-glycanase in 0.2 M citrate buffer, pH 4.5, for 3 h at 37 °C. Samples were analyzed by electrophoresis on 5% SDS-polyacrylamide gels. Proteins in the gel were blotted onto nitrocellulose membranes, and the membranes were incubated with MY-174. Localization of MY-174 binding was performed using a peroxidase-conjugated secondary antibody. Molecular weights of the protein bands on the SDS gel were estimated from the migration positions of protein standards (Bio-Rad Laboratories, Hercules, CA). An image analysis program (IMAGE, National Institutes of Health) was used to measure the expression of SPACR in each sample.

N-terminal Amino Acid Sequence of the MY-174-positive Band in Two-dimensional Electrophoresis-- A 10× volume of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 7 M urea, and 0.5% Triton X-100 was added to PBS-insoluble samples from retinas. Samples were applied to DEAE-Sephacel (Amersham Biosciences, Buckinghamshire, England) in the above urea solution. Bound proteins were eluted with a 0-0.5 M NaCl gradient. MY-174-positive fractions (0.2-0.25 M NaCl) were dialyzed against 4 M guanidinium chloride, 0.5% Triton X-100, 50 mM Tris-HCl, pH 8.0. Samples were subjected to a Sephacryl S-300 HR column (Amersham Biosciences) after reduction with 10 mM dithiothreitol (37 °C for 30 min). Partially purified MY-174 antigen obtained from the column was subjected two-dimensional gel electrophoresis (Mini-Protean II 2-D system, Bio-Rad Laboratories). Prior to isoelectric focusing, samples were solubilized at a protein concentration of ~1 mg/ml in 1.6% Bio-Lyte 5/7 ampholyte (Bio-Rad Laboratories), 0.4% Bio-Lyte 3/10 ampholyte (Bio-Rad Laboratories), 9.5 M urea, 2.0% Triton X-100, and 5% beta -mercaptoethanol. Isoelectric focusing of 30-µl samples took place in 0.9- x 57-mm tube gels (4% acrylamide (C = 3.0%) containing 9.2 M urea, 2.0% Triton X-100, 1.6% Bio-Lyte 5/7 ampholyte, and 0.4% Bio-Lyte 3/10 ampholyte). The first-dimension isoelectric focusing was performed at 500 V for 15 min and then at 750 V for 3 h. After the isoelectric focusing, the tube gels were extruded and placed on top of a 7.5% polyacrylamide gel (C = 3.0%) prepared in a Mini Protein Cell (Bio-Rad Laboratories). After equilibration for about 5 min in the presence of 0.5 ml of transfer buffer (62.5 mM Tris-HCl, pH 6.8, 10% (w/v) glycerol, 2.3% (w/v) SDS, 5.0% (v/v) beta -mercaptoethanol, and an aliquot of bromphenol blue), the second-dimension SDS-PAGE took place at a constant 100 V for 2 h. The protein separated by SDS-PAGE was then electrotransferred onto a ProBlott polyvinylidene difluoride membrane (Applied Biosystems, Foster City, CA). The transferred protein was visualized with Coomassie Brilliant Blue R-250. The amino acid sequences of the separated proteins were analyzed with a Model Procise 494 cLC protein sequencing system (Applied Biosystems). Another transferred membrane from a sample similar to that used in the Coomassie Brilliant Blue staining was developed with the MY-174 antibody. The blot was then reprobed. The membrane was incubated at 50 °C for 30 min in 0.05 M sodium phosphate, pH 6.5-10 mM SDS, 0.1 M beta -mercaptoethanol and then washed with PBS-Tween for removing the reaction solution. The stripping membrane was used for O46-F and O47-F antibodies and biotin-hyaluronan binding. Localizations of O46-F and O47-F antibody binding were performed using a peroxidase-conjugated protein A (ICN Pharmaceuticals, Inc., Aurora, OH), and the bound biotin-hyaluronan was developed with a peroxidase-conjugated streptavidin (Amersham Biosciences).

Chick SPACR Cloning-- Based on the N-terminal amino acid sequence of the purified MY-174 antigen, primers were designed to perform a sense strand as indicated in Table I. PCR was performed on a chick newborn retinal cDNA library. Extension of the known sequence at both the 5'- and 3'-ends revealed a coding sequence of 2784 base pairs. The full-length gene was amplified from the chick newborn cDNA library using forward (5'-CTCGGGAAGCGCGCCATTGTGTTGGT-3') and reverse (5'-ATACGACTCACTATAGGGCGAATTGGC-3') primers designed according to the plasmid sequence. The cDNA was cloned into pUC118 DNA vector (Takara Biomedicals, Kyoto, Japan), and both strands were sequenced on an ABI sequencer (Applied Biosystems).

Antibodies for Chick SPACR-- The DNA sequences were analyzed using GENETYX-MAC computer programs (Software Development Co., Ltd., Tokyo, Japan). According to the predicted amino acid sequence, two polypeptides (785-KQLEILNFRNGSVI-798 and 838-SYSLDIEPADQADPC-852) were selected and synthesized for making polyclonal antibodies against rabbit (termed them O46-F and O47-F, respectively).

Biotin-labeled Hyaluronan-- Biotin-labeled hyaluronan was prepared by dissolving hyaluronan (0.7 mg/0.5 ml) in 0.1 M sodium borate, pH 8.8, incubating it with N-hydroxysuccinimide biotin (hyaluronan:biotin, 50:1, Pierce) prepared in Me2SO (7.5 mg/ml) at room temperature for 4 h, and reacting the resulting products with 25 µl of 1 M ammonium chloride for 10 min. The reacted solution was dialyzed against PBS, pH 7.2.

Northern Blot Analysis-- Total RNA from each retinal sample was prepared and transferred to nylon filter as described previously (20). Reverse transcriptional reaction was performed using SuperScript II reverse transcriptase (Invitrogen, Groningen, Netherlands). A cDNA probe (0.4 kb) corresponding to chick SPACR N terminus was amplified from the newborn retinal cDNA template using forward (5'-ATGCATTTGAAAACTGGATT-3') and reverse (5'-TTTCCCTCTGGCAGGCAGTA-3') primers and then used for hybridization.

Measurements of Retinal Adhesion-- To quantify the degree of adherence of retinal pigment epithelium to the neural retina, a previously reported assay was used (11, 21, 22). In brief, enucleated eyecups of newborn chicks were rapidly cut into strips. The retina was peeled manually from the retinal pigment epithelium under Hanks' solution at room temperature. All observations were made within 3 min because retinal adhesiveness changes after enucleation (23). The strength of retinal adhesion was estimated by measuring the area of retina that was covered by adherent retinal pigment epithelium pigment using a computer video image analysis system (100% adherent pigment indicated firm adhesion; 0% adherent pigment indicated weak adhesion). About 10% retinal area of each strip was detached when the forceps were inserted. All statistical results are given as mean ± S.E.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of the Retinal MY-174 Antigen at the Interphotoreceptor Matrix-- MY-174 antigen was localized by immunofluorescent staining of radial sections of adult retina. Bright fluorescence was specifically detected in the photoreceptor layer (Fig. 1A, arrowhead) but not in the other layers of the retina. Fig. 1B shows a higher magnification of an oblique section of the photoreceptor layer, illustrating the clear honeycomb-like pattern of fluorescence that corresponds to the interphotoreceptor matrix (IPM) (Fig. 1B). Immunoelectron microscopy of cross-sections of the photoreceptor layer confirmed the localization of MY-174-reactive antigen in the matrix surrounding the inner segments of photoreceptor cells (Fig. 1C, arrowheads).


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  		Fig. 1.   Localization of retinal MY-174 antigen. A, radial sections of adult retina stained with MY-174. Fluorescence is specifically detected at the photoreceptor layer (arrowhead). Hematoxylin and eosin section is at the right. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PL, photoreceptor layer; RPE, retinal pigment epithelium. B, a higher magnification of the oblique section of the photoreceptor layer stained with MY-174. C, immunoelectron micrograph of a cross-section through the photoreceptor layer. MY-174 antigen (arrowheads) is detected in the matrix surrounding the inner segments (IS) of photoreceptor cells. Scale bars: 50 µm (A); 5 µm (C).

Identification of Retinal MY-174 Antigen as Chick SPACR-- The IPM consists of PBS-soluble and -insoluble molecules. To determine whether retinal MY-174 antigen was PBS-soluble or -insoluble, we performed immunoblot analysis on both PBS-soluble and -insoluble IPM samples prepared from adult chick retina. A distinct 150-kDa band was detected in the PBS-insoluble sample but not in the PBS-soluble sample (Fig. 2A, arrows), indicating that this antigen is entirely PBS-insoluble.


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  		Fig. 2.   Characterization of retinal MY-174 antigen. A, Western blot analyses of PBS-soluble and -insoluble IPM samples stained with MY-174 and O46-F antibodies. A distinct 150-kDa band is detected by MY-174 and O46-F in PBS-insoluble sample (arrows and arrowheads, respectively). B, Western blot analyses on the PBS-insoluble samples stained with MY-174 before and after digestions with chondroitinase ABC, N- or O-glycanases, and neuraminidase. The mobility of the 150-kDa band shows no change after chondroitinase ABC or O-glycanase treatment alone. N-Glycanase or neuraminidase treatment decreased mobility by 10 and 30 kDa, respectively. A smeared 120-kDa band yielded by the neuraminidase treatment was abolished by further treatment with O-glycanase. An arrowhead and horizontal bars indicate the positions of the 150-kDa original band for retinal MY-174 antigen. MY-174 weakly stains a nonspecific 160-kDa band in every lane (asterisks). MY-174 also stains the 160- and 130-kDa bands corresponding to O-glycanase itself (lanes having O-glycanase). +, -, and E indicate the samples with or without digestion by each enzyme, and the lane containing enzyme alone, respectively. C, effects of the combined enzyme treatments on the immunofluorescent staining of the retinal sections. Neuraminidase treatment alone did not change the staining pattern, but a combination of neuraminidase and O-glycanase treatments eliminates the fluorescence at the photoreceptor layer (PL) except around the outer segments of photoreceptor cells (arrowhead). D, Western blot analysis on the IPM samples from adult chick, human, and rat. A 150-kDa band is detected only in the lane for chick sample (arrowhead).

We examined whether the retinal antigen of MY-174 was modified by glycoconjugates. Retinal samples were subjected to membrane blot analyses before and after digestions with chondroitinase ABC, N- or O-glycanases, and neuraminidase in the presence of protease inhibitors, and the membrane was stained with MY-174 (Fig. 2B). When treated with chondroitinase ABC or O-glycanase alone, the mobility of the 150-kDa band showed no significant change. However, when treated with N-glycanase or neuraminidase, mobility increased by 10 and 30 kDa, respectively. The neuraminidase treatment yielded a smeared 120-kDa band regardless of the amount of neuraminidase used (data not shown). Further treatment with O-glycanase after the neuraminidase treatment abolished the stained band (Fig. 2B, Neuraminidase + O-glycanase). This abolishment by O-glycanase treatment was not observed without predigestion with neuraminidase (Fig. 2B, O-glycanase), a requirement that has been well documented for other glycoproteins (24). These results suggest that the retinal antigen of MY-174 does not have chondroitin sulfate chains but has sialylated N- and O-linked glycoconjugates, which have a molecular mass of more than 30 kDa, and the latter may be included at least in part in the epitope structure for MY-174. MY-174 weakly stained a 160-kDa band in every lane, which may be due to staining with some contaminating proteins (Fig. 2B, asterisks; see arrowheads in Fig. 7A). MY-174 also stained rather strongly the 160- and 130-kDa bands corresponding to O-glycanase itself (Fig. 2B, lanes having O-glycanase), which might be due to the presence of cross-reactive structures in the enzyme.

We further examined the effects of enzymes on the immunofluorescent staining of the retinal sections. The sections pretreated with neuraminidase were further treated with or without O-glycanase and then stained with MY-174 (Fig. 2C). Neuraminidase or O-glycanase treatment alone did not show any change to the staining (Fig. 2C and data not shown), but a combination of neuraminidase and O-glycanase treatments eliminated the fluorescence at the photoreceptor layer except around the outer segments of photoreceptor cells (Fig. 2C, arrowhead). MY-174 was originally established as a monoclonal antibody against PG-M/versican and has been shown to be chick-specific (1). To investigate if the MY-174 epitope structure was specific to chick or not, we performed Western blot analysis on IPM samples obtained from adult human and rat. Interestingly, a 150-kDa band was only detected in the lane of the chick sample (Fig. 2D, arrowhead), suggesting that the epitope structure might include chick-specific O-glycoconjugates.

The retinal MY-174 antigen, having a molecular mass of 150 kDa, was partially purified by ion exchange chromatography in conjunction with gel filtration chromatography. The partially purified sample was then subjected to a two-dimensional gel electrophoresis system as described under "Experimental Procedures." The separated protein on a polyvinylidene difluoride membrane was visualized with Coomassie Brilliant Blue staining (Fig. 3A). Another immunoblot membrane obtained after a similar sample separation was developed with MY-174 (Fig. 3B). The four spots, having a molecular mass of 150 kDa, were similarly detected by both Coomassie Brilliant Blue staining and MY-174 antibody (Fig. 3, A and B, arrowheads). One of the four spots was cut out to analyze the amino acid sequence (Fig. 3, A and B, asterisks). According to the obtained N-terminal sequences, a combination of oligonucleotide primers had been designed (Table I). PCR was performed on the newborn retinal cDNA library to determine the full length of the nucleotide sequences as described under "Experimental Procedures." The 2787-base pair open reading frame encoded a 928-amino acid protein (Fig. 4). BLAST analysis of public databases revealed human SPACR to be its most homologous relatively. This nucleotide sequence contains seven potential N-linked glycosylation sites, numerous potential O-linked glycosylation sites, and an EGF-like domain near the C terminus like human and mouse SPACRs. It has 56 and 54% nucleotide sequence identities with human and mouse SPACRs, respectively. Fig. 5 shows the deduced primary amino acid sequences of chick SPACR compared with human and mouse SPACR. The deduced amino acid sequence shares 52 and 42% similarities with the human and mouse deduced sequences, respectively, suggesting that they are indeed orthologs. The polyclonal antibodies against synthesized peptides were newly established. The O46-F antibody reacted with MY-174-positive spots (Figs. 2A and 3C, arrowheads). Another antibody, O47-F, established based on another determined amino acid sequence also showed similar positive spots (data not shown). These four spots corresponding to retinal MY-174 antigen showed the binding activity to biotin-hyaluronan (Fig. 3D, arrowheads). These results suggested that these spots were derived from the same molecule that reacted with both MY-174 and peptide antibodies. Taken together, we concluded that the retinal antigen of MY-174 in the interphotoreceptor matrix was the chick ortholog of human SPACR.


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  		Fig. 3.   Retinal MY-174 antigen and its reactivity to biotin-hyaluronan. A, Coomassie Brilliant Blue-stained gel after two-dimensional gel electrophoresis using partially purified MY-174 antigen from chick retinas. The four spots correspond to retinal MY-174 antigen (arrowheads, see arrowheads in Fig. 3B). Molecular mass marker positions are indicated on the left. PI is indicated at the top. Isoelectric focusing in the horizontal dimension with the anode is on the left. B, the immunoblot from a separation similar to the one shown in A and developed with MY-174 diluted 1:10,000. One of the spots was cut out to analyze amino acid sequence (asterisks in A and B). C, the immunoblot of the same membrane in B after stripping and developed with O46-F antibody diluted 1:1,000. The four spots react with O46-F antibody (arrowheads). D, the immunoblot of the same membrane in B after stripping and developed with biotin-hyaluronan diluted 1:500. The four spots also react with biotin-hyaluronan (arrowheads).

                              
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Table I
N-terminal peptide sequences of the purified MY-174 antigen and the predicted nucleotide sequences


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  		Fig. 4.   Nucleotide and deduced peptide sequences of chick SPACR (GenBankTM accession number AB070714). The deduced protein contains 928 amino acids. Seven potential N-linked glycosylation sites are underlined. Numerous potential O-linked glycosylation sites are present. An EGF-like domain (in boldface from residues Cys874 to Cys888) is present near the C terminus. Two residue regions (Lys785 to Ile798 and Ser838 to Cys852) were selected for making polyclonal antibodies (underlined with dotted lines, O46-F and O47-F, respectively). Consensus sites for GAG attachment are boxed.


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  		Fig. 5.   An alignment of the predicted amino acid sequences of chick (c), human (h), and mouse (m) SPACRs were created with the GENETYX-MAC computer programs. Amino acids are numbered. Identical amino acids are boxed.

SPACR Expression and Retinal Adhesiveness during Development-- Immunofluorescent staining of retinas at embryonic day 5 (E5), 9 (E9), 14 (E14), newborn (Nb) and adult (Ad) showed that the expression of retinal MY-174 antigen is developmentally regulated. No fluorescence was observed at photoreceptor layers in any of the embryonic retinas examined (Fig. 6, E5-E14). However, significant staining of the matrix in photoreceptor layers became detectable in newborn retinas (Fig. 6, arrowhead in Nb), and the highest expression was seen in adult retina (Fig. 6, arrowhead in Ad). This suggested that MY-174 antigen synthesis was initiated at the later embryonic stages between E14 and birth.


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  		Fig. 6.   Immunofluorescent staining of SPACR at embryonic days 5 (E5), 9 (E9), and 14 (E14) and newborn (Nb) and adult (Ad) with MY-174. No staining is seen in any photoreceptor layer of embryonic retinas examined (E5-E14). However, strong staining of the photoreceptor layer is detected in newborn and adult retinas (Nb and Ad, arrowheads). H & E sections are on the right. The positions of retinal pigment epithelium (RPE) are shown. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PL, photoreceptor layer. Scale bar, 50 µm.

Immunoblot analysis was performed to assess the expression of chick SPACR (Fig. 7, A and B). PBS-insoluble IPM samples obtained from chick retinas at various stages (E14-Nb) were electrophoresed on 5% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and stained with MY-174 or O46-F. Distinct 150-kDa bands, corresponding to retinal MY-174 antigen (Fig. 7A, arrow; see Fig. 2A) or O46-F antigen (Fig. 7B, arrow) were measured by using the image analysis program IMAGE (National Institutes of Health). At E16, a specific 150-kDa band appeared. Expressions steeply increased at E18 and E17 in MY-174 and O46-F antigens, respectively. In every lane, a 160-kDa band was detected (Fig. 7A, arrowheads) but was not band-specific to the staining of the photoreceptor layer, because it was not detected at E14 (see E14 in Fig. 6 and asterisks in Fig. 2B). In adult, the expression of the 150-kDa MY-174-reactive antigen increased 1.87-fold over newborn levels (data not shown). Amounts of SPACR mRNA were quantified using Northern blot analysis (Fig. 7C). After 10 µg/lane of total RNA, prepared from the retinas of each embryonic stage, were electrophoresed and then transferred to nylon filter, they were hybridized with radiolabeled probes derived from cDNA for chick SPACR. The expression of SPACR in each sample was measured using IMAGE. At E15, a specific 6.0-kb single band was first detected and increased with development.


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  		Fig. 7.   Occurrences of chick SPACR and retinal adhesiveness during development. A, SPACR expression was measured with MY-174 on E14 to newborn (Nb) retinal samples. At E16, a 150-kDa band first appears, and expression increases with developmental age (arrow). The densities at the positions of 150-kDa bands in E14 and newborn retinas are defined as 0 and 100%, respectively. In every lane, a 160-kDa band is detected (arrowheads) but is not band-specific to the photoreceptor (see "Results"). B, SPACR expression was measured with O46-F antibody on E14 to newborn (Nb) retinal samples. C, the 6.0-kb mRNA corresponding to chick SPACR was analyzed by Northern blot analysis. At E15, a band is first detected, and expression increases with developmental age. A 10-µg total RNA prepared from retina on each stage was transferred to a nylon filter and hybridized with a radiolabeled N terminus cDNA (0.4 kb) of chick SPACR. D, retinal adhesiveness of these samples was measured. Retinal adhesiveness is initially detected at E16 and increases with developmental age. Homogenized samples of peeled retina corresponding to these days are also shown. The amounts of pigmentation derived from retinal pigment epithelium in homogenized samples demonstrate the retinal adhesiveness.

Retinal adhesiveness was measured by peeling the retina from the retinal pigment epithelium and observing the amount of adherent pigment (Fig. 7D). Retinal adhesiveness was initially detected at E16 and increased with development. There was no difference between the retinal adhesiveness of adult and newborn retinas (data not shown). The amounts of pigmentation derived from retinal pigment epithelium in homogenized samples of peeled retina applied onto wells demonstrated the retinal adhesiveness corresponding to these stages.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we showed that a MY-174 antigen in the IPM is the ortholog of human SPACR and that there is an increase in SPACR expression during chick development. We also showed that the expression increases in parallel with retinal adhesiveness, implying that SPACR might be involved in the adhesion between neural retina and retinal pigment epithelium.

We showed that sialylated O-linked glycoconjugates are involved in the epitope structure recognized by MY-174, which was developed as a monoclonal antibody to the core protein of PG-M/versican. The antigen was prepared by mild alkali treatment of the core protein of PG-M/versican, and the treatment eliminated glycoconjugates from the core protein (1). However, when we made fusion proteins for the full length of PG-M/versican (V0) by Escherichia coli, MY-174 showed no immunoreactivity to the fusion core protein (data not shown). No immunoreactive clones could be detected by a MY-174 in screening of the chick retina cDNA library (data not shown). Acharya et al. (16) mentioned that the major glycoconjugate on SPACR is the O-linked carbohydrate, NeuAcalpha 2-3Galbeta 1-3GalNAc and that the O-linked sugar in SPACR could have a structure similar to that present in bone sialoprotein and aggrecan. At this stage we speculate that any residual O-glycans left on the core protein of PG-M/versican, specific but similar to those characteristic of SPACR, were the antigen for MY-174.

Because our previous study using a polyclonal antibody that recognizes all PG-M/versican forms showed no staining in the photoreceptor layers of adult chick retinas (5), we also thought it unlikely that PG-M/versican would be a target for MY-174 in retina. We have repeated this published observation with 2B1, a monoclonal antibody that recognizes all human PG-M/versican forms (25), but again no specific staining of the human photoreceptor layer could be detected (data not shown). Furthermore, no specific staining at an adult mouse photoreceptor layer could be detected using polyclonal antibodies against mouse PG-M/versican (data not shown).

MY-174 staining showed resistance to a combination of neuraminidase and O-glycanase treatments around the outer segments of photoreceptor cells. Tien et al. (15) also reported that wheat germ agglutinin staining of SPACR is resistant to neuraminidase around the outer segments of photoreceptor cells in retinal sections. The reason for the resistance to enzymatic treatments is unclear, but the IPM seems to have different properties around inner or outer segments of photoreceptor cells.

Acharya et al. (17) showed human SPACR has a functional hyaluronan-binding domain. This domain (RHAMM-type hyaluronan binding motif) corresponded to 280KEIHVLGFK288 in chick SPACR. It showed high consensus with human and mouse SPACR. This is a candidate for the RHAMM-type hyaluronan binding motif, because a single acidic group can be present in one residue inside either flanking basic residue in a B7XB RHAMM-type hyaluronan binding motif (26). Our data showed chick SPACR can bind hyaluronan. Mutagenesis studies of the putative hyaluronan binding motif will be required to establish the site of hyaluronan binding in chick SPACR. Because hyaluronan is a prominent constituent of the IPM in all species studied except mouse (27, 28), we speculate that SPACR and hyaluronan form an adhesive complex in the matrix between the neural retina and retinal pigment epithelium. Some reports have indeed shown that the insoluble-IPM is involved in the adhesion. A histochemical study using experimentally detached retinas showed that the insoluble cone matrix sheath was closely associated with both the cone photoreceptor and the apical surface of the retinal pigment epithelium, suggesting that this sheath mediated attachment (29). Insoluble IPM constituents could be found in vitreous from eyes suffering from rhegmatogenous retinal detachment (30).

Fig. 7 (A-C) shows the expression levels of mRNA, core protein, and sialylated O-glycan that were analyzed by using labeled cDNA-probe, O-46F, and MY-174, respectively. Successively, each expression increased in a comprehensible order during the development, because the translation processes were followed by a glycosylation process. There are some different expression patterns between mRNA and the core protein. One possibility is that the processes for transcription and translation and the glycosylation that follows are regulated in different manners in SPACR expression. Another possibility is that, at earlier developmental stages, there might be an increased turnover of the protein, because the protein levels at E15-E16 are significantly lower than the corresponding mRNA levels at the same developmental stages.

Adler and Gibson (23) showed that neural retinal adhesiveness starts at E17-E18 and that this is coincident with maturation of photoreceptor outer segments. In this study, we examined retinal adhesion using a peeling method (11, 21, 22). Although there is no adhesion at E15, we detected substantial adhesiveness at E16 by this method. The expression of SPACR in adult retina is 1.9 times the newborn level, but retinal adhesiveness of adult retina is almost equal to the newborn one. We suggest that adhesiveness does not increase after a threshold SPACR expression level is reached.

Chondroitin sulfates are major constituents of the IPM (31). Chick SPACR is not a chondroitin-type proteoglycan, because the mobility of the chick SPACR band showed no significant change after chondroitinase ABC treatment. We used the antibodies for Delta Di6S-, Delta Di4S-, and Delta Di0S-chondroitin epitopes exposed following chondroitinase ABC digestion to determine whether a chick SPACR core protein is released. That there were no specific bands for each epitope suggested that chick SPACR is not a chondroitin-type proteoglycan (data not shown), whereas Fig. 4 showed SG residues, SGD residues, and DGS residues as candidates for chondroitin sulfate attachment consensus sequences. Intracellular blocking of the attachment of chondroitin sulfate chains with xyloside prevented the secretion of proteoglycans and resulted in retinal detachment (13). Retinal adhesiveness was also weakened by chondroitinase ABC (11). These reports suggest that chondroitin sulfate proteoglycans are involved in the adhesion. However, our report implicates a different type of molecule in this process.

Our present study showed that SPACR has a pericellular distribution reminiscent of cell membranes (Fig. 1C). To clarify whether SPACR is associated with the cell membrane, we tried a similar immunohistochemical study using O46-F and O47-F. However, these antibodies were not available for immunohistochemical study. Two SEA modules (32) corresponding to 231-348 and 728-853 in amino acid sequence positions of chick SPACR are also conserved in human and mouse SPACR (Pfam data base). The SEA module found in a number of heavily O-linked glycosylated membrane-associated adhesive proteins is considered to regulate receptor-ligand alliance by proteolytic cleavage (33). Bishop et al. (34) showed sialylated glycans at the IPM and photoreceptor plasmalemmata by histochemical study. There is no evidence regarding the association of SPACR with the cell membrane, but SPACR is a molecule potentially associated with the cell membrane. To clarify the retinal adhesion mechanism and other biological functions of SPACR, we must establish an in vivo system.

In summary, the present findings demonstrate that the MY-174 antigen at the photoreceptor layer is identical to chick SPACR. The O-glycans on the core protein of PG-M/versican are similar to those characteristics of chick SPACR and are considered to be the antigen for MY-174. The correlation of the appearance of SPACR and the development of retinal adhesiveness suggests that SPACR may be a functional adhesive between neural retina and retinal pigment epithelium.

    FOOTNOTES

* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB070714.

§ To whom correspondence should be addressed. Tel.: 81-52-264-4811 (ext. 2181); Fax: 81-561-63-7255; E-mail: zako@aichi-med-u.ac.jp.

Published, JBC Papers in Press, May 3, 2002, DOI 10.1074/jbc.M201279200

    ABBREVIATIONS

The abbreviations used are: IPM, interphotoreceptor matrix; SPACR, sialoprotein associated with cones and rods; PBS, phosphate-buffered saline; EGF, epidermal growth factor; E, embryonic day; Nb, newborn; Ad, adult; RHAMM, receptor for hyaluronic acid-mediated motility.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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