August 2000
Volume 41, Issue 9
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Retinal Cell Biology  |   August 2000
Isolation and Characterization of Mucinlike Glycoprotein Associated with Photoreceptor Cells
Author Affiliations
  • Fumiyuki Uehara
    From the Departments of Ophthalmology and
  • Norio Ohba
    From the Departments of Ophthalmology and
  • Masayuki Ozawa
    Biochemistry, Kagoshima University, Faculty of Medicine, Japan.
Investigative Ophthalmology & Visual Science August 2000, Vol.41, 2759-2765. doi:
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      Fumiyuki Uehara, Norio Ohba, Masayuki Ozawa; Isolation and Characterization of Mucinlike Glycoprotein Associated with Photoreceptor Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2759-2765.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Although previous lectin-histochemical studies have shown that O-linked glycoproteins are distributed in cone pedicles and rod spherules, as well as in photoreceptors, including associated interphotoreceptor matrices (IPM), attention has been directed only to those in the IPM. In this study, cloning of the O-linked glycoproteins not only in the IPM but also in the region including the cone pedicles and rod spherules was attempted.

methods. The cDNA for the core protein of the O-linked glycoprotein in the bovine retina was isolated by screening a bovine retinal cDNA library using a polyclonal antibody against the jacalin (a lectin specific for O-linked sugar residues)-binding glycoproteins (JBGPs) in the whole bovine retina. The expression of the JPGP core protein in the retina was examined by means of in situ hybridization histochemistry and immunohistochemistry.

results. The cDNA was isolated and found to encode an entire core protein[ predicted molecular mass (Mr): 101 kDa; rich in Ser and Thr; mucin-like] for the JBGPs with Mr of 120 and 135 kDa. The mRNA was expressed in both cone and rod photoreceptor cells. This protein was distributed in the cone pedicles and rod spherules as well as the photoreceptor layer.

conclusions. Mucinlike glycoproteins with Mr of 120 and 135 kDa may be synthesized in the cone and rod photoreceptor cells, respectively, and distributed not only in the photoreceptor layer (probably including the IPM) but also in the cone pedicles and rod spherules.

Glycoproteins on the cell surface and extracellular matrix are involved in a variety of cellular functions and intercellular interactions. 1 In the retina, various kinds of N-linked glycoproteins, including rhodopsin, peripherin, and interphotoreceptor retinoid binding protein, have been the subject of research, and their functions and relation to retinal diseases have been well defined. 2 Regarding O-linked glycoproteins, attention has been directed recently only to those in the interphotoreceptor matrix (IPM) 3 4 in relation to retinal adhesion, although the previous lectin histochemical studies strongly suggested that O-linked glycoproteins are distributed in cone pedicles 5 6 and rod spherules 6 as well as in cone 5 6 7 and rod 6 photoreceptors, including the IPM. 4 8 9 10 The biochemical analysis of the O-linked glycoprotein in these synaptic terminals is also important for understanding the physiology and pathology of the photoreceptor cells as well as the IPM. 
Jacalin, a lectin from jackfruit (Artocarpus integrifolia), exhibits a specific affinity for galactose β1,3 N-acetyl-d-galactosamine residues (Galβ1,3GalNAc; O-linked sugar residues), 11 whose binding is not prevented in the presence of sialic acids. Therefore, jacalin-binding glycoproteins (JBGPs), consisting of both asialo- and sialo-O-linked glycoproteins, probably associated with cones and rods, 6 12 respectively, can be isolated by lectin affinity chromatography on a jacalin-Sepharose column (Vector Laboratories, Burlingame, CA). When whole retina extracts are applied to this column, not only the JBGPs of the IPM, but also those of the other regions (including the cone pedicles and rod spherules) will be isolated. For cDNA screening using a cDNA expression library, we can use a polyclonal antibody against a mixture of the O-linked glycoproteins. With the combination of these procedures, therefore, it may be possible to obtain the cDNAs for the core proteins of the JBGPs in different regions including the cone pedicles and rod spherules. 
We report the successful cloning of cDNA for a JPGP core protein through the screening of a cDNA expression library using a polyclonal antibody against the JBGPs from the whole bovine retina. We also report the expression of the core protein in the retina by means of in situ hybridization histochemistry and immunohistochemistry, showing that a common core protein is present in the photoreceptors (probably also including the IPM) and the synaptic terminals (cone pedicles and rod spherules). 
Materials and Methods
Purification of Anti-JBGP Antibody for cDNA Screening
Twenty-six bovine retinas were homogenized in 100 ml of 2% Triton X-100 in 0.01 M Tris-HCl buffer (pH 7.6) containing 0.15 M NaCl, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF; extraction buffer), followed by centrifugation at 140,000g for 1 hour. The supernatant (Triton X-100 extracted fraction) was decanted and applied to a jacalin-Sepharose column (volume: 2.5 ml) equilibrated with 0.1% Triton X-100 in 0.01 M Tris-HCl buffer (pH 7.6) containing 0.15M NaCl, 2 mM EDTA, and 1 mM PMSF (equilibration buffer). The column was washed with 50 ml of the equilibration buffer, and then the glycoconjugates bound to the jacalin-Sepharose were eluted with 10 ml of the equilibration buffer containing 0.1 M d-galactose, which is the haptenic sugar for jacalin. Approximately 384 mg of JBGPs was purified from the 26 bovine retinas (14.8 mg/retina), determined with a protein assay (DC; Bio-Rad, Hercules, CA). Therefore, to obtain enough glycoproteins for the immunization of two rabbits and affinity purification of the antibodies, we purified 5.2 g of JBGPs from 351 bovine retinas by seven repetitive cycles of the procedure, using a 5-ml column volume each time. Although the JBGPs were separated into three bands of glycoconjugates with different molecular sizes on Western blot analysis (data shown in the Results section), we considered that the polyclonal antibody produced against a mixture of the glycoconjugates could be convenient for the isolation of cDNAs coding different types of core proteins by cDNA screening using a cDNA expression library at the same time. Therefore, by a method previously described, 12 rabbit antisera were raised against the JBGPs by immunizing two New Zealand White rabbits, and then the antibody was affinity purified on a column of the JBGPs coupled to Affi-Gel 10 Support (Bio-Rad). 
Cloning of cDNA for JPGP Core Protein
A cDNA library of the bovine retina (Uni-ZAP XR; Stratagene, La Jolla, CA) was screened with the anti-JBGP antibody according to the manufacturer’s instructions. Five positive plaques were detected in screening approximately 1.5 × 106 phages of the cDNA expression library. The ExAssist/SOLR system (Stratagene) was used to allow efficient excision of the phagemid (Bluescript SK; Stratagene) from the Uni-ZAP XR vector, and SOLR cells containing positive clones were isolated. Each phagemid isolated was demonstrated on agarose gel electrophoresis to contain a cDNA insert of approximately 3.5 kbp. These DNAs were sequenced on both strands, using T3 and T7 universal primers (Stratagene) according to the protocol for use of the terminator cycle sequencer (ABI Prism BigDye; Perkin–Elmer Applied Biosystems, Foster City, CA). In all five positive clones, the same nucleotide sequences containing polyA signals were detected at the 3′ ends of the insert cDNAs, showing that only one kind of cDNA was isolated from the cDNA library in this study. According to the protocol for the oligo-capping method, 13 the cDNA of the JBGP core protein containing the 5′ end was amplified by reverse transcription–polymerase chain reaction (RT-PCR) using a pair of oligonucleotides, 5′-GAGAGACACATATGGGATCCGAGAG-3′ and 5′-GGGTGTAGTTGAGGATTTCGTTGAGC-3′, as PCR primers and the first-strand cDNAs synthesized from the 5′ end-labeled mRNAs as templates. The PCR products were isolated and cloned into a vector (Bluescript II SK+; Stratagene). After ligation, the DNA was sequenced on both strands, using T3 and T7 universal primers according to the manufacturer’s protocol (Perkin–Elmer Applied Biosystems). 
In Situ Hybridization Histochemistry
Digoxigenin-labeled cRNA probes for in situ hybridization histochemistry were prepared according to the standard protocol for an RNA labeling kit (Boehringer–Mannheim, Mannheim, Germany). In brief, after linearization of the template DNA with XbaI and XhoI, antisense and sense cRNA probes were transcribed with T7 and T3 RNA polymerases on linearized DNA templates, respectively. Digoxigenin-UTP was used as a substrate and incorporated into the transcript. The lengths of the cRNA probes were shortened to approximately 120 nucleotides by mild hydrolysis. The expression of mRNA for the core protein of the JBGPs in the retina was examined by the procedure described in the previous report 14 using digoxigenin-labeled cRNA probes. 
Purification of Anti-Core Protein Antibody
The cDNA fragment of 1.4 kbp (833-2261) encoding a 60% portion of the entire core protein was excised with EcoRI and cloned into the equivalent site in a maltose-binding protein (MBP) fusion vector (pMALc2; New England Biolabs, Beverly, MA) or glutathione S-transferase (GST) fusion vector (pGEX-4T; Amersham Pharmacia, Piscataway, NJ). The plasmid DNA with the inserted cDNA was introduced into Escherichia coli BL21 cells (Amersham Pharmacia). Bacterial cells containing a fusion plasmid were cultured, and the fusion protein was induced, isolated, and affinity purified on a column of amylose-resin (New England Biolabs; for MBP fusion protein) or glutathione-Sepharose (Amersham Pharmacia; for GST fusion protein), according to the manufacturer’s instructions. Rabbit antisera were raised against the MBP–core protein–fusion protein using a New Zealand White rabbit, and the antibody against the core protein was affinity purified on a column of the GST–core protein–fusion protein coupled to Affi-Gel 10 Support (Bio-Rad) by a method previously described. 15  
Immunohistochemistry
Following the procedures described in previous articles, 6 12 tissue sections of bovine, human, monkey, and rat eyes were prepared for immunohistochemistry and reacted with the antibody against the bovine JBGP core protein (1:100 diluted with phosphate-buffered saline [PBS]) for 1 hour at room temperature. After extensive washing, the tissue sections were incubated with biotinylated anti-rabbit IgG (Vector Laboratories; 1:100 diluted with PBS) for 1 hour at room temperature. Binding was visualized with the reagents of an ABC kit (Vectastain Elite; Vector Laboratories) and diaminobenzidine as the peroxidase substrate, according to the manufacturer’s instructions. 
Western Blot Analysis
Because JBGPs may contain both asialo- and sialo-O-linked glycoproteins, it is useful for the analysis of JBGP glycans to compare samples of JBGPs with or without neuraminidase treatment by Western blot analysis. Therefore, JBGPs (38 mg/ml in equilibration buffer), isolated by the same method as described in the first part of this section, were diluted with 0.2 M acetate buffer (pH 5.0) to a concentration of 1 mg/ml. One milliliter of the diluted JBGPs were poured into a bottle containing 1 unit of lyophilized neuraminidase from Arthrobacter ureafaciens (Nacalai Tesque, Kyoto, Japan), followed by incubation in a water bath at 37°C. After neuraminidase digestion for 1, 6, or 17 hours, 100 μl of the solution was serially removed from each bottle, ethanol precipitated, and dissolved in 100 μl Laemmli’s sample buffer (62.5 mM Tris-HCl [pH 6.8], containing 25% glycerol, 2% sodium dodecyl sulfate [SDS] and 0.01% bromphenol blue). 16 One hundred microliters of the diluted JBGPs without neuraminidase treatment was also ethanol precipitated and dissolved in 100 μl Laemmli’s sample buffer. 
Western blot analysis was performed essentially as previously described. 12 In brief, the blotted JBGPs, with or without neuraminidase digestion, were incubated with the anti-JBGP core protein antibody (1:100 diluted with PBS) for 1 hour at room temperature, extensively washed, and then incubated with biotinylated anti-rabbit IgG (Vector Laboratories; 1:100 diluted with PBS) for 1 hour at room temperature. The blotted JBGPs with or without neuraminidase digestion were also incubated with a biotinylated lectin, jacalin, Maackia amurensis lectin II (MAL II; specific for sialic acidα2,3Gal), 17 Erythrina cristagalli agglutinin (ECA; specific for Galβ1,4GlcNAc: N-glycan), 18 or peanut agglutinin (PNA; specific for Galβ1,3GalNAc: O-glycan; all from Vector), 19 at 10 μg/ml in PBS for 1 hour at room temperature instead of the antibodies. Binding was visualized by the same procedure as described for immunohistochemistry. 
All animal procedures conformed to the Guidelines of the Kagoshima University Faculty of Medicine for Animal Experiments, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Results
cDNA and Predicted Amino Acid Sequence of JPGP Core Protein
The nucleotide sequence of the bovine cDNA for the core protein of the JBGPs is shown in Figure 1 . The cDNA that was initially isolated from the cDNA library contained the putative translation initiation start codon ATG, conforming to the Kozak consensus sequence, 20 close to the 5′ end and polyA signals at the 3′ end. The 5′-extending cDNA (97 nucleotides) that was isolated by the oligo-capping method 13 contained two in-frame termination codons at nucleotides −60 and −153. 
The predicted amino acid sequence of the bovine JBGP core protein deduced from the cDNA is also shown in Figure 1 . A search of GenBank revealed that the predicted amino acid sequence encoded by the cDNA showed 72% nucleotide similarity with that encoded by the human cDNA of IPM proteoglycan 1 (IMPG1; AF017776). 3 21 The predicted molecular size of the bovine core protein of the JBGPs (794 sequences) was 103,755 Da. In the predicted sequence, a signal–anchor sequence, Val8-Leu16, was detected. When the signal sequence of Met1-Gly20 was excluded, the predicted molecular size decreased to 100,997 Da. The predicted amino acid sequence contained two hyaluronan-binding motifs, 22 Lys50-Arg58 and Lys285-Arg293. The sequence had clusters of cysteine residues at both the N- and C-terminals. A number of serine and threonine residues, which are potential O-linked glycosylation sites and five potential N-linked glycosylation sites were detected in the sequence. The differences in the molecular sizes (19 and 34 kDa) between the bands (120 and 135 kDa) determined on Western blot analysis and the predicted one (101 kDa) may be due to these sugar residues. 
In Situ Hybridization Histochemistry
Weak hybridization signals were detected using an antisense cRNA probe on the basal portions of the cone and rod photoreceptor inner segments in the adult retina of all species examined (Figs. 2A 2B : monkey retina is shown, because discrimination between cones and rods was easy). These signals on the inner segments were not observed using a sense cRNA probe (Fig. 2C) , implying that they were specifically induced. On the contrary, weak staining of red blood cells in the blood vessels, which was observed in the inner nuclear layer and ganglion cell layer, was regarded as being nonspecifically induced, because this labeling was detected in both tissue sections using an antisense and a sense cRNA probe (Figs. 2A 2C) . These observations show that the mRNA for the JPGP core protein is selectively expressed in the cone and rod photoreceptor cells. 
Immunohistochemistry
By means of immunohistochemistry, the antibody against the bovine JPGP core protein bound diffusely to the photoreceptor layer, whereas a punctate, widely scattered binding pattern was observed in the outer plexiform layer of the bovine, rat, and human adult retinas (Figs. 3A 3B 3C ). Weak staining was also observed on the cell surface in the outer nuclear layer (Figs. 3A 3B 3C) . These labeling patterns were almost completely inhibited by preincubation of the antibody with the GST–core protein fusion protein (Fig. 3D) , suggesting that these reactions were specifically induced by the antibody. When a monkey retina with good morphologic preservation was incubated with the antibody, the immunostaining was more precisely localized. In the photoreceptor layer, the surfaces of the inner and outer segments of both cones and rods were stained (Fig. 3E) , whereas the cone pedicles and rod spherules reacted with the antibody in the outer plexiform layer (Fig. 3F)
Western Blot Analysis
On Western blot analysis, two major bands (180 and 135 kDa) and one minor band (120 kDa) were detected with biotinylated jacalin 11 in the JBGPs without neuraminidase treatment (Fig. 4A ). When the blotted JBGPs with neuraminidase digestion were incubated with jacalin, a different binding pattern was observed: a 160-kDa band was detected instead of the 180- and 135-kDa bands, although the 120-kDa band was observed at the same position (Fig. 4B : JBGPs after neuraminidase digestion for 17 hours), implying that the 180- and 135-kDa band materials decreased in molecular size to 160- and 120-kDa bands, respectively, on neuraminidase digestion. These findings showed that the JBGPs of 180 and 135 kDa are sialo-glycoproteins, whereas that of 120 kDa is an asialo-glycoprotein. When the JBGPs without neuraminidase treatment was incubated with the anti-core protein antibody, 135- and 120-kDa bands intensely and weakly reacted with the antibody, respectively, whereas the 180-kDa band did not react with it (Figs. 4C 4D) . In this antibody reaction, the 120-kDa band was more visible in a blot of a large amount of the JBGPs (Fig. 4C : 15 μg) than in one of a small amount (Fig. 4D : 5 μg). On digestion of the JBGPs with neuraminidase for 1 hour, the antibody binding to the 135-kDa band decreased in both intensity and molecular size, whereas that to the 120-kDa band increased (Fig. 4E) . The 135-kDa band was faintly and scarcely detected after neuraminidase digestion for 6 and 17 hours, respectively, whereas the 120-kDa band was constantly detected (Figs. 4F 4G) . On reaction with the antibody preincubated with GST–core protein fusion protein, no band was observed for the JBGPs, with or without neuraminidase digestion (Fig. 4H : JBGPs without neuraminidase digestion), implying that both the 135- and 120-kDa bands were specific for binding to the anti-core protein antibody. These findings suggested that the terminal sialic acids on the sugar chains may have caused the different mobilities in the electrophoresed gel of the glycoproteins in the 135- and 120-kDa bands, the core structures of which, including the core protein, may be the same. In contrast, the JBGP of 180 kDa was considered to be a sialo-glycoprotein with a different core protein, the cDNA of which should be isolated in a future study. 
The glycans of the JBGPs were more precisely analyzed using other biotinylated lectins. When the blotted JBGPs without neuraminidase treatment were incubated with MAL II, 17 180- and 135-kDa bands were positively detected (Fig. 5A ), whereas their molecular sizes and intensities on binding by MAL II decreased on neuraminidase digestion of the JBGPs (Fig. 5B : 1-hour digestion with neuraminidase). In contrast, a 120-kDa band was positively detected with biotinylated ECA 18 for the JBGPs without neuraminidase treatment (Fig. 5C) , whereas two bands corresponding to MAL II-positive bands were detected in addition to the 120-kDa band after neuraminidase digestion of the JBGPs (Fig. 5D : 1 hour digestion with neuraminidase). With biotinylated PNA, 19 almost the same binding patterns as in the case of ECA were observed for the JBGPs, with or without neuraminidase digestion (Fig. 5E : without neuraminidase treatment; Fig. 5F : 1-hour digestion with neuraminidase). All these lectin-stained bands either decreased or disappeared when the haptenic sugars were premixed with the lectins before the incubation (Fig. 5G : JBGPs without neuraminidase digestion incubated with a mixture of biotinylated PNA and 0.1 M d-galactose), implying that the bands detected earlier were specific for lectin binding. These findings showed that all three bands of JBGPs contain both N-glycans (recognized by ECA 18 ) and O-glycans (recognized by PNA, 19 ), although their terminal sugar structures are different: the 180- and 135-kDa JBGPs have sialo-glycans, whereas the 120-kDa JBGP has asialo-glycans. 
Discussion
In the present study, we succeeded in cloning the cDNA for the common core protein of 135-kDa sialo-JBGP and 120-kDa asialo-JBGP, both of which reacted with the anti-core protein antibody. The experiment involving neuraminidase treatment of the JBGPs showed that these two glycoproteins may share not only the core protein but also the core glycans. The existence of sialic acids at the termini of both O- and N-glycans in the 135-kDa sialo-JBGP may be the cause of the lower mobility on electrophoresis than that of the 120-kDa asialo-JBGP. Some type of conformational change occurring along with the loss of sialic acid residues may also be concerned with the higher mobility of the 120-kDa asialo-JBGP in addition to the decrease in molecular weight due to a removal of sialic acid residues. 
In situ hybridization histochemistry showed that the mRNA for the JBGP core protein is expressed in both cone and rod photoreceptor cells. Immunohistochemistry showed that the JPGP core protein is present not only in the photoreceptor layer but also in the cone pedicles and rod spherules. These findings suggest that the JPGP core protein is produced in both the cone and rod photoreceptor cells, followed by modification processes including glycosylation, and then transported to both the photoreceptor and synaptic terminals of the cells. The predicted amino acid sequence of the JPGP core protein has N-terminal signal peptides and clusters of cysteine residues at both the N- and C-terminals, whereas it does not have membrane-spanning regions consisting of hydrophobic amino acids, implying that the JBGPs are extracellularly secreted into the subretinal space and synaptic cleft as the IPM and the synaptic matrix (SM), respectively. 
Taking these findings together, i.e., the identification of sialo- and asialo-JBGP, the presence of the JPGP core protein in both cones and rods, and the previous lectin histochemical observations 6 10 revealing the terminal carbohydrate difference between cones and rods, we can conclude that sialo-JBGP and asialo-JBGP are present in the extracellular matrices (IPM and SM) associated with rod photoreceptor cells and those with cone photoreceptor cells, respectively. Because the average densities of photoreceptors in the bovine retina 23 are 15 to 27 × 103 cones per mm2 and 2.3 to 2.7 × 105 rods per mm2, the sialo-JBGP (135 kDa) associated with rods may be dominantly detected on Western blot analysis with the anti-core protein antibody. These findings have now confirmed our previous speculation that the terminal presence of sialyl residues is the major difference between rods and cones. 6 12  
It is noteworthy that a common glycoprotein is distributed in the subretinal space and the synaptic cleft. This glycoprotein may be involved in similar physiological functions in these different regions, offering a structure suitable for the intercellular adhesion and exchange of metabolites including retinoids and/or neurotransmitters. The glycoproteins, bovine JBGPs, examined in this study are considered to be identical with that previously named IMPG1 3 21 and SPACR, 4 although they have been investigated as components of the human IPM, and their molecular weight (150 kDa) is somewhat more than those of the bovine JBGPs (120 and 135 kDa). The predicted amino acid sequences of all these core proteins contain hyaluronan-binding consensus sequences in the N-terminal domain. The hyaluronan binding of SPACR has been confirmed by in vitro experiments. 4 The interactions between the glycoprotein and hyaluronan may be involved in organization of the insoluble structure of the IPM and/or synapses. The cysteines and N-linked glycans present in the N-terminal and C-terminal domains may also be involved in this organization through disulfide cross-linking and interaction with galectins, which have been shown to be distributed in the apical villi of Müller cells, 24 respectively. 
In contrast, the predicted amino acid sequence of the core protein contains a number of serine (Ser) and threonine (Thr) residues in the central domain when we divide this protein into three domains mainly based on the presence of N-linked glycosylation sites for convenience. The Ser/Thr-content of the central domain (Thr301-Gly540), which has no N-linked glycosylation sites, is 26.3%. The contents of Ser/Thr in the N-terminal domain (Met1-Gly300) and the C-terminal one (Pro541-Asn794), both of which have several N-linked glycosylation sites, are 12.7% and 11.4%, respectively. The central domain, which has a high content of Ser/Thr, can be called a mucinlike domain, although it is neither a mucin nor a mucin-type glycoprotein in the strict sense. Mucins are highly O-glycosylated glycoproteins implicated in the protection of cells from extracellular agents. 25 The term mucin has more recently been expanded to include cell surface glycoproteins with high contents of O-linked oligosaccharides. 25 The genes for human mucins have been numbered sequentially, MUC1 26 through MUC8, 27 , as they have been identified. For example, the MUC1 protein, which is an epithelial cell mucin, was the first mucin to be characterized by cloning. 26 Recent studies have suggested that mucins play important roles in not only normal tissue functions but also tumor progression. 26 The O-linked oligosaccharides are thought to be responsible for the rheologic properties of mucus. 25 The central domain of the core protein in the present study may be responsible for the rheologic properties, similar to other mucins and mucin-like glycoproteins. This mucinlike domain may be useful for smooth movement of metabolites including retinoids and/or neurotransmitters, functioning as a lubricant in the subretinal space and/or synaptic cleft where this glycoprotein is present. 
Finally, we propose the term, mucinlike glycoprotein associated with photoreceptor cells (MLGAPC), rather than JBGP, to more exactly reflect the distribution and nature of the protein reported herein. 
 
Figure 1.
 
Complete amino acid sequence of the JPGP core protein inferred from the nucleotide sequence of the JPGP core protein cDNA. The proposed signal sequence, Val8-Leu16, is bold underlined. Two hyaluronan-binding motifs, 18 Lys50-Arg58 and Lys285-Arg293, are double underlined. Potential N-linked glycosylation sites with the sequence Asn-X-Thr/Ser are boxed.
Figure 1.
 
Complete amino acid sequence of the JPGP core protein inferred from the nucleotide sequence of the JPGP core protein cDNA. The proposed signal sequence, Val8-Leu16, is bold underlined. Two hyaluronan-binding motifs, 18 Lys50-Arg58 and Lys285-Arg293, are double underlined. Potential N-linked glycosylation sites with the sequence Asn-X-Thr/Ser are boxed.
Figure 2.
 
In situ hybridization histochemistry. (A, B) Weak hybridization signals are detected using an antisense cRNA probe for the mRNA of the JPGP core protein on the basal portions of cone (c) and rod (r) photoreceptor inner segments (monkey retina). (C) Almost no signals are observed in the inner segments using a sense cRNA probe (monkey retina). (A, C) Weak staining of red blood cells is observed in the inner nuclear and ganglion cell layers independent of the type of cRNA probe. rpe, retinal pigment epithelium; os, outer segment; is, inner segment; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bars, (A, C) 25 μm; (B) 10 μm.
Figure 2.
 
In situ hybridization histochemistry. (A, B) Weak hybridization signals are detected using an antisense cRNA probe for the mRNA of the JPGP core protein on the basal portions of cone (c) and rod (r) photoreceptor inner segments (monkey retina). (C) Almost no signals are observed in the inner segments using a sense cRNA probe (monkey retina). (A, C) Weak staining of red blood cells is observed in the inner nuclear and ganglion cell layers independent of the type of cRNA probe. rpe, retinal pigment epithelium; os, outer segment; is, inner segment; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bars, (A, C) 25 μm; (B) 10 μm.
Figure 3.
 
Immunohistochemistry with the anti-JBGP core protein antibody. (A, B, and C) A diffuse, intense binding pattern, and a punctate, widely scattered one are observed in the photoreceptor layer (pl) and the outer plexiform layer (opl), respectively, with weak staining of the cell surface in the outer nuclear layer (onl) (A) bovine retina; (B) rat retina; (C) human retina. (D) Only faint staining is observed throughout the retina after preincubation of the antibody with the GST–core protein fusion protein (human retina). (E) In the monkey retina, the surfaces of the inner segments (is) and outer segments (os) of both cones (c) and rods (r) are stained with the antibody. (F) In the monkey retina, punctate bindings are detected with the antibody in the cone pedicles (arrowhead) and the rod spherules (arrow). Scale bars, (A through D) 25 μm; (E, F) 10 μm.
Figure 3.
 
Immunohistochemistry with the anti-JBGP core protein antibody. (A, B, and C) A diffuse, intense binding pattern, and a punctate, widely scattered one are observed in the photoreceptor layer (pl) and the outer plexiform layer (opl), respectively, with weak staining of the cell surface in the outer nuclear layer (onl) (A) bovine retina; (B) rat retina; (C) human retina. (D) Only faint staining is observed throughout the retina after preincubation of the antibody with the GST–core protein fusion protein (human retina). (E) In the monkey retina, the surfaces of the inner segments (is) and outer segments (os) of both cones (c) and rods (r) are stained with the antibody. (F) In the monkey retina, punctate bindings are detected with the antibody in the cone pedicles (arrowhead) and the rod spherules (arrow). Scale bars, (A through D) 25 μm; (E, F) 10 μm.
Figure 4.
 
Western blot analysis of the JBGPs, with or without neuraminidase digestion, using either jacalin or anti-core protein antibody. (A) Two major bands (180 and 135 kDa) and one minor one (120 kDa) are detected with biotinylated jacalin in the nondigested JBGPs. (B) Bands of 160 and 120 kDa are detected with jacalin for the JBGPs after digestion for 17 hours. (C, D) The 135- and 120-kDa bands are intensely and weakly detected, respectively, with the anti-core protein antibody for JBGPs without treatment (JBGPs applied: C, 15 μg; D, 5 μg). (E, F, and G) After digestion of JBGPs, the major positive band corresponding to the 135-kDa band in (D) gradually decreases in molecular weight, whereas the 120-kDa band is more prominently detected (digestion time: E, 1 hour; F, 6 hours; G, 17 hours). (H) No band is detected with the antibody preincubated with the GST–core protein fusion protein for JBGPs without digestion.
Figure 4.
 
Western blot analysis of the JBGPs, with or without neuraminidase digestion, using either jacalin or anti-core protein antibody. (A) Two major bands (180 and 135 kDa) and one minor one (120 kDa) are detected with biotinylated jacalin in the nondigested JBGPs. (B) Bands of 160 and 120 kDa are detected with jacalin for the JBGPs after digestion for 17 hours. (C, D) The 135- and 120-kDa bands are intensely and weakly detected, respectively, with the anti-core protein antibody for JBGPs without treatment (JBGPs applied: C, 15 μg; D, 5 μg). (E, F, and G) After digestion of JBGPs, the major positive band corresponding to the 135-kDa band in (D) gradually decreases in molecular weight, whereas the 120-kDa band is more prominently detected (digestion time: E, 1 hour; F, 6 hours; G, 17 hours). (H) No band is detected with the antibody preincubated with the GST–core protein fusion protein for JBGPs without digestion.
Figure 5.
 
Western blot analysis of JBGPs with or without neuraminidase digestion using lectins. (A) Two bands of 180 and 135 kDa are detected with biotinylated MAL II in the nondigested JBGPs. (B) Two bands, weaker and smaller than in (A), are detected with MAL II for JBGPs digested for 1 hour. (C) A 120-kDa band is detected with biotinylated ECA for JBGPs without treatment. (D) Two bands are detected with ECA in addition to the 120-kDa band in JBGPs digested for 1 hour. (E) A 120-kDa band is detected with biotinylated PNA for JBGPs without treatment. (F) Two bands are detected with PNA in addition to the 120-kDa band in JBGPs digested for 1 hour. (G) Almost no band is detected with a mixture of biotinylated PNA and 0.1 M d-galactose in nondigested JBGPs.
Figure 5.
 
Western blot analysis of JBGPs with or without neuraminidase digestion using lectins. (A) Two bands of 180 and 135 kDa are detected with biotinylated MAL II in the nondigested JBGPs. (B) Two bands, weaker and smaller than in (A), are detected with MAL II for JBGPs digested for 1 hour. (C) A 120-kDa band is detected with biotinylated ECA for JBGPs without treatment. (D) Two bands are detected with ECA in addition to the 120-kDa band in JBGPs digested for 1 hour. (E) A 120-kDa band is detected with biotinylated PNA for JBGPs without treatment. (F) Two bands are detected with PNA in addition to the 120-kDa band in JBGPs digested for 1 hour. (G) Almost no band is detected with a mixture of biotinylated PNA and 0.1 M d-galactose in nondigested JBGPs.
The authors thank Yoshiko Maeda for technical assistance. 
Sharon N, Lis H. Glycoproteins. Research booming on long-ignored, ubiquitous components. Chem Eng News. 1981;59:21–44.
Molday RS. Photoreceptor membrane proteins, phototransduction, and retinal degenerative diseases. Invest Ophthalmol Vis Sci. 1998;39:2493–2513.
Kuehn MH, Stone EM, Hageman GS. Molecular analyses of IPM150, a photoreceptor cell-specific proteoglycan [ARVO Abstract]. Invest Ophthalmol Vis Sci. 1997;38:S599.Abstract nr 2788.
Acharya S, Rodriguez IR, Moreira EF, et al. SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan. J Biol Chem. 1998;273:31599–31606. [CrossRef] [PubMed]
Blanks JC, Johnson LV. Specific binding of peanut lectin to a class of retinal photoreceptor cells. Invest Ophthalmol Vis Sci. 1984;25:546–557. [PubMed]
Uehara F, Muramatsu T, Sameshima M, Kawano K, Koide H, Ohba N. Effects of neuraminidase on lectin binding sites in photoreceptor cells of monkey retina. Jpn J Ophthalmol. 1985;29:54–62. [PubMed]
Uehara F, Muramatsu T, Sameshima M, Ohba N. Localization of fluorescence-labeled lectin binding sites on photoreceptor cells of the monkey retina. Exp Eye Res. 1983;36:113–123. [CrossRef] [PubMed]
Johnson LV, Hageman GS, Blanks JC. Interphotoreceptor matrix domains ensheath vertebrate cone photoreceptor cells. Invest Ophthalmol Vis Sci. 1986;27:129–135. [PubMed]
Sameshima M, Uehara F, Ohba N. Specialization of the interphotoreceptor matrices around cone and rod photoreceptor cells in the monkey retina, as revealed by lectin cytochemistry. Exp Eye Res. 1987;45:845–863. [CrossRef] [PubMed]
Uehara F, Yasumura D, LaVail MM. Rod- and cone-associated interphotoreceptor matrix in the rat retina. Invest Ophthalmol Vis Sci. 1991;32:285–292. [PubMed]
Hortin GL, Trimpe BL. Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: Application of jacalin-agarose. Anal Biochem. 1990;188:271–277. [CrossRef] [PubMed]
Uehara F, Muramatsu T, Ozawa M, Koide H, Sameshima M, Ohba N. Purification of antibody against peanut agglutinin-receptors of bovine interphotoreceptor matrix. Jpn J Ophthalmol. 1985;30:56–62.
Maruyama K, Sugano S. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene. 1994;138:171–174. [CrossRef] [PubMed]
Uehara F, Ohba N, Nakashima Y, Yanagita T, Ozawa N, Muramatsu T. A fixative suitable for in situ hybridization histochemistry. J Histochem Cytochem. 1993;41:947–953. [CrossRef] [PubMed]
Ozawa M, Terada H, Pedraza C. The fourth armadillo repeat of plakoglobin (-catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cadherin Dsg2, and the tumor suppressor APC protein. J Biochem. 1995;118:1077–1082. [CrossRef] [PubMed]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. [CrossRef] [PubMed]
Sata T, Lackie PM, Taatjes DJ, Peumans W, Roth J. Detection of the Neu5Ac (α2,3) Gal(β1,4)GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid inα2,3- andα2,6-linkage. J Histochem Cytochem. 1989;37:1577–1588. [CrossRef] [PubMed]
De Boeck H, Loontiens FG, Lis H, Sharon N. Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand. Arch Biochem Biophys. 1984;234:297–304. [CrossRef] [PubMed]
Lotan R, Skutelsky E, Danon D, Sharon N. The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea). J Biol Chem. 1975;250:8518–8523. [PubMed]
Kozak M. Interpreting cDNA sequences: some insights from studies on translation. Mammal Genome. 1996;7:563–574. [CrossRef]
Felbor U, Gehrig A, Sauer G, et al. Genomic organization and chromosomal localization of the interphotoreceptor matrix proteoglycan-1 (IMPG1) gene: a candidate for 6q-linked retinopathies. Cytogenet Cell Genet. 1998;81:12–17. [CrossRef] [PubMed]
Yang B, Yang BL, Savani RC, Turley EA. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J. 1994;13:286–296. [PubMed]
Krebs W, Friedrich I. Quantitative morphology of bovine retina. Hollyfield JG eds. The Structure of the Eye. 1982;175–182. Elsevier Biomedical New York.
Maldonado CA, Castagna LF, Rabinovich Ga, Landa CA. Immunocytochemical study of the distribution of a 16-kDa galectin in the chicken retina. Invest Ophthalmol Vis Sci. 1999;40:2971–2977. [PubMed]
Carraway KL, Hull SR. Cell surface mucin-type glycoproteins and mucin-like domains. Glycobiology. 1991;1:131–138. [CrossRef] [PubMed]
Gendler SJ, Lancaster CA, Taylor–Papadimitriou J, et al. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem. 1990;265:15286–15293. [PubMed]
Shankar V, Pichan P, Eddy RL, Jr, et al. Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus. Am J Respir Cell Mol Biol. 1997;16:232–241. [CrossRef] [PubMed]
Figure 1.
 
Complete amino acid sequence of the JPGP core protein inferred from the nucleotide sequence of the JPGP core protein cDNA. The proposed signal sequence, Val8-Leu16, is bold underlined. Two hyaluronan-binding motifs, 18 Lys50-Arg58 and Lys285-Arg293, are double underlined. Potential N-linked glycosylation sites with the sequence Asn-X-Thr/Ser are boxed.
Figure 1.
 
Complete amino acid sequence of the JPGP core protein inferred from the nucleotide sequence of the JPGP core protein cDNA. The proposed signal sequence, Val8-Leu16, is bold underlined. Two hyaluronan-binding motifs, 18 Lys50-Arg58 and Lys285-Arg293, are double underlined. Potential N-linked glycosylation sites with the sequence Asn-X-Thr/Ser are boxed.
Figure 2.
 
In situ hybridization histochemistry. (A, B) Weak hybridization signals are detected using an antisense cRNA probe for the mRNA of the JPGP core protein on the basal portions of cone (c) and rod (r) photoreceptor inner segments (monkey retina). (C) Almost no signals are observed in the inner segments using a sense cRNA probe (monkey retina). (A, C) Weak staining of red blood cells is observed in the inner nuclear and ganglion cell layers independent of the type of cRNA probe. rpe, retinal pigment epithelium; os, outer segment; is, inner segment; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bars, (A, C) 25 μm; (B) 10 μm.
Figure 2.
 
In situ hybridization histochemistry. (A, B) Weak hybridization signals are detected using an antisense cRNA probe for the mRNA of the JPGP core protein on the basal portions of cone (c) and rod (r) photoreceptor inner segments (monkey retina). (C) Almost no signals are observed in the inner segments using a sense cRNA probe (monkey retina). (A, C) Weak staining of red blood cells is observed in the inner nuclear and ganglion cell layers independent of the type of cRNA probe. rpe, retinal pigment epithelium; os, outer segment; is, inner segment; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bars, (A, C) 25 μm; (B) 10 μm.
Figure 3.
 
Immunohistochemistry with the anti-JBGP core protein antibody. (A, B, and C) A diffuse, intense binding pattern, and a punctate, widely scattered one are observed in the photoreceptor layer (pl) and the outer plexiform layer (opl), respectively, with weak staining of the cell surface in the outer nuclear layer (onl) (A) bovine retina; (B) rat retina; (C) human retina. (D) Only faint staining is observed throughout the retina after preincubation of the antibody with the GST–core protein fusion protein (human retina). (E) In the monkey retina, the surfaces of the inner segments (is) and outer segments (os) of both cones (c) and rods (r) are stained with the antibody. (F) In the monkey retina, punctate bindings are detected with the antibody in the cone pedicles (arrowhead) and the rod spherules (arrow). Scale bars, (A through D) 25 μm; (E, F) 10 μm.
Figure 3.
 
Immunohistochemistry with the anti-JBGP core protein antibody. (A, B, and C) A diffuse, intense binding pattern, and a punctate, widely scattered one are observed in the photoreceptor layer (pl) and the outer plexiform layer (opl), respectively, with weak staining of the cell surface in the outer nuclear layer (onl) (A) bovine retina; (B) rat retina; (C) human retina. (D) Only faint staining is observed throughout the retina after preincubation of the antibody with the GST–core protein fusion protein (human retina). (E) In the monkey retina, the surfaces of the inner segments (is) and outer segments (os) of both cones (c) and rods (r) are stained with the antibody. (F) In the monkey retina, punctate bindings are detected with the antibody in the cone pedicles (arrowhead) and the rod spherules (arrow). Scale bars, (A through D) 25 μm; (E, F) 10 μm.
Figure 4.
 
Western blot analysis of the JBGPs, with or without neuraminidase digestion, using either jacalin or anti-core protein antibody. (A) Two major bands (180 and 135 kDa) and one minor one (120 kDa) are detected with biotinylated jacalin in the nondigested JBGPs. (B) Bands of 160 and 120 kDa are detected with jacalin for the JBGPs after digestion for 17 hours. (C, D) The 135- and 120-kDa bands are intensely and weakly detected, respectively, with the anti-core protein antibody for JBGPs without treatment (JBGPs applied: C, 15 μg; D, 5 μg). (E, F, and G) After digestion of JBGPs, the major positive band corresponding to the 135-kDa band in (D) gradually decreases in molecular weight, whereas the 120-kDa band is more prominently detected (digestion time: E, 1 hour; F, 6 hours; G, 17 hours). (H) No band is detected with the antibody preincubated with the GST–core protein fusion protein for JBGPs without digestion.
Figure 4.
 
Western blot analysis of the JBGPs, with or without neuraminidase digestion, using either jacalin or anti-core protein antibody. (A) Two major bands (180 and 135 kDa) and one minor one (120 kDa) are detected with biotinylated jacalin in the nondigested JBGPs. (B) Bands of 160 and 120 kDa are detected with jacalin for the JBGPs after digestion for 17 hours. (C, D) The 135- and 120-kDa bands are intensely and weakly detected, respectively, with the anti-core protein antibody for JBGPs without treatment (JBGPs applied: C, 15 μg; D, 5 μg). (E, F, and G) After digestion of JBGPs, the major positive band corresponding to the 135-kDa band in (D) gradually decreases in molecular weight, whereas the 120-kDa band is more prominently detected (digestion time: E, 1 hour; F, 6 hours; G, 17 hours). (H) No band is detected with the antibody preincubated with the GST–core protein fusion protein for JBGPs without digestion.
Figure 5.
 
Western blot analysis of JBGPs with or without neuraminidase digestion using lectins. (A) Two bands of 180 and 135 kDa are detected with biotinylated MAL II in the nondigested JBGPs. (B) Two bands, weaker and smaller than in (A), are detected with MAL II for JBGPs digested for 1 hour. (C) A 120-kDa band is detected with biotinylated ECA for JBGPs without treatment. (D) Two bands are detected with ECA in addition to the 120-kDa band in JBGPs digested for 1 hour. (E) A 120-kDa band is detected with biotinylated PNA for JBGPs without treatment. (F) Two bands are detected with PNA in addition to the 120-kDa band in JBGPs digested for 1 hour. (G) Almost no band is detected with a mixture of biotinylated PNA and 0.1 M d-galactose in nondigested JBGPs.
Figure 5.
 
Western blot analysis of JBGPs with or without neuraminidase digestion using lectins. (A) Two bands of 180 and 135 kDa are detected with biotinylated MAL II in the nondigested JBGPs. (B) Two bands, weaker and smaller than in (A), are detected with MAL II for JBGPs digested for 1 hour. (C) A 120-kDa band is detected with biotinylated ECA for JBGPs without treatment. (D) Two bands are detected with ECA in addition to the 120-kDa band in JBGPs digested for 1 hour. (E) A 120-kDa band is detected with biotinylated PNA for JBGPs without treatment. (F) Two bands are detected with PNA in addition to the 120-kDa band in JBGPs digested for 1 hour. (G) Almost no band is detected with a mixture of biotinylated PNA and 0.1 M d-galactose in nondigested JBGPs.
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