Interphotoreceptor retinoid-binding protein (IRBP) - Journal of Cell ...

Journal of Cell Science 105, 7-21 (1993) Printed in Great Britain © The Company of Biologists Limited 1993

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Interphotoreceptor retinoid-binding protein (IRBP), a major 124 kDa glycoprotein in the interphotoreceptor matrix of Xenopus laevis Characterization, molecular cloning and biosynthesis Federico Gonzalez-Fernandez1,2,3,*, Karen L. Kittredge1, Mary E. Rayborn5, Joe G. Hollyfield5, Robert A. Landers5, Margaret Saha4 and Robert M. Grainger3,4 Departments of 1Ophthalmology, 2Pathology (Neuropathology), 3Graduate Program in Neuroscience and 4Biology of the University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA 5Department of Ophthalmology and Division of Neuroscience, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030, USA *Author for correspondence

SUMMARY We have demonstrated that the neural retina of Xeno pus laevis secretes into the extracellular matrix surrounding the inner and outer segments of its photoreceptors a glycoprotein containing hydrophobic domains conserved in mammalian interphotoreceptor retinoidbinding proteins (IRBPs). The soluble extract of the interphotoreceptor matrix contains a 124 kDa protein that cross-reacts with anti-bovine IRBP immunoglobulins. In vitro [3H]fucose incorporation studies combined with in vivo light and electron microscopic autoradiographic analysis, showed that the IRBP-like glycoprotein is synthesized by the neural retina and secreted into the interphotoreceptor matrix. A 1.2 kb Xenopus IRBP cDNA was isolated by screening a stage 42 (swimming tadpole) lZap II library with a human IRBP cDNA under low-stringency conditions. The cDNA hybridizes with a 4.2 kb mRNA in adult Xenopus neural retina, tadpole heads as well as a less-abundant mRNA of the

same size in brain. During development, IRBP and opsin mRNA expression correlates with photoreceptor differentiation. The translated amino acid sequence of the Xenopus IRBP clone has an overall 70% identity with the fourth repeat of the human protein. Sequence alignment with the four repeats of human IRBP showed three highly conserved regions, rich in hydrophobic residues. This focal conservation predicts domains important to the protein’s function, which presumably is to facilitate the exchange of 11-cis retinal and all-trans retinol between the pigment epithelium and photoreceptors, and to the transport of fatty acids through the hydrophilic interphotoreceptor matrix.

INTRODUCTION

arisen from the quadruplication of an ancestral gene (Borst et al., 1989; Liou et al., 1989, 1991; Si et al., 1989; Fong et al., 1990; Nickerson et al., 1991). IRBP appears to function as a hydrophobic ligand-binding protein and may have a critical role during retinal development. IRBP carries endogenous vitamin A in a lightdependent manner as well as six to seven fatty acid equivalents (Liou et al., 1982; Fong et al., 1984b; Bazan et al., 1985; Saari et al., 1982). Possible functions of IRBP include: transport of 11-cis retinal and all-trans retinol between the photoreceptors and pigment epithelium during the visual cycle (Lai et al., 1982; Lin et al., 1989; Flannery et al., 1990; Okajima et al., 1990; Adler and Spencer, 1991; Carlson and Bok, 1992), buffering excess vitamin A in the interphotoreceptor matrix (Ho et al., 1989) and protecting retinoids from degradation (Crouch et al., 1992).

Interphotoreceptor retinoid-binding protein (IRBP) is the major soluble component of the interphotoreceptor matrix (for review see Chader et al., 1986). This important extracellular matrix surrounds the outer and inner segments of the photoreceptors and separates the neural retina from the pigment epithelium. IRBP is a large glycolipoprotein (135 kDa in man), which is secreted into this matrix by both rods and cones (Gonzalez-Fernandez et al., 1984, 1992; Hollyfield et al ., 1985a; Porrello et al., 1991; Yokoyama et al., 1992; Hessler et al., 1993). In bony fish (Osteichthyes) IRBP is about half the size (67,600 ± 2,700 Da; Bridges et al., 1986, 1984; Hessler et al., 1993) of that in higher vertebrates. These observations and the four repeat structure of the mammalian protein suggest that IRBP may have

Key words: interphotoreceptor retinoid-binding protein, Xenopus laevis, interphotoreceptor matrix, retinoidbinding proteins

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F. Gonzalez-Fernandez and others

Interactions between the neural retina and pigment epithelium are critical to retinal differentiation and growth (Hollyfield and Witkovsky, 1974; Vollmer and Layer, 1986; Hunter et al., 1992). It is therefore interesting that the expression of IRBP is up-regulated relatively early during the period of retinal differention (Eisenfeld et al., 1985; Johnson et al., 1985; Carter-Dawson et al., 1986; Gonzalez-Fernandez and Healy, 1990; Liou et al., 1991; Hauswirth et al., 1992; Wang et al., 1992; Gonzalez-Fernandez et al., 1993). Its temporal control of expression and the critical interphase that it occupies suggest that IRBP could mediate important interactions between the pigment epithelium and neural retina during development. The appearance of IRBP during retinal development is likely to be linked to one of the proposed functions for IRBP described above. Possible additional roles of IRBP during retinal development could be: (1) to transport from the pigment epithelium hydrophobic morphogens such as retinoic acid; (2) to provide a transport vehicle for fatty acids, particularly docosahexanoic acid, an essential fatty acid that is important for normal retinal development, and outer segment structure and function (Scott and Bazan, 1989); or (3) to provide a role unrelated to its hydrophobic ligand-binding properties. Such functions could be adhesion, maintenance of structure or cell/cell communication. Xenopus laevis can provide a unique experimental system to study the role of the interphotoreceptor matrix in development as well as identify functionally conserved domains within the protein. We have previously shown that molecules may be introduced into the future interphotoreceptor matrix of the Xenopus embryo through optic vesicle microinjection (Hollyfield and Ward, 1974; Gonzalez-Fernandez and Kittredge, 1992). This technique, which does not disturb the normal relationships between the pigment epithelium and the neural retina, could allow studies of the fate of recombinant IRBP and the effect of immunological inactivation of IRBP. Furthermore, the sequence of nonmammalian IRBPs will be invaluable for identifying functionally important domains and understanding the evolution of its gene. Despite the potential advantages of lower vertebrates for experimental studies and phylogenetic comparisons, most studies to date have examined only mammalian IRBP. In the present paper, we have characterized the biosynthesis of the Xenopus homologue of IRBP and isolated cDNAs encoding it. Preliminary reports of portions of this work have been published in abstract form (Rayborn et al., 1984; Kittredge et al., 1992). MATERIALS AND METHODS Animals For the metabolic studies, juvenile Xenopus laevis (Xenopus I, Ann Arbor, MI) 2-3 cm in length were housed under cyclic lighting conditions of 12 h light, followed by 12 h darkness at 19°C for several weeks prior to utilization. Adult Xenopus laevis were mated, and embryos collected and dejellied as described by Henry and Grainger (1987). Embryos were reared in 20% Steinberg’s solution (Rugh, 1962) containing 50 µg/ml gentamycin sulfate. Embryos were anesthetized with 3aminobenzoic acid ethyl ester (Sigma) prior to dissection.

Extraction of interphotoreceptor matrix and western blot analysis For the western blot analysis, interphotoreceptor matrix was extracted from adult Xenopus eyes. The anterior segment including the ciliary body and lens were first removed by a circumferential limbal incision. Under phosphate buffered saline (PBS; 5 mM sodium phosphate, pH 7.4, 0.15 M NaCl) in the presence of 1.0 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor, the neural retinal was gently detached from the pigment epithelium. The two tissues were completely separated by severing the optic nerve. The retina and eye cup were incubated together for 5 min in PBS on ice and the apical surface of the pigment epithelium was gently irrigated with this saline. The eye cup and neural retina were removed and the PBS extract was centrifuged at 50,000 g for 30 min at 4°C. The supernatant was precipitated with an equal volume of acetone at −20°C, treated with dithiothreitol, sodium dodecyl sulfate (SDS) and fractionated by polyacrylamide gradient (5% to 12%) gel electrophoresis in a discontinuous buffer system (Laemmli, 1970). The proteins were electrophoretically transferred to nitrocellulose and probed with rabbit anti-bovine IRBP immunoglobulins (Fong et al., 1984a) followed by peroxidase-conjugated goat anti-rabbit IgG as previously described (Gonzalez-Fernandez et al., 1985).

Isolation of a Xenopus IRBP cDNA The cDNA library used in this study was prepared from poly(A)+ mRNA from whole stage 42 embryos (swimming tadpoles; refer to staging system of Nieuwkoop and Faber, 1956). A full description of the preparation of this library is described elsewhere (Saha and Grainger, 1993). The library was screened with a full-length human IRBP cDNA provided by Dr C. David Bridges (Purdue University). This probe was labeled with [a-32P]dCTP by the method of random primed labeling (Feinberg and Vogelstein, 1983). The bacteriophage l plaques were immobilized on Nytran membranes (Schleicher and Schuell, Inc., Keene, NH). Duplicate filters were made from each primary master plate so that genuine signals could be distinguished from spurious spots. The l phage were lysed by steam treatment using the method of G. Struhl as described by Sambrook et al. (1989) and cross-linked to the Nytran paper by ultraviolet irradiation. Prehybridization was carried out overnight at 42°C in 30% formamide, 1 M NaCl, 100 µg/ml denatured salmon sperm DNA, 1% SDS, 10 mM Tris-HCl, pH 7.5. Hybridization was carried out overnight under the same conditions with 106 c.p.m./ml−1 denatured probe. Following hybridization, the membranes were washed twice in 2×SSC (1×SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1% SDS twice at room temperature and twice at 50°C. Each wash was 30 min in duration. The filters were exposed to X-ray film (X-AR, Kodak) at −70°C with an intensifying screen. Synthetic oligonucleotides corresponding to the Xenopus IRBP sequence were used as sequencing primers.

RNA analysis Total RNA was isolated from adult Xenopus tissues or embryos by the method of Chomczynski and Sacchi (1987) as described by Gonzalez-Fernandez and Healy (1990). Glyoxal was used to denature RNA before electrophoresis in 1.0% Seakem GTG agarose (FMC, Rockland, ME). The RNA was transferred to Nytran paper (Schleicher and Schuell) and cross-linked by ultraviolet irradiation. Prehybridization and hybridization were carried out at 42°C in 50% formamide, 5×SSPE (SSPE at a 1×concentration is 0.18 M NaCl; 10 mM sodium phosphate, pH 7.7; 1 mM EDTA), 5×Denhardt’s solution (0.1% Ficoll; 0.1% polyvinylpyrrolidone; 0.1% BSA), 1% SDS, and 100 µg/ml denatured salmon sperm DNA. The excised cDNA inserts were gel purified and labeled with [a-32P]dCTP by the random primer

Xenopus laevis IRBP method (Feinberg and Vogelstein, 1983). The blots were hybridized with 106 c.p.m. of radiolabeled probe per ml of the above buffer. D.D. Oparian (Brandeis University) provided the bovine opsin cDNA, which was nearly full-length (1.0 kb in size starting at nucleotide number 234; Nathans and Hogness, 1983). Following hybridization, the blots were washed twice at room temperature in 6×SSPE, 0.5% SDS for 15 min, twice at 37°C in 1×SSPE, 0.5% SDS for 15 min and finally for 30 min at 65°C in 1×SSPE, 0.5% SDS. The conditions for the northern blot of Fig. 13 (bottom) where RNA probes were used has been described by Gonzalez-Fernandez and Healy (1990). For all northern blots, XAR film (Kodak) was exposed against an intensifier screen at −80°C.

Metabolic labelling In the in vivo studies, L-[6-3H]fucose (30 Ci/mmol; ICN, Irvine, CA) was evaporated to dryness to remove the ethanol carrier and was resuspended in amphibian Ringer’s solution at a concentration of 2.0 mCi/ml. Ten animals were given dorsal subcutaneous injections of approximately 12 µCi. The injected animals were rinsed repeatedly for the next several hours and the eyes were removed the following day. The eyes were placed in a small drop of incubation medium, Ringer’s bicarbonate-pyruvate buffer (RBP; Hollyfield and Anderson, 1982), and the anterior segment removed. Some of the eyes were fixed with the retina and the pigment epithelium left intact, while others were dissected free of each other and fixed after extensive rinses in Ringer buffer. All tissues were fixed for 1.5 h in an ice-cold mixture of 1% OsO4, 1.65% glutaraldehyde in 0.075 M cacodylate buffer (pH 7.4). Tissues were dehydrated with ethanol and embedded in Epon. For autoradiography, 1 µm thick sections were cut for light microscopic autoradiography and placed on glass slides. After dipping in Kodak NTB liquid emulsion diluted 1:1 with distilled water, the slides were dried and exposed in the dark at 4°C for 5 to 25 days. Electron microscopic autoradiographic methods employed Ilford L4 emulsion and Phenadon developing as described in detail previously (Hollyfield, 1979). For biochemical analysis, retinas were dissected from the pigment epithelium/choroid complex as described above and rinsed extensively in the incubation medium. The proteins in the rinse medium, retinal cytosol, pigment epithelial cytosol, retinal membranes and pigment epithelial/choroid membranes were prepared for polyacrylamide gel electrophoresis and fluorography as previously described (Gonzalez-Fernandez et al., 1985). For the in vitro experiments, retinas and pigment epithelium/choroid from 40 dark-adapted eyes were collected separately in RBP/Wolf-Quimby media (Fliesler et al., 1985) on ice. Metabolic labeling was initiated by placing the two groups of tissue in separate tubes containing 5 ml of medium with 200 µCi/ml L-[6- 3H]fucose (30 Ci/mmol). The tissues were maintained in the dark supplied with 95% O2/5% CO2 at 21°C with gentle agitation. After 4 h, the incubation medium and the tissues were collected by the addition of 1.0 mM PMSF. The tissues were homogenized in PBS and centrifuged at 100,000 g to obtain soluble and membrane fractions. Aliquots of each fraction were examined by SDS-polyacrylamide gel electrophoresis (SDSPAGE) and fluorography.

RESULTS Identification and cloning of Xenopus IRBP The rabbit anti-bovine IRBP immunoglobulins, which cross-react with human and rat IRBPs (Fong et al., 1984a; Gonzalez-Fernandez et al., 1984), recognize a 124 kDa pro-

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Mr × 10−3

Fig. 1. Comparison of Xenopus and bovine IRBPs by western blot analysis. Interphotoreceptor matrix from Xenopus retina (lane 1) and bovine retina (lane 2) were subjected to SDS-PAGE and transferred to nitrocellulose paper. Immunological visualization of IRBP was carried out by incubating the transfer with rabbit anti-bovine IRBP immunoglobulins followed by peroxidase-conjugated goat anti-rabbit IgG. Transfers that had been treated with preimmune serum displayed no reaction (data not shown).

tein in the soluble extract of adult Xenopus interphotoreceptor matrix. Although smaller than mammalian IRBPs (bovine and rat, 144 kDa; human IRBP, 135 kDa), the Xenopus homolog is similar in size to Rana pipiens IRBP (125 kDa, Fong et al., 1986) and approximately twice the size of IRBP in bony fish (Bridges et al., 1984, 1987; Wagenhorst et al., 1993). The fact that its size is similar to that of IRBPs in higher vertebrates suggests that Xenopus IRBP probably has a four-repeat structure similar to that described for human and bovine IRBPs (Nickerson et al., 1991). In order to characterize better the structure of the Xeno pus homolog of IRBP, cDNAs for this protein were isolated. We screened a stage 42 (swimming tadpole) cDNA library under low-stringency conditions (see Materials and Methods) with a human IRBP cDNA. Seven putative Xeno pus IRBP cDNA clones were isolated, four of which were partially characterized. The sizes of the inserts were determined by digesting the rescued bluescript plasmid with EcoRI. Based on the restriction pattern, the four cloned cDNAs had a similar size (1.2 kb), contained an internal EcoRI site and probably corresponded to a similar portion of the mRNA, presumably the 3′ end. One of the clones, termed XenIRBP.B1, was sequenced. Fig. 2 shows the translated amino acid sequence of XenIRBP.B1. Synthetic nested oligomers were used to generate the staggered sequence readings. Both strands of the cDNA were sequenced entirely (arrows summarize the sequencing strategy). The 1.2 kb clone consisted of 893 bp of open reading frame (filled bar) followed by 336 bp of 3′-untranslated region (UTR, open bar) ending in a typical signal polyadenlyation motif (AATAAA) located 25 bases upstream from a poly(A) tail (Wickens and Stephenson, 1984). Since the mRNA for Xenopus IRBP is 4.2 kb (major form), the clone corresponds to 29% of the mRNA. The computer program, LAWRENCE (Lawrence and Goldman, 1988), identified homology domains between the Xenopus

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F. Gonzalez-Fernandez and others Table 1. Nucleic acid and amino acid sequence identity between XenIRBP.B1 and human IRBP Homology SD domain* score† I II III IV

I II

III IV

Fig. 2. Sequence analysis of Xenopus IRBP clone XenIRBP.B1. The clone was isolated by low-stringency screening of a λZapII stage 42 (swimming tadpole) cDNA library using a human IRBP cDNA. Top: map of the clone (Bluescript KS−). Arrows summarize the sequencing strategy. Filled bar corresponds to the coding region and the open bar to the 3′-untranslated UTR region. Bottom: nucleotide sequence of the Xenopus IRBP cDNA and deduced amino acid sequence. The nucleotide sequence of the cDNA is 1.2 kb in size. A signal polyadenylation site (bold and underlined) is located 25 bases upstream from the poly(A) tail. Restriction endonucleases: E, EcoRI; B, BglII, P, PvuII. This sequence is available under accession number X69469 XLIRBPA in the European Molecular Biology Laboratory Nucleotide Sequence Database.

IRBP cDNA and each of the four repeats of human IRBP. Of the four homology domains, the degree of similarity is greatest within the fourth repeat. In this region, the maximum percentage identity of the nucleic and amino acid sequences between Xenopus and human IRBPs is 70% (Table 1). Although the overall sequence identity between Xenopus and mammalian IRBPs is low compared to opsin (see Discussion), focal regions are highly conserved. Conserved regions within IRBP probably form domains important to the protein’s function. Fig. 3 shows the amino acid alignment of XenIRBP.B1 with each of the four repeats of human IRBP. Of the 100 amino acids that are non-identical between the fourth repeats of Xenopus and human IRBP, 75 are conservative substitutions. The boxed regions within this alignment demonstrate a high degree of conservation between the XenIRBP.B1 and each of the four repeats of human IRBP. Conservative substitutions are defined as: I=L=V=M; K=R; D=E. Three regions stand out from this alignment. The first two of these regions have the

% Identity over similarity Length domain (nucleotides)

Region of homology domain Xenopus

Human

28.8 6.9 5.9 22.1 8.7 53.5

Nucleic acid sequence 52.4 429 277→705 48.4 221 256→476 66.2 65 627→691 47.9 758 113→870 68.8 77 1→77 69.1 758 108→865

477→905 1383→1603 1757→1821 2152→2909 2943→3019 3044→3801

3.8 14.3 3.1 3.9 10.2 6.6 3.4 16.7 4.0 28.9

Translated amino acid sequence 20.5 88 2→89 38.8 165 92→256 51.9 27 263→289 30.6 36 22→57 35.4 96 63→158 37.1 70 161→230 53.6 28 263→290 34.3 286 4→289 53.6 28 1→28 69.6 260 31→290

32→119 121→285 294→320 350→385 401→496 500→569 604→631 646→931 944→971 972→1231

The computer program LAWRENCE was used to identify regions of homology of the partial Xenopus IRBP sequence to human IRBP at both the nucleic acid and amino acid levels. The table shows that although the isolated clone is homologous with each of the four human IRBP repeats, its identity is greatest with the fourth repeat of human IRBP. *Homology domain refers to the internal repeats I-IV within human IRBP. †The “SD score” is equivalent to the number of standard deviations by which the similarity found in a region of two sequences is greater than the similarity expected by chance alone.

invariant sequences OGYOROD (residues 113-119) and OOODOR (136-141), where O represents a hydrophobic residue. The fact that these segments contain amino acids perfectly conserved not only in the four repeats of human IRBP, but also in Xenopus, and have a high proportion of hydrophobic residues predicts that these domains may be important for vitamin A or fatty acid-binding. In addition to these two domains, a third region showing a high degree of conservation was also seen (OOGE; 237-240). The length of the 3′-UTR of Xenopus IRBP mRNA (322 bp) is similar to that of the human IRBP mRNA 3′-UTR (416 bp) and is significantly smaller than that of the 3′UTR of bovine IRBP mRNA, which is 1,988 bp. There is a 47.4% sequence identity within the last 342 bp of the human 3′-UTR and the Xenopus 3′-UTR. These observations are consistent with the hypothesis that the longer 3′UTR of bovine IRBP is the result of a large insertion that has been made in the 3′-UTR of the ancestor of the bovine sequence. Northern blot analysis Fig. 4 is a northern blot of total RNA from adult Xenopus retina probed with both XenIRBP.B1 and bovine opsin cDNA probes. The mRNA sizes for Xenopus IRBP and opsin are 4.2 kb and 2.1 kb, respectively. A longer exposure of the autoradiogram revealed a less-abundant 6.0 kb

Xenopus laevis IRBP

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Fig. 3. Alignment of the XenIRBP.B1 sequence with the four repeats of human IRBP. In lines showing human repeat sequences upper case shows aligned non-identical amino acids, lower case unaligned amino acids; (−) represents aligned identical amino acids; (.) are gaps. Boxed regions represent amino acids that are identical or conservative substitutions in the Xenopus sequence and all four domains of human IRBP. Conservative substitutions are: I=L=V=M; K=R; D=E (Dayhoff et al., 1983). Regions with the invariant sequences OGYOROD, OGDOR and OOGE, where O represents a hydrophobic residue, are indicated (see text). Proline-rich regions are underlined.

Fig. 4. Characterization of the mRNA for IRBP from adult Xenopus neural retina. Ethidium bromide-stained agarose gel showing molecular mass standards (lane S) and total RNA (14 µg) isolated from adult male Xenopus neural retina. 20 µg of RNA, run on the same gel but not stained was transferred directly to Nytran paper. These identical blots were probed with either the Xenopus IRBP cDNA (lanes B and C) or bovine opsin cDNA (lanes D and E). The autoradiograms were exposed at −80°C with an intensifying screen for approximately 4 h (lanes B and C) or overnight (lanes C and E). The longer exposure brought out a lessabundant 6.0 kb IRBP mRNA transcript but failed to identify additional rod opsin mRNAs. Arrowheads: 6.0 kb, 4.2 kb, 2.0 kb.

IRBP transcript but failed to identify additional sizes for the opsin mRNA. Multiple IRBP mRNAs have been observed in a number of mammals (Inouye et al., 1989; Gonzalez-Fernandez and Healy, 1990) and in rat are due to different sizes of the 3′-UTR (Gonzalez-Fernandez et al.,

Fig. 5. Tissue distribution of the mRNA for IRBP. Northern blot of Xenopus laevis neural retina (8 µg), brain (4.6 µg) and liver (8 µg) total RNA in lanes A, B and C, respectively. The blot was probed with the 32P-labeled Xenopus IRBP cDNA under highstringency conditions. The autoradiograms in the left and right panels were exposed for 2.75 h and 85 h, respectively, at −80°C with an intensifying screen. Arrowheads correspond to the major mRNA IRBP band at 4.2 kb and minor band at 6.0 kb, which is difficult to discern in the photograph. In the longer exposure of the brain RNA (right panel, lane B), the mRNA for IRBP was detected.

1993). Although multiple forms of the mRNA for rod opsin have been documented in a variety of mammals (Al-Ubaidi et al., 1990), only one form of the opsin mRNA is present Xenopus. The sequence of Xenopus rod opsin has been described by Saha and Grainger (1993). In the northern blot of Fig. 5, adult Xenopus retina, brain and liver total RNA were probed with XenIRBP.B1 under high-stringency conditions. This blot demonstrates that the XenIRBP.B1 probe does not bind to the upper ribosomal

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Mr × 10−3

Fig. 6. Secretion of IRBP by the Xenopus neural retina but not RPE/choroid. Adult Xenopus isolated neural retina and RPE/choroid were incubated in the presence of [3H]fucose and the membrane and cytosolic fractions, and incubation medium was analyzed by SDS-PAGE and fluorography. Left: Coomassie bluestained acrylamide gel. Right: Fluorogram of the same gel. Lanes: s, molecular mass standards; 1, retina membranes; 2, retina cytosol; 3, retina incubation medium; 4, RPE/choroid incubation medium; 5, RPE/choroid cytosol; 6, RPE/choroid membranes. Arrowhead is Mr 124×10−3.

subunit and that brain expresses low levels of the mRNA for IRBP. The source of the mRNA for IRBP in the Xeno pus brain may be the pineal gland, which is known to express this retinal protein (Chader et al., 1986; Bridges et al., 1987; Gonzalez-Fernandez et al., 1993; Lopez et al., unpublished results). Secretion of Xenopus IRBP into the interphotoreceptor matrix The experiments in this section utilized biochemical, and light and electron microscopic autoradiographic analysis of [3H]fucose in vitro and in vivo radiolabelling to localize IRBP and further define its site of synthesis. Fucose was selected for these studies because it is present in the oligosaccharide of mammalian IRBP is not metabolized to other compounds, and is not incorporated into glycosaminoglycans. We separately incubated isolated adult neural retinas and eye-cups (pigment epithelium/choroid) in the presence of [ 3H]fucose for 4 h. The incubation medium, cytosolic and membrane fractions from the neural retinas and pigment epithelium/choroid were analyzed by SDSPAGE and fluorography (Fig. 6). A radioactive 124 kDa glycoprotein was observed in the incubation medium of the neural retina, but not in the medium of the pigment epithelium/choroid. A faint 124 kDa band was identified in the neural retinal cytosol. This experiment, coupled with the immunoblotting data presented in Fig. 1, demonstrates that Xenopus IRBP is fucosylated and secreted by the neural retina and not the pigment epithelium. In order to show that the radiolabeled IRBP secreted into the incubation media normally accumulates in the interphotoreceptor matrix, we performed a similar experiment in vivo. [3H]fucose was injected intraperitoneally and the eyes were removed after 24 h. The tissues were either fixed

Mr × 10−3

Fig. 7. In vivo synthesis of fucosylated interphotoreceptor matrix proteins. Interphotoreceptor matrix was isolated 24 h after intraperitoneal injection of [3H]fucose. The matrix preparation was subjected to SDS-PAGE and fluorography. The acrylamide gel stained with Coomassie blue is shown in A (left lane, molecular mass standards; right lane, crude matrix preparation) and the fluorogram in B. Arrow corresponds to 124×10−3 Mr.

for light and electron microscopic autoradiography or analyzed biochemically. For the latter studies, the neural retina was gently detached from the eye-cup, and the saline washes of the neural retina and apical surface of the pigment epithelium were combined and subjected to SDSPAGE and fluorography. Fig. 7 shows that radiolabelled IRBP could be identified in the interphotoreceptor matrix extract. Besides IRBP, an additional radioactive band at approximately 183 kDa was noted. This band probably does not represent a matrix component secreted into the subretinal space because it was not detected in the in vitro experiment of Fig. 6. This band more likely represents a labeled protein that leaked into the matrix extract from neural retinal cells damaged as this tissue layer was mechanically separated from the pigment epithelium. Consistent with this interpretation is the fact the retina cytosol normally contains a prominently radiolabelled protein of this size (compare with Fig. 6, lane 2). Light and electron microscopic autoradiography were performed in order to confirm the compartmentalization of the radiolabelled protein. The light microscopic autoradiographs in Fig. 8 show the distribution of silver grains across the outer retina from one of the recovered eyes. The silver grains were present over the inner segments of the photoreceptors, in the interphotoreceptor matrix and extensively over the pigment epithelium. When the retina was slightly teased from the pigment epithelium and immediately fixed, an extensive cloud of silver grains was present

Xenopus laevis IRBP

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Fig. 8. Light microscopic autoradiographic analysis of in vivo incorporation of [3H]fucose by the Xenopus retina. (A) Intact retina demonstrating grains within the neural retina, pigment epithelium and surrounding outer segments. (B) The neural retina was gently teased from the pigment epithelium immediately before fixation. (C) After PBS wash grains surrounding outer segments have disappeared.

over the extracellular compartment between the retina and pigment epithelium. Our interpretation is that these silver grains represent radiolabelled matrix glycoconjugates that have become dispersed throughout the expanded volume occupied by the matrix at the detachment site. This prepa-

ration was not rinsed but the retina and pigment epithelium were slightly separated at the margin of the eye cup before fixation. In the lower panel of Fig. 8, an isolated retina is shown that was rinsed prior to fixation. Little radioactivity is asso-

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Fig. 9. Electron microscopic autoradiographic analysis of in vivo incorporation of [3H]fucose at the level of the external limiting membrane. Here radioactivity is associated with the apical termination of the villous processes, cytoplasm of the Muller cells, and inner segments.

ciated with the interphotoreceptor matrix. Electron microscopy was used to study the localization of the radioactivity still remaining in the retina. Fig. 9 shows that at the outer limiting membrane, radioactivity is associated with both the apical termination of the villous processes and the cytoplasm of the Muller cells. Additional radioactivity is associated with what appears to be Golgi apparatus in the inner segments of the two side-by-side photoreceptors. Fig. 10 demonstrates that the flocculent interphotoreceptor matrix material is also radioactive, as indicated by the silver grains associated with this extracellular compartment. In this figure, a band of radioactivity is evident at the base of one of the rod outer segments shown. This represents one of the minor rods, which has a very rapid rate of membrane turnover. Hollyfield et al. (1984) has shown that there are two populations of rods in Xeno pus, which have different rates of outer segment renewal and utilization of fucose. The absence of the band in the

right photoreceptor in this figure suggests that this outer segment belongs to a principal rod and the presence of a band on the photoreceptor to the left probably reflects this minor rod. Fig. 11 shows that when the pigment epithelium is rinsed and prepared for autoradiography, silver grains are distributed over the cytoplasm of the pigment epithelium and the apical microvilli of these cells. In view of the fact that IRBP is synthesized by the photoreceptors (at least in mammals) the silver grains over the outer segments, pigment epithelium, and Muller cells probably represent glycoconjugates newly synthesized by these cells. The possibility that some of the radiolabel may represent uptake of IRBP cannot be excluded (see Hollyfield et al., 1985b). Expression of IRBP during development Fig. 12 compares the expression of the mRNAs for IRBP and opsin during development. In the top panels of this figure, the mRNA for both proteins at stage 43 (swimming

Xenopus laevis IRBP

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Fig. 10. Electron microscopic autoradiographic analysis of in vivo incorporation of [3H]fucose. (A) The interphotoreceptor matrix is extensively labelled. (B) Demonstration of grains within the interphotoreceptor matrix and base of a minor rod outer segment.

tadpole) are shown to be restricted to the head, absent from the body and have a similar electrophoretic mobility to that of the mRNAs for these proteins in the adult retina. In the bottom panel of this figure, 23 µg of total RNA from whole embryos at various developmental stages from neurula to tadpole were subjected to northern blot analysis. This blot was first probed with antisense Xenopus IRBP transcripts generated from the bacteriophage T7 promoter using BamHI-linearized plasmid (the BamHI site is contained

within the multiple cloning segment 5′ to the insert). The transfer was then reprobed with a Xenopus opsin antisense probe. The mRNAs for both IRBP and opsin could be detected first at stage 40 (3 days old, recently hatched). At this stage photoreceptor outer segments are just beginning to form (Kinney and Fisher, 1978b). Between stages 40 and 45/46 there was a marked up-regulation of both IRBP and opsin mRNA expression. During this period there is further growth of the outer segments (outer segments attain

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Fig. 11. Electron microscopic autoradiographic analysis of in vivo incorporation of [3H]fucose in the pigment epithelium. Following thorough washing of the eye-cup, significant radioactivity remained associated with the pigment epithelium and its apical processes.

their adult length at stage 53/54; Kinney and Fisher, 1978b). DISCUSSION Our long-term goals are to determine IRBP’s structural requirements for ligand-binding and role during retinal development. Xenopus laevis will provide a valuable system in which to study both of these questions through phylogenetic comparisons of protein sequence and embryological manipulation of the interphotoreceptor matrix possible in this species. Preliminary data from other groups have led us to believe that a protein homologous to IRBP probably exists in the Xenopus retina. Using antisera raised against bovine or primate IRBPs, IRBP-like proteins have been identified in all six major vertebrate classes, emphasizing

its importance to visual function (Bridges et al., 1986). Bovine and human IRBP cDNAs hybridize with the mRNA for IRBP in a variety of mammals but not birds (Inouye et al., 1989; Liou et al., 1991). In contrast, IRBP has been identified in the chicken retina by western blotting (Bridges et al.,1986). This apparent discrepancy is probably due to the greater evolutionary constraints on the amino acid sequence than on the nucleic acid sequence because of evolutionary pressure to preserve functionally important domains in the protein. Another possibility is that some non-mammals may utilize another protein in the interphotoreceptor matrix rather than IRBP to transport vitamin A. In this regard, it has been noted that the chicken IPM contains purpurin, a retinol-binding protein with no apparent homology with IRBP (Schubert et al., 1986). Fong et al. (1986) showed that the N-terminal sequence of a 125 kDa glycoprotein from the soluble extract of the interphotore-

Xenopus laevis IRBP

A

B

Fig. 12. Northern blot analysis of the expression of IRBP and opsin during development. (A) Lanes A and B received 14 µg each of total RNA extracted from stage 43 heads and bodies respectively. Left panel: ethidium bromide-stained gel showing molecular mass standards and ribosomal RNA before blotting. Center panel: autoradiogram of blot probed with Xenopus IRBP cDNA. Right panel: same blot stripped and reprobed with bovine opsin cDNA. Lane S corresponds to the molecular mass standards. (B) Each lane received 23 µg of total RNA extracted from whole embryos. The developmental stage of the embryo is indicated above the lane. Top panel: the blot was probed with antisense Xenopus IRBP probe and exposed for 24 h. Bottom panel:the blot was then stripped and reprobed with a Xenopus opsin (Saha and Grainger, 1993) antisense probe and exposed for 6 h.

ceptor of Rana pipiens is highly conserved compared to IRBPs from nine other vertebrate species (see also Schneider et al., 1986; Wood et al., 1984). Liou et al. (1991) were able to detect in the retina but not liver of Rana pipiens a 4.4 kb band by probing a northern blot of poly(A)+ RNA with a human cDNA under reduced stringency conditions (5×SSC, in 50% formamide at 37°C). In order to isolate a cDNA for Xenopus IRBP, we utilized the technique of low-stringency screening. Based on the size of the mRNA for IRBP in Xenopus, and the homology with the fourth repeat of human IRBP, the cDNA isolated in this study represents all but the eight N-terminal amino acids of the fourth repeat. Because the present cDNA spans virtually the entire fourth repeat of mammalian IRBP, we were able to align the translated XenIRBP.B1 with the four repeats of human IRBP. Although XenIRBP.B1 is most homologous with the fourth repeat, at least two regions within the clone were highly conserved between all four

17

repeats of human IRBP. 70% of the nucleotides and amino acids between Xenopus and human IRBPs are identical. In contrast, the nucleotide and amino acid sequences of Xeno pus rod opsin have percentage identities of 76% and 85%, respectively with human rodopsin (Saha and Grainger, 1993). Although the overall percentage identity between Xenopus and human IRBPs is low compared to opsin, focal regions are highly conserved. This supports the idea that specific regions within IRBP participate in the formation of functionally important domains. Two regions in particular, which were noted by Liou et al. (1991) to be conserved between each of the four repeats of human IRBP, stand out when Xenopus and human IRBPs are compared. These regions have the invariant sequences OGYOROD and OOODOR, where O represents a hydrophobic residue. The fact that these segments contain amino acids perfectly conserved not only between the four repeats of human IRBP, but also in Xenopus, and have a high proportion of hydrophobic residues suggests that these regions are important to the formation of the ligand-binding domains. Evidence that these domains have hydrophobic binding activity comes from the observation that they are present in Tsp, a tail-specific protease that selectively degrades proteins with nonpolar C termini in Escherichia coli (Silber et al., 1992). A striking feature of these two conserved regions is the presence in both of an arginine residue that is conserved between Xenopus IRBP and each of the four repeats of human IRBP. This amino acid, in other hydrophobic ligandbinding proteins, confers specificity for fatty and retinoic acids by providing its a-guanidinium group to stabilize the carboxyl moiety of these ligands (Cheng et al., 1991; Stump et al., 1991). The presence of two arginines per repeat suggests that the whole protein should be able to bind eight fatty acids. Bazan et al. (1985) found that IRBP carries four fatty acids noncovalently. The number of endogenous fatty acids may be lower than our predicted stoichiometry because the native protein used in those studies may not have been saturated with ligand. An alternative model would be that more than one arginine participates in the stabilization of the carboxyl group of each bound fatty acid. Although the relationship between IRBP’s retinol and fatty acid-binding sites is not known, preliminary studies suggest that one but not both of IRBP’s retinol-binding sites can be blocked by palmitic acid (Hazard et al., 1991). Perhaps the third domain, OOGE, which does not contain arginine, is more important to retinol than to fatty acid-binding. Mutageneis studies aimed at understanding the role of these conserved arginines are in progress in our laboratory (Van Niel et al., 1993). In any event, we predict that the above three conserved regions form the ligand-binding domain(s) and the conserved arginines in the first two regions are important for fatty acid-binding. The smaller size of the 3′-UTR of human IRBP mRNA (416 bp) compared to the 3′-UTR of the bovine IRBP mRNA (1,988 bp) is due either to a large insertion into the ancestor of the bovine gene or a large deletion from the ancestor of the human gene (Si et al., 1989). We found that the final 342 bp of the human 3′-UTR has a 46% sequence identity with the 3′-UTR of the Xenopus IRBP mRNA. This high degree of conservation, which is not present between

18


the 3′-UTRs of Xenopus and human rod opsins (Saha and Grainger, 1992), may suggest an importance of the IRBP 3′-UTR in promoting mRNA stability and translational control. The sequence homology, taken together with the similar size of the of Xenopus IRBP 3′-UTR (322 bp), suggests that the mechanism responsible for the larger bovine IRBP 3′-UTR was an insertion into the bovine ancestor rather than a deletion from the human ancestor gene. The physiological significance of the longer 3′-UTRs of mammalian IRBPs is unknown. Two forms of the mRNA for Xenopus IRBP were noted, a major form at 4.2 kb and a less-abundant 6.0 kb transcript. Two forms of the mRNA have been observed in several species, particularly rat, which displays clear bands at 5.2 kb and 6.4 kb (Gonzalez-Fernandez and Healy, 1990; Inouye et al., 1989). Different lengths of the 3′-UTR are responsible for the two forms of the rat IRBP mRNA (Gonzalez-Fernandez et al., 1993). Opsin displayed only one size for its mRNA, although multiple forms of the mRNA for rod opsin are common in mammals and have been shown to be due to multiple signal polyadenylation sites in the 3′UTR (Al-Ubaidi et al., 1990). Our in vitro and in vivo [3H]fucose-labelling studies demonstrate that IRBP is synthesized by the neural retina and secreted into the interphotoreceptor matrix. Fucose was used for these studies because it is present in the oligosaccharide component of mammalian IRBP (Taniguchi et al., 1986). Although this has not been formally demonstrated for IRBPs from other vertebrate classes, Rana pipiens IRBP has been shown to bind concanavalin A (Bridges et al., 1986). In the present study, we used in vitro [3H]fucoselabelling to show that the isolated Xenopus neural retina, but not the pigment epithelium, synthesizes and secretes a protein that has identical electrophoretic mobility to the 124 kDa interphotoreceptor matrix protein, which crossreacts with anti-bovine IRBP immunoglobulins (Fig. 1). We showed by in vivo [3H]fucose-labelling studies employing light and electron microscopic autoradiography that this glycoprotein is normally secreted into the extracellular matrix surrounding the rod outer and inner segments. These metabolic studies, taken together with the western blot analysis, suggest that the homolog of IRBP in Xenopus laevis is synthesized by the neural retina and secreted into the interphotoreceptor matrix. The cell types within the neural retina that border the interphotoreceptor matrix are the photoreceptors and the Muller cells. Which of these two cells is responsible for the synthesis of IRBP cannot be determined from the data presented here. Studies using immunohistochemistry (Chader et al., 1986; Rodrigues et al; 1986; Carter-Dawson and Burroughs, 1992; Gonzalez-Fernandez et al., 1992), mutant rats with photoreceptor specific degeneration (Gonzalez-Fernandez et al., 1985), cell culture systems (Hollyfield et al., 1985a) and in situ hybridization (Veen et al., 1986; Porello et al., 1991; Hessler et al., 1993; Bukelman et al., 1993; Hessler et al., 1993; Wagenhorst et al., 1993) indicate that the photoreceptors and not the Muller cells are responsible for IRBP synthesis. During development, the mRNAs for IRBP and opsin were first detected at stage 40 and increased markedly by stage 45/46. The developing Xenopus neural retina segregates into layers at stage 33/34. The postmitotic cells find

themselves in different microenvironments, leading to adoption of distinct cellular fates (Holt et al., 1988). Outer segments first develop at stage 37/38-40 and reach their adult length by stage 53-54 (Kinney and Fisher, 1978). The up-regulation of these genes coincides with the emergence of the visual cycle in the Xenopus embryo (Azuma et al., 1990; Bridges et al., 1987). In the developing rodent retina, the mRNA for IRBP is up-regulated before that of opsin (Gonzalez-Fernandez and Healy, 1990; Gonzalez-Fernandez et al., 1993; Wang et al., 1992). In the present study, differences in the temporal expression of these two genes may not have been apparent because our Northern blot includes only two time points with photoreceptor gene expression. Furthermore, cellular differentiation is less synchronous in the amphibian retina compared to that of rodents because the former continues to grow throughout life by continual cell division. In situ hybridization studies, in progress in our laboratory, should help to address these issues. We have demonstrated that Xenopus laevis will provide useful information towards understanding the structural requirements of vitamin A-binding and allow future studies to exploit the potential of this system to study the role of IRBP in development. Using a combination of biochemical, genetic and [3H]fucose-radiolabeling techniques, we have demonstrated that the Xenopus neural retina secretes into the interphotoreceptor matrix a soluble 124 kDa glycoprotein homologous to mammalian IRBPs. Our study demonstrates that low-stringency screening may also be useful to isolate cDNAs for proteins homologous to IRBP in species distant from man, and that comparative studies could provide clues to the location of the protein’s ligandbinding domains. Future studies from our laboratory will utilize the cDNA described here to express Xenopus IRBP in order define the specific amino acids involved in creating hydrophobic ligand-binding pocket(s). Xenopus will be particularly valuable in studying the role of IRBP in development, since optic vesicle microinjection provides a way of introducing molecules into the embryonic subretinal space (Gonzalez-Fernandez and Kittredge, 1992). This approach will allow us to study the fate of IRBP and the effect of immunological inactivation of IRBP on eye development. This work was supported by The Thomas F. Jeffress and Kate Miller Memorial Trust (F.G.-F.); grant IN149H from the American Cancer Society (F.G.-F.); an Ophthalmology Research Grant from The Knight’s Templar Eye Foundation Inc. (F.G.-F.); grant T32 NS 7236 from the NINCDS (F.G.-F.); a post-doctoral fellowship from the Fight for Sight Research Division of the National Society to Prevent Blindness (K.L.K.); a development grant from Research to Prevent Blindness; a gift from Elizabeth Jones to the University of Virginia for pediatric eye research; NIH grant EY06675 (R.M.G.) and NSF grant DCB9005468 (R.M.G.); NIH grant EY02633 (J.G.H.); a Retina Research Foundation Grant (J.G.H.), a Senior Investigator Award from Research to Prevent Blindness (J.G.H.) and an award from Alcon Research Institute (J.G.H.).

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characterization of lectin binding sites of retinal photoreceptor outer segments and interphotoreceptor matrix. J. Comp. Neurol. 228, 299-307. Yokoyama, T., Liou, G. I., Caldwell, R. B. and Overbeek, P. A. (1992). Photoreceptor-specific activity of the human interphotoreceptor retinoidbinding protein (IRBP) promoter in transgenic mice. Exp. Eye Res. 55, 225-238. (Received 16 December 1992 - Accepted 25 January 1993)

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Note added in proof In situ hybridization studies performed in our laboratory demonstrate that in the Xenopus eye, IRBP is synthesized uniquely by both the rod and cone photoreceptors (Bukelman et al., 1993; Hessler et al., 1993).