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Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

ABSTRACT: Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling.The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each approximately 300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 microM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites. However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules.Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle.

SUBMITTER: Gonzalez-Fernandez F

PROVIDER: S-EPMC2000878 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

Gonzalez-Fernandez Federico F Baer Claxton A CA Ghosh Debashis D

BMC biochemistry 20070804

<h4>Background</h4>Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were ...[more]

PMID: 17683573

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Project description:Despite decades of investigation, the function of interphotoreceptor retinoid binding protein (IRBP), the most abundant protein in the interphotoreceptor matrix of vertebrates, remains enigmatic. Roles for IRBP in the visual cycle of rod photoreceptors and in the independent visual cycle of cone photoreceptors have been suggested, yet very little is known of the biology of IRBP in cone-dominant retinas, such as those of diurnal birds. Our aim was to identify and characterize expression of the IRBP of the cone-dominant chicken (Gallus gallus domesticus).Chicken IRBP mRNA was identified by PCR cloning. Primary protein structure, genomic organization, and phylogenies were determined through comparative sequence analyses. Expression of IRBP mRNA was characterized by northern analysis and by in situ hybridization on cryosectioned chicken retina. Expression of the IRBP protein was characterized by western blotting and by indirect immunofluorescence on cryosectioned retina and on retinal whole mounts.The chicken IRBP gene encodes a secreted protein with a predicted 1,252 amino acid length. The gene structure for chicken IRBP resembles that of most other vertebrates, with four homologous, modular repeats and introns within only the fourth module. Each module is more homologous with the corresponding module in other species than it is with the remaining chicken modules. Chicken retinal tissue contains a single IRBP mRNA transcript of approximately 4.8 kb and western analysis of chicken retina shows a single major band of 140 kDa. Chicken IRBP mRNA is expressed exclusively by retinal photoreceptor cells and the intensity of the hybridization signal shows light/dark rhythmicity. The IRBP protein is localized to the interphotoreceptor matrix of the chicken retina and to intracellular regions of photoreceptors, with a spatial distribution indicating an association with cone outer segments.The high degree of conservation of IRBP's primary structure, genomic organization, and cell-specific expression within the retinas of all vertebrates examined to date, including those with cone-dominant retinas, implies a conserved role for IRBP in photoreceptor function and/or health. Expression of chicken IRBP and its mRNA are functionally regulated. This report provides a necessary first step to explore a specific function for IRBP in the cone visual cycle.

Project description:The interphotoreceptor retinoid-binding protein (IRBP) gene possesses an unusual structure, encoding multiple Repeats, each consisting of about 300 amino acids. Our goals were to gain insight into the function of IRBP, and to test the current model for the evolution of IRBP, in which Repeats were replicated from a simpler ancestral gene.We employed a bioinformatics approach to analyze IRBP loci in recently completed or near-complete genome sequences of several vertebrates and nonvertebrate chordates. IRBP gene expression in zebrafish was evaluated by reverse transcriptase PCR (RT-PCR) and in situ mRNA hybridizations with gene-specific probes.Patterns of exons and introns in the IRBP genes of tetrapods were highly similar, as were predicted amino acid sequences and Repeat structures. IRBP gene structure in teleost fish was more variable, and we report a new gene structure for two species, the Japanese puffer fish (Takifugu rubripes) and the zebrafish (Danio rerio). These teleost genomes contain a two-gene IRBP locus arranged head-to-tail in which the first gene, Gene 1, is intronless and contains a single large exon encoding three complete Repeats. It is followed by a second gene, Gene 2, which corresponds to the previously reported gene consisting of two Repeats spread across four exons and three introns. Each of the two zebrafish genes is transcribed. Gene 2 is expressed in the photoreceptors and RPE, and Gene 1 is expressed in the inner nuclear layer and weakly in the ganglion cell layer.The tetrapod IRBP gene structure is highly conserved while the teleost fish gene structure was a surprise: It appears to be a two-gene locus with distinct Repeat organization in each open reading frame. This gene structure and gene expression data are consistent with possible neofunctionalization or sub-function partitioning of Gene 1 and Gene 2 in the zebrafish. We suggest that the two-gene locus in teleost fish arose as a consequence of either the known whole genome duplication or single gene tandem duplication.

Dataset Information

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

Publications

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

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