The interphotoreceptor retinoid-binding protein (IRBP) of the chicken (Gallus gallus domesticus)

Abstract

Purpose: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Translated amino acid sequence of chicken IRBP. The protein consists of four modules, each about 300 amino acid residues in length. The corresponding nucleotide sequence repeats are colored red, blue, dark purple, and turquoise. ATG and TAG start and termination codons are orange. The signal secretion sequence is blocked in green. Conserved tryptophan (W) residues with putative ligand-binding function [13] are purple. Glycosylation consensus sequences (N-X-S/T) are indicated in brown. An asterisk (*) indicates the position of the additional 82 amino acids in the jungle fowl IRBP protein. The nucleic acid sequence for chicken IRBP is available in GenBank (AY994153).

Figure 2

Genomic and phylogenetic analysis of chicken IRBP. A: Genomic structure of chicken IRBP compared to human [66] and zebrafish [11] IRBP; exons are represented by boxes and introns are represented by lines. B: Phylogenetic analysis of vertebrate IRBP modules. Unrooted, neighbor-joining tree was prepared in Biology Workbench using amino acid sequence alignments and default parameters for PHYLIP [53,79].

Figure 3

Northern and western blot analysis of chicken IRBP expression. A: Northern analysis of chicken (C) IRBP mRNA reveals a predominant single transcript of about 4.8 kb. B: The Coomassie blue stained SDS-PAGE of soluble interphotoreceptor matrix fractions obtained from chicken and rat retina. C: Western blot comparing chicken (C) and rat (R) IRBPs; autoradiograms of blots from identical unstained gels probed with rabbit anti-X4IRBP (the same “anti-Xenopus IRBP (fourth module)” as used in Figure 5) serum (I) or pre-immune serum (PI) followed by goat [¹²⁵I]antirabbit IgG. Arrowheads point to chicken IRBP (140 kDa) and rat IRBP (144 kDa) bands. Lane “S” contains molecular weight standards.

Figure 4

In situ hybridizations of IRBP and opsins on chicken retinal cryosections. A–C: Sections obtained from chickens sacrificed 2.4 h after light-onset (2.4 h L), hybridized with probes corresponding to IRBP (A), rod opsin (B), and red cone opsin (C). D–G: Sections obtained from chickens sacrificed 7 h after light-onset (7 h L), hybridized with probes corresponding to IRBP (D), rod opsin (E), red cone opsin (F), and a combination of IRBP and rod opsin (G). In G, IRBP expression is visualized with a red color product and rod opsin expression is visualized with a dark color product. The position of the outer limiting membrane (olm) in each image is indicated by thin black line; the outer segments (os), inner segments (is), outer nuclear layer (onl), and inner nuclear layer (inl) are also identified. The scale bar represents 10 μm.

Figure 5

Immunocytochemistry of IRBP. Sections were obtained from chickens sacrificed 7 h after light-onset (7 h L; A) and 6 h after light-offset (6 h D; B), were stained with a polyclonal anti-Xenopus IRBP (fourth module; red fluorescence), and with a monoclonal anti-bovine IRBP (green fluorescence). The position of the outer limiting membrane (olm) in each image is indicated by a thin white line; the outer segments (os) and inner segments (is) are also identified. The scale bar represents 10 μm.

Figure 6

Topographic expression pattern of IRBP. A: Sketch of whole mounted retina showing regions scored for comparative intensity of IRBP immunofluorescence (using the monoclonal anti-bovine IRBP antibody). Three naïve observers scored several, 1 mm² regions. Boxed areas indicate locations of three of these regions and correspond to panels B–D. In B–D, small profiles (arrows) and larger profiles (arrowheads) are specified at higher magification. E–G: Double labeling with anti-IRBP antibody (E) and peanut lectin (F), with merged image in G. IRBP-positive profiles, including smallest profiles (arrows) are also peanut-lectin-positive (arrowheads). Some peanut-lectin-positive profiles are not IRBP-positive (arrowhead with asterisk in G). Scale bar in B represents 10 μm and applies to B–D; scale bar in inset of B represents 5 μm and applies to all insets and E–G.