XB-ART-22636J Cell Sci May 1, 1993; 105 ( Pt 1) 7-21.
Interphotoreceptor retinoid-binding protein (IRBP), a major 124 kDa glycoprotein in the interphotoreceptor matrix of Xenopus laevis. Characterization, molecular cloning and biosynthesis.
We have demonstrated that the neural retina of Xenopus laevis secretes into the extracellular matrix surrounding the inner and outer segments of its photoreceptors a glycoprotein containing hydrophobic domains conserved in mammalian interphotoreceptor retinoid-binding 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) lambda Zap 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.
PubMed ID: 8360278
Article link: J Cell Sci
Genes referenced: grb10 rbp3
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|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).|
|Fig. 2. Sequence analysis of Xenopus IRBP clone XenIRBP.B1. The clone was isolated by low-stringency screening of a lZapII 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.|
|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. 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.|
|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.|
|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.|
|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.|
|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.|
|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.|
|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.|
|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.|