Molecular Cloning and Characterization of Chick SialoproteinAssociated with Cones and Rods, a Developmentally RegulatedGlycoprotein of Interphotoreceptor Matrix*

Received for publication, February 7, 2002, and in revised form, May 2, 2002Published, JBC Papers in Press, May 3, 2002, DOI 10.1074/jbc.M201279200

Masahiro Zako‡§, Masayoshi Iwaki‡, Masahiko Yoneda¶, Osamu Miyaishi�, Jinsong Zhao‡,Yasuhiko Suzuki**, Makoto Takeuchi‡, Goichiro Miyake‡, Hiroshi Ikagawa‡, and Koji Kimata‡‡

From the Departments of ‡Ophthalmology and �Pathology, the ‡‡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

MY-174 is an IgM class monoclonal antibody originallyestablished against chick PG-M/versican. The antibodyspecifically stains the photoreceptor layer, where werecently reported an absence of PG-M/versican. In thisstudy, we re-characterized the antibody and identifiedthe molecule that reacts to MY-174 at the photoreceptorlayer. Immunohistochemistry localized the antigen tothe matrix surrounding photoreceptors. A variety of gly-cosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylated N- and O-linkedglycoconjugates having a molecular mass of more than30-kDa. The peptide sequences obtained from purifiedMY-174 antigen showed we had sequenced a full-lengthcDNA with an open reading frame of 2787 base pairs,encoding a polypeptide of 928 amino acids, with 56 and54% identities to human and mouse sialoprotein associ-ated with cones and rods (SPACRs), respectively, andwith the structural features observed in SPACRs. Thespecific sialylated O-glycoconjugates here are involvedin the epitope structure for MY-174. SPACR first ap-peared by embryonic days 15–16, and expression in-creased with developmental age, paralleling the adhe-sion between neural retina and retinal pigmentepithelium. Thus, we concluded that the MY-174 antigenat the photoreceptor layer, a developmentally regulatedglycoprotein, is identical to chick SPACR and may beinvolved in a novel system mediating adhesion betweenneural retina and retinal pigment epithelium.

MY-174, a monoclonal antibody, has been shown to recognizechick PG-M/versican (1), a member of the hyaluronan-bindingchondroitin 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 thechick photoreceptor layer using a polyclonal antibody that rec-ognizes all alternatively spliced forms of PG-M/versican (5).These inconsistent results suggest that MY-174 does not rec-

ognize PG-M/versican but another molecule in the photorecep-tor layer.

The IPM (interphotoreceptor matrix),1 resides in an extra-cellular compartment between the outer limiting membrane ofthe neural retina and apical surface of the retinal pigmentepithelium and is composed of proteins, glycoproteins, proteo-glycans, and glycosaminoglycans (6, 7). A variety of importantreactions relating to vision, including visual pigment chro-mophore exchange, metabolite trafficking, photoreceptor align-ment, and membrane turnover, is thought to be mediated bythe IPM (8). However, one of the most essential functions ofIPM is that of a biological glue for retinal adhesion through itsviscous adhesive properties (9, 10). Retinal adhesiveness can beweakened by treatments with enzymes such as neuraminidase,hyaluronidase, and chondroitinase ABC (11, 12). Intracellularblocking of glycosylation with xyloside also prevents the secre-tion of proteoglycans and results in retinal detachment (13).These results suggest that the proteoglycans, glycosaminogly-cans, or glycoproteins of the IPM are involved in retinal adhe-sion. However, the specific IPM molecule that mediates adhe-sion has not been identified.

SPACR (sialoprotein associated with cones and rods) is ahyaluronan-binding glycoprotein newly identified in adult hu-man PBS (phosphate-buffered saline)-insoluble IPM (14–17).This 147- to 150-kDa glycoprotein was purified by wheat germagglutinin-affinity chromatography and characterized (17). Ithas been shown that (a) SPACR is heavily sialylated, (b) bothN- and O-linked glycoconjugates are present in the molecule,and (c) glycoconjugates account for �30% of the molecularmass (17). Recently, mouse SPACR was also cloned and char-acterized (18). Both human and mouse SPACRs have a largecentral mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain nearthe C-terminal, and several potential hyaluronan-binding mo-tifs in common. Interestingly, biochemical studies showed thatSPACR is a glycoprotein in human and a proteoglycan inmouse. Except for the ability of SPACR to bind hyaluronan(17), other properties or functional roles of SPACR remainunknown.

In the present study, we first examined the localizations ofthe retinal antigen that binds to MY-174 in the photoreceptor

* This work was supported by a grant-in-aid for scientific researchfrom the Ministry of Education, Culture, Sports, Science and Technol-ogy, Japan. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto 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.

1 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-mediatedmotility.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 28, Issue of July 12, pp. 25592–25600, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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layer. We then determined a full-length cDNA of MY-174 an-tigen at photoreceptor layer and showed that the antigen cor-responded to chick SPACR. Finally, to investigate the biologi-

cal significance of SPACR, we compared the expressions ofchick SPACR and the occurrence of adhesion between neuralretina and retinal pigment epithelium during development.

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

FIG. 2. Characterization of retinal MY-174 antigen. A, Western blot analyses of PBS-soluble and -insoluble IPM samples stained withMY-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 treatmentalone. N-Glycanase or neuraminidase treatment decreased mobility by 10 and 30 kDa, respectively. A smeared 120-kDa band yielded by theneuraminidase treatment was abolished by further treatment with O-glycanase. An arrowhead and horizontal bars indicate the positions of the150-kDa original band for retinal MY-174 antigen. MY-174 weakly stains a nonspecific 160-kDa band in every lane (asterisks). MY-174 also stainsthe 160- and 130-kDa bands corresponding to O-glycanase itself (lanes having O-glycanase). �, �, and E indicate the samples with or withoutdigestion by each enzyme, and the lane containing enzyme alone, respectively. C, effects of the combined enzyme treatments on the immunoflu-orescent staining of the retinal sections. Neuraminidase treatment alone did not change the staining pattern, but a combination of neuraminidaseand 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 chicksample (arrowhead).

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EXPERIMENTAL PROCEDURES

Materials—Fertilized eggs from white leghorns were maintained in ahumidified incubator at 37 °C, and embryos at the different develop-mental stages were obtained according to the standards used byHamburger and Hamilton (19) and their retinas used for study. Retinasfrom adult white leghorns and Sprague-Dawley rats were also used. Allexperimental procedures in this study conformed to the Association forResearch in Vision and Ophthalmology Statement for the Use of Ani-mals in Ophthalmic and Vision Research.

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

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

Immunofluorescent Microscopy—Chick eyes were cut into two piecesand then fixed in 3.7% (w/v) formaldehyde solution neutralized withcalcium carbonate in 0.1 M sodium phosphate buffer, pH 6.8, for 1 h atroom temperature with gentle shaking. Fixed retinas were rinsed inPBS containing 0.1% (w/v) glycine at 4 °C overnight and then embeddedin OCT compound (Miles Scientific, Naperville, IL) on petroleum ether-dry ice and dissected. Cryostat sections (6–8 �m) were incubated withMY-174 in 10% (w/v) normal swine serum for 30 min at room temper-ature. After rinsing three times with PBS for 3 min for each rinse, thesections were incubated with goat antibody against mouse IgM (TAGO,

Burlingame, CA) and then with fluorescein isothiocyanate-conjugatedswine antibody against goat IgG (TAGO) in PBS containing 10% (w/v)normal swine serum. Finally, the sections were rinsed in PBS andmounted in mounting media (Shandon Lipshaw, Detroit, MI). Immu-nolabeled tissue sections were observed using a fluorescence microscope(Olympus, Tokyo, Japan). Photographs were taken using Kodak Tri-Xpan film (Eastman Kodak Co., Rochester, NY). Immunohistochemicalstaining was performed on 20 different eyes, and reproducible resultswere achieved. A control for nonspecific staining omitted the primaryantibody and replaced it with mouse nonimmune serum at the sameprotein concentration. No staining was observed in control sections.

In some cases, enzymatic treatments were performed prior to immu-nostaining. Acylneuraminyl hydrolase and O-glycosidase digestionswere 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-�-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 smallpieces 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 at4 °C. The fixed tissues were rinsed overnight at 4 °C in PBS containing1% glycine. Then the tissues were treated with 10% (w/v) sucrose inPBS followed with 20% (w/v) sucrose in PBS for 2 h at 4 °C. The frozentissues embedded in OCT compound were cut into sections 10–15 �mthick. After treatment with 5% (w/v) bovine serum albumin for 1 h, thesections were immunostained using MY-174 antibody. Then the sec-tions 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 dehydrationwith ethanol, the sections were embedded in Epon resin and observedusing an electron microscope.

Western Blot Analyses—PBS-soluble and -insoluble IPM samplesfrom retinas were prepared according to reported procedures (16, 17).PBS-insoluble IPM samples were also prepared from human and ratretinas. Each sample (10 �g of protein) was digested with enzymes inthe presence of protease inhibitors. N-Glycosidase digestion was per-formed with recombinant N-glycanase in 0.5% SDS, 50 mM �-mercap-toethanol, and 0.2 M Tris-HCl, pH 8.0, for 3 h at 37 °C. Acylneuraminylhydrolase digestion was performed with neuraminidase (sialidase) in0.5 M Tris-HCl, pH 6.5, for 1 h at 37 °C. O-Glycosidase digestion wasperformed with O-glycanase in 0.2 M citrate buffer, pH 4.5, for 3 h at37 °C. Samples were analyzed by electrophoresis on 5% SDS-polyacryl-amide gels. Proteins in the gel were blotted onto nitrocellulose mem-branes, and the membranes were incubated with MY-174. Localizationof MY-174 binding was performed using a peroxidase-conjugated sec-ondary antibody. Molecular weights of the protein bands on the SDS gelwere 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 ofSPACR in each sample.

N-terminal Amino Acid Sequence of the MY-174-positive Band inTwo-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 reti-nas. Samples were applied to DEAE-Sephacel (Amersham Biosciences,Buckinghamshire, England) in the above urea solution. Bound proteinswere 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 toa Sephacryl S-300 HR column (Amersham Biosciences) after reductionwith 10 mM dithiothreitol (37 °C for 30 min). Partially purified MY-174antigen obtained from the column was subjected two-dimensional gelelectrophoresis (Mini-Protean II 2-D system, Bio-Rad Laboratories).Prior to isoelectric focusing, samples were solubilized at a proteinconcentration of �1 mg/ml in 1.6% Bio-Lyte 5/7 ampholyte (Bio-RadLaboratories), 0.4% Bio-Lyte 3/10 ampholyte (Bio-Rad Laboratories),

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

TABLE IN-terminal peptide sequences of the purified MY-174 antigen and the

predicted nucleotide sequences

N-terminal sequencesa

Ser Pro Phe Phe Ser Thr Gly Val Lys IleCCT TTT TTT TCI ACI GGI GTI AAA ATC C C AGT GA AGCG

5�- -3�a I, deoxyionosine.

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9.5 M urea, 2.0% Triton X-100, and 5% �-mercaptoethanol. Isoelectricfocusing of 30-�l 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 andthen at 750 V for 3 h. After the isoelectric focusing, the tube gels wereextruded and placed on top of a 7.5% polyacrylamide gel (C � 3.0%)prepared in a Mini Protein Cell (Bio-Rad Laboratories). After equilibra-tion 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) �-mer-captoethanol, and an aliquot of bromphenol blue), the second-dimensionSDS-PAGE took place at a constant 100 V for 2 h. The protein separatedby SDS-PAGE was then electrotransferred onto a ProBlott polyvinyli-dene difluoride membrane (Applied Biosystems, Foster City, CA). Thetransferred protein was visualized with Coomassie Brilliant BlueR-250. The amino acid sequences of the separated proteins were ana-lyzed with a Model Procise 494 cLC protein sequencing system (AppliedBiosystems). Another transferred membrane from a sample similar tothat used in the Coomassie Brilliant Blue staining was developed withthe 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 6.5–10mM SDS, 0.1 M �-mercaptoethanol and then washed with PBS-Tweenfor removing the reaction solution. The stripping membrane was usedfor O46-F and O47-F antibodies and biotin-hyaluronan binding. Local-izations of O46-F and O47-F antibody binding were performed using aperoxidase-conjugated protein A (ICN Pharmaceuticals, Inc., Aurora,OH), and the bound biotin-hyaluronan was developed with a peroxi-dase-conjugated streptavidin (Amersham Biosciences).

Chick SPACR Cloning—Based on the N-terminal amino acid se-quence of the purified MY-174 antigen, primers were designed to per-form a sense strand as indicated in Table I. PCR was performed on achick newborn retinal cDNA library. Extension of the known sequenceat both the 5�- and 3�-ends revealed a coding sequence of 2784 basepairs. The full-length gene was amplified from the chick newborn cDNAlibrary using forward (5�-CTCGGGAAGCGCGCCATTGTGTTGGT-3�)and reverse (5�-ATACGACTCACTATAGGGCGAATTGGC-3�) primersdesigned according to the plasmid sequence. The cDNA was cloned intopUC118 DNA vector (Takara Biomedicals, Kyoto, Japan), and bothstrands were sequenced on an ABI sequencer (Applied Biosystems).

Antibodies for Chick SPACR—The DNA sequences were analyzed

FIG. 4. Nucleotide and deduced peptide sequences of chick SPACR (GenBankTM accession number AB070714). The deduced proteincontains 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 GAGattachment are boxed.

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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-SYSLDIE-PADQADPC-852) were selected and synthesized for making polyclonalantibodies against rabbit (termed them O46-F and O47-F, respectively).

Biotin-labeled Hyaluronan—Biotin-labeled hyaluronan was pre-pared 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 tempera-ture for 4 h, and reacting the resulting products with 25 �l of 1 M

ammonium chloride for 10 min. The reacted solution was dialyzedagainst PBS, pH 7.2.

Northern Blot Analysis—Total RNA from each retinal sample wasprepared and transferred to nylon filter as described previously (20).Reverse transcriptional reaction was performed using SuperScript IIreverse transcriptase (Invitrogen, Groningen, Netherlands). A cDNAprobe (0.4 kb) corresponding to chick SPACR N terminus was amplifiedfrom the newborn retinal cDNA template using forward (5�-ATGCAT-TTGAAAACTGGATT-3�) and reverse (5�-TTTCCCTCTGGCAGGCAG-TA-3�) primers and then used for hybridization.

Measurements of Retinal Adhesion—To quantify the degree of adher-ence of retinal pigment epithelium to the neural retina, a previouslyreported assay was used (11, 21, 22). In brief, enucleated eyecups ofnewborn chicks were rapidly cut into strips. The retina was peeledmanually from the retinal pigment epithelium under Hanks’ solution atroom temperature. All observations were made within 3 min becauseretinal adhesiveness changes after enucleation (23). The strength ofretinal adhesion was estimated by measuring the area of retina thatwas covered by adherent retinal pigment epithelium pigment using acomputer video image analysis system (100% adherent pigment indi-cated firm adhesion; 0% adherent pigment indicated weak adhesion).About 10% retinal area of each strip was detached when the forcepswere inserted. All statistical results are given as mean � S.E.

RESULTS

Expression of the Retinal MY-174 Antigen at the Interphotore-ceptor Matrix—MY-174 antigen was localized by immunofluores-cent staining of radial sections of adult retina. Bright fluores-cence was specifically detected in the photoreceptor layer (Fig.1A, arrowhead) but not in the other layers of the retina. Fig. 1Bshows a higher magnification of an oblique section of the photo-receptor layer, illustrating the clear honeycomb-like pattern offluorescence that corresponds to the interphotoreceptor matrix(IPM) (Fig. 1B). Immunoelectron microscopy of cross-sections ofthe photoreceptor layer confirmed the localization of MY-174-reactive antigen in the matrix surrounding the inner segments ofphotoreceptor cells (Fig. 1C, arrowheads).

Identification of Retinal MY-174 Antigen as Chick SPACR—The IPM consists of PBS-soluble and -insoluble molecules. Todetermine 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 chickretina. 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.

We examined whether the retinal antigen of MY-174 wasmodified by glycoconjugates. Retinal samples were subjected tomembrane blot analyses before and after digestions with chon-droitinase ABC, N- or O-glycanases, and neuraminidase in thepresence of protease inhibitors, and the membrane was stainedwith MY-174 (Fig. 2B). When treated with chondroitinase ABCor O-glycanase alone, the mobility of the 150-kDa band showed

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

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no significant change. However, when treated with N-gly-canase or neuraminidase, mobility increased by 10 and 30 kDa,respectively. The neuraminidase treatment yielded a smeared120-kDa band regardless of the amount of neuraminidase used(data not shown). Further treatment with O-glycanase afterthe neuraminidase treatment abolished the stained band (Fig.2B, Neuraminidase � O-glycanase). This abolishment by O-glycanase treatment was not observed without predigestionwith neuraminidase (Fig. 2B, O-glycanase), a requirement thathas been well documented for other glycoproteins (24). Theseresults suggest that the retinal antigen of MY-174 does nothave chondroitin sulfate chains but has sialylated N- and O-linked glycoconjugates, which have a molecular mass of morethan 30 kDa, and the latter may be included at least in part inthe epitope structure for MY-174. MY-174 weakly stained a160-kDa band in every lane, which may be due to staining withsome contaminating proteins (Fig. 2B, asterisks; see arrow-heads 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 thepresence of cross-reactive structures in the enzyme.

We further examined the effects of enzymes on the immunoflu-orescent staining of the retinal sections. The sections pretreatedwith neuraminidase were further treated with or without O-glycanase and then stained with MY-174 (Fig. 2C). Neuramini-dase or O-glycanase treatment alone did not show any change tothe staining (Fig. 2C and data not shown), but a combination ofneuraminidase and O-glycanase treatments eliminated the fluo-rescence at the photoreceptor layer except around the outer seg-ments of photoreceptor cells (Fig. 2C, arrowhead). MY-174 wasoriginally established as a monoclonal antibody against PG-M/versican and has been shown to be chick-specific (1). To investi-gate if the MY-174 epitope structure was specific to chick or not,we performed Western blot analysis on IPM samples obtainedfrom adult human and rat. Interestingly, a 150-kDa band wasonly detected in the lane of the chick sample (Fig. 2D, arrow-head), suggesting that the epitope structure might include chick-specific O-glycoconjugates.

The retinal MY-174 antigen, having a molecular mass of 150kDa, was partially purified by ion exchange chromatography inconjunction with gel filtration chromatography. The partiallypurified sample was then subjected to a two-dimensional gelelectrophoresis system as described under “Experimental Pro-cedures.” The separated protein on a polyvinylidene difluoridemembrane was visualized with Coomassie Brilliant Blue stain-ing (Fig. 3A). Another immunoblot membrane obtained after asimilar sample separation was developed with MY-174 (Fig.3B). The four spots, having a molecular mass of 150 kDa, weresimilarly detected by both Coomassie Brilliant Blue stainingand MY-174 antibody (Fig. 3, A and B, arrowheads). One of thefour spots was cut out to analyze the amino acid sequence (Fig.3, A and B, asterisks). According to the obtained N-terminalsequences, a combination of oligonucleotide primers had beendesigned (Table I). PCR was performed on the newborn retinalcDNA library to determine the full length of the nucleotidesequences as described under “Experimental Procedures.” The2787-base pair open reading frame encoded a 928-amino acidprotein (Fig. 4). BLAST analysis of public databases revealedhuman SPACR to be its most homologous relatively. This nu-cleotide sequence contains seven potential N-linked glycosyla-tion sites, numerous potential O-linked glycosylation sites, andan EGF-like domain near the C terminus like human andmouse SPACRs. It has 56 and 54% nucleotide sequence iden-tities with human and mouse SPACRs, respectively. Fig. 5shows the deduced primary amino acid sequences of chickSPACR compared with human and mouse SPACR. The de-duced amino acid sequence shares 52 and 42% similarities withthe human and mouse deduced sequences, respectively, sug-gesting that they are indeed orthologs. The polyclonal antibod-ies against synthesized peptides were newly established. TheO46-F antibody reacted with MY-174-positive spots (Figs. 2Aand 3C, arrowheads). Another antibody, O47-F, establishedbased on another determined amino acid sequence also showedsimilar positive spots (data not shown). These four spots cor-responding to retinal MY-174 antigen showed the binding ac-tivity to biotin-hyaluronan (Fig. 3D, arrowheads). These re-

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

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sults suggested that these spots were derived from the samemolecule that reacted with both MY-174 and peptide antibod-ies. Taken together, we concluded that the retinal antigen ofMY-174 in the interphotoreceptor matrix was the chick or-tholog of human SPACR.

SPACR Expression and Retinal Adhesiveness during Devel-opment—Immunofluorescent staining of retinas at embryonicday 5 (E5), 9 (E9), 14 (E14), newborn (Nb) and adult (Ad)showed that the expression of retinal MY-174 antigen is devel-opmentally regulated. No fluorescence was observed at photo-receptor layers in any of the embryonic retinas examined (Fig.6, E5–E14). However, significant staining of the matrix inphotoreceptor layers became detectable in newborn retinas(Fig. 6, arrowhead in Nb), and the highest expression was seenin adult retina (Fig. 6, arrowhead in Ad). This suggested thatMY-174 antigen synthesis was initiated at the later embryonicstages between E14 and birth.

Immunoblot analysis was performed to assess the expressionof chick SPACR (Fig. 7, A and B). PBS-insoluble IPM samplesobtained from chick retinas at various stages (E14–Nb) wereelectrophoresed on 5% SDS-polyacrylamide gels, transferred toa nitrocellulose membrane, and stained with MY-174 or O46-F.Distinct 150-kDa bands, corresponding to retinal MY-174 an-tigen (Fig. 7A, arrow; see Fig. 2A) or O46-F antigen (Fig. 7B,arrow) were measured by using the image analysis programIMAGE (National Institutes of Health). At E16, a specific 150-kDa band appeared. Expressions steeply increased at E18 andE17 in MY-174 and O46-F antigens, respectively. In every lane,a 160-kDa band was detected (Fig. 7A, arrowheads) but was notband-specific to the staining of the photoreceptor layer, becauseit was not detected at E14 (see E14 in Fig. 6 and asterisks inFig. 2B). In adult, the expression of the 150-kDa MY-174-reactive antigen increased 1.87-fold over newborn levels (datanot shown). Amounts of SPACR mRNA were quantified usingNorthern blot analysis (Fig. 7C). After 10 �g/lane of total RNA,prepared from the retinas of each embryonic stage, were elec-trophoresed and then transferred to nylon filter, they werehybridized with radiolabeled probes derived from cDNA forchick SPACR. The expression of SPACR in each sample wasmeasured using IMAGE. At E15, a specific 6.0-kb single bandwas first detected and increased with development.

Retinal adhesiveness was measured by peeling the retinafrom the retinal pigment epithelium and observing the amountof adherent pigment (Fig. 7D). Retinal adhesiveness was ini-tially detected at E16 and increased with development. Therewas no difference between the retinal adhesiveness of adultand newborn retinas (data not shown). The amounts of pigmen-tation derived from retinal pigment epithelium in homogenizedsamples of peeled retina applied onto wells demonstrated theretinal adhesiveness corresponding to these stages.

DISCUSSION

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

We showed that sialylated O-linked glycoconjugates are in-volved in the epitope structure recognized by MY-174, whichwas developed as a monoclonal antibody to the core protein ofPG-M/versican. The antigen was prepared by mild alkali treat-ment of the core protein of PG-M/versican, and the treatmenteliminated 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 immuno-reactivity to the fusion core protein (data not shown). No im-

FIG. 7. Occurrences of chick SPACR and retinal adhesivenessduring development. A, SPACR expression was measured with MY-174 on E14 to newborn (Nb) retinal samples. At E16, a 150-kDa bandfirst appears, and expression increases with developmental age (arrow).The densities at the positions of 150-kDa bands in E14 and newbornretinas are defined as 0 and 100%, respectively. In every lane, a 160-kDa band is detected (arrowheads) but is not band-specific to thephotoreceptor (see “Results”). B, SPACR expression was measured withO46-F antibody on E14 to newborn (Nb) retinal samples. C, the 6.0-kbmRNA corresponding to chick SPACR was analyzed by Northern blotanalysis. At E15, a band is first detected, and expression increases withdevelopmental age. A 10-�g total RNA prepared from retina on eachstage was transferred to a nylon filter and hybridized with a radiola-beled N terminus cDNA (0.4 kb) of chick SPACR. D, retinal adhesive-ness of these samples was measured. Retinal adhesiveness is initiallydetected at E16 and increases with developmental age. Homogenizedsamples of peeled retina corresponding to these days are also shown.The amounts of pigmentation derived from retinal pigment epitheliumin homogenized samples demonstrate the retinal adhesiveness.

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munoreactive clones could be detected by a MY-174 inscreening of the chick retina cDNA library (data not shown).Acharya et al. (16) mentioned that the major glycoconjugate onSPACR is the O-linked carbohydrate, NeuAc�2–3Gal�1–3GalNAc and that the O-linked sugar in SPACR could have astructure similar to that present in bone sialoprotein and ag-grecan. At this stage we speculate that any residual O-glycansleft on the core protein of PG-M/versican, specific but similar tothose characteristic of SPACR, were the antigen for MY-174.

Because our previous study using a polyclonal antibody thatrecognizes all PG-M/versican forms showed no staining in thephotoreceptor layers of adult chick retinas (5), we also thoughtit unlikely that PG-M/versican would be a target for MY-174 inretina. We have repeated this published observation with 2B1,a monoclonal antibody that recognizes all human PG-M/versi-can forms (25), but again no specific staining of the humanphotoreceptor layer could be detected (data not shown). Fur-thermore, no specific staining at an adult mouse photoreceptorlayer could be detected using polyclonal antibodies againstmouse PG-M/versican (data not shown).

MY-174 staining showed resistance to a combination ofneuraminidase and O-glycanase treatments around the outersegments of photoreceptor cells. Tien et al. (15) also reportedthat wheat germ agglutinin staining of SPACR is resistant toneuraminidase around the outer segments of photoreceptorcells in retinal sections. The reason for the resistance to enzy-matic treatments is unclear, but the IPM seems to have differ-ent properties around inner or outer segments of photoreceptorcells.

Acharya et al. (17) showed human SPACR has a functionalhyaluronan-binding domain. This domain (RHAMM-type hya-luronan binding motif) corresponded to 280KEIHVLGFK288 inchick SPACR. It showed high consensus with human andmouse SPACR. This is a candidate for the RHAMM-type hya-luronan binding motif, because a single acidic group can bepresent in one residue inside either flanking basic residue in aB7XB RHAMM-type hyaluronan binding motif (26). Our datashowed chick SPACR can bind hyaluronan. Mutagenesis stud-ies of the putative hyaluronan binding motif will be required toestablish the site of hyaluronan binding in chick SPACR. Be-cause hyaluronan is a prominent constituent of the IPM in allspecies studied except mouse (27, 28), we speculate thatSPACR and hyaluronan form an adhesive complex in the ma-trix between the neural retina and retinal pigment epithelium.Some reports have indeed shown that the insoluble-IPM isinvolved in the adhesion. A histochemical study using experi-mentally detached retinas showed that the insoluble cone ma-trix sheath was closely associated with both the cone photore-ceptor and the apical surface of the retinal pigment epithelium,suggesting that this sheath mediated attachment (29). Insolu-ble IPM constituents could be found in vitreous from eyessuffering from rhegmatogenous retinal detachment (30).

Fig. 7 (A–C) shows the expression levels of mRNA, coreprotein, and sialylated O-glycan that were analyzed by usinglabeled cDNA-probe, O-46F, and MY-174, respectively. Succes-sively, each expression increased in a comprehensible orderduring the development, because the translation processeswere followed by a glycosylation process. There are some dif-ferent expression patterns between mRNA and the core pro-tein. One possibility is that the processes for transcription andtranslation and the glycosylation that follows are regulated indifferent manners in SPACR expression. Another possibility isthat, at earlier developmental stages, there might be an in-creased turnover of the protein, because the protein levels atE15–E16 are significantly lower than the corresponding mRNAlevels at the same developmental stages.

Adler and Gibson (23) showed that neural retinal adhesive-ness starts at E17–E18 and that this is coincident with matu-ration of photoreceptor outer segments. In this study, we ex-amined retinal adhesion using a peeling method (11, 21, 22).Although there is no adhesion at E15, we detected substantialadhesiveness at E16 by this method. The expression of SPACRin adult retina is 1.9 times the newborn level, but retinaladhesiveness of adult retina is almost equal to the newbornone. We suggest that adhesiveness does not increase after athreshold SPACR expression level is reached.

Chondroitin sulfates are major constituents of the IPM (31).Chick SPACR is not a chondroitin-type proteoglycan, becausethe mobility of the chick SPACR band showed no significantchange after chondroitinase ABC treatment. We used the an-tibodies for �Di6S-, �Di4S-, and �Di0S-chondroitin epitopesexposed following chondroitinase ABC digestion to determinewhether a chick SPACR core protein is released. That therewere no specific bands for each epitope suggested that chickSPACR is not a chondroitin-type proteoglycan (data notshown), whereas Fig. 4 showed SG residues, SGD residues, andDGS residues as candidates for chondroitin sulfate attachmentconsensus sequences. Intracellular blocking of the attachmentof chondroitin sulfate chains with xyloside prevented the secre-tion of proteoglycans and resulted in retinal detachment (13).Retinal adhesiveness was also weakened by chondroitinaseABC (11). These reports suggest that chondroitin sulfate pro-teoglycans are involved in the adhesion. However, our reportimplicates a different type of molecule in this process.

Our present study showed that SPACR has a pericellulardistribution reminiscent of cell membranes (Fig. 1C). To clarifywhether SPACR is associated with the cell membrane, we trieda similar immunohistochemical study using O46-F and O47-F.However, these antibodies were not available for immunohis-tochemical study. Two SEA modules (32) corresponding to 231–348 and 728–853 in amino acid sequence positions of chickSPACR are also conserved in human and mouse SPACR (Pfamdata base). The SEA module found in a number of heavilyO-linked glycosylated membrane-associated adhesive proteinsis considered to regulate receptor-ligand alliance by proteolyticcleavage (33). Bishop et al. (34) showed sialylated glycans atthe IPM and photoreceptor plasmalemmata by histochemicalstudy. There is no evidence regarding the association of SPACRwith the cell membrane, but SPACR is a molecule potentiallyassociated with the cell membrane. To clarify the retinal adhe-sion mechanism and other biological functions of SPACR, wemust establish an in vivo system.

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

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Yasuhiko Suzuki, Makoto Takeuchi, Goichiro Miyake, Hiroshi Ikagawa and Koji KimataMasahiro Zako, Masayoshi Iwaki, Masahiko Yoneda, Osamu Miyaishi, Jinsong Zhao,

MatrixCones and Rods, a Developmentally Regulated Glycoprotein of Interphotoreceptor

Molecular Cloning and Characterization of Chick Sialoprotein Associated with

doi: 10.1074/jbc.M201279200 originally published online May 3, 20022002, 277:25592-25600.J. Biol. Chem. 

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