Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas.

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) was quantitatively assessed in retinas of normal and rd mutant mice using short-term organ culture with [35S]methionine. Retinas were studied at ages P9-P12, time points prior to and immediately after the onset of the degeneration of the rd retina. Soluble proteins of the retinal pellet and the incubation medium were subjected to SDS-polyacrylamide gel electrophoresis. Analysis of labeled protein bands utilized a radioactivity scanning system to quantify [35S]methionine incorporation into newly synthesized IRBP. The synthesis and secretion into the incubation medium of IRBP by rd mouse retinas was comparable to normal retinas at P9-P10 but decreased by more than 50% by P12. IRBP mRNA levels were evaluated in retinas of normal and rd mice ages P7-P14. Although IRBP mRNA expression increased in the rd mouse through P10, it decreased markedly thereafter. Previously reported immunocytochemical studies suggested that IRBP was not secreted in the rd mouse retina. The results of this study indicate, however, that rd mouse retinas, when removed from the eye, have the capacity to synthesize and secrete IRBP.     

Interphotoreceptor retinoid-binding protein (IRBP) in the postnatal developing rds mutant mouse retina: EM immunocytochemical localization

The distribution of IRBP was examined in postnatal developing retinas of rds (020/A) mutant mice and Balb/c controls by EM immunocytochemistry. Light labeling for IRBP was detected in mutant and control retinas by postnatal day 9 (P9) largely in the interphotoreceptor matrix (IPM). At P14, some photoreceptors in the rds retina showed a higher density of label in the Golgi for IRBP than neighboring cells and those of controls processed simultaneously. This high density of label for IRBP was observed also in the Golgi of a small population of photoreceptor cells at P18, P19 and P21 in rds retinas. These cells were found to represent approximately 3-5% of the photoreceptor population. The density of label for IRBP at the apical RPE region was obviously low in the rds retinas by P18, P19 and P21. However, this same region in controls of the same ages was densely labeled for IRBP. The low density of labeling at the apical RPE region in the rds retinas may indicate a change in the rate of synthesis, secretion, distribution and/or degradation in the IPM. The high density of intracellular labeling in a small population of cells may be indicative of impaired secretion, an increase in IRBP synthesis or the initiation of photoreceptor deterioration. Whether the differences observed in the distribution of IRBP in the rds mutant are primary or secondary effects of the genetic lesion remains undetermined.     

Interphotoreceptor retinoid-binding protein (IRBP) is rapidly cleared from the Xenopus interphotoreceptor matrix

The interphotoreceptor matrix (IPM) is a highly-organized extracellular matrix critical to retinal development and function. Although the concentrations of its components are carefully regulated, little is known about the mechanisms of this regulation. Interphotoreceptor retinoid-binding protein (IRBP) is the most abundant soluble protein component of the IPM. Although its rate of clearance is thought to be an important factor regulating the concentration of IRBP within the IPM, no study has measured the rate of its extracellular turnover. Here we determine the rate of turnover of matrix IRBP in Xenopus. The rate of IRBP turnover was estimated by measuring the loss of radioactivity from protein labeled by a single injection of a radiolabeled protein precursor. To provide an estimate of the rate of IRBP turnover, we have examined the following issues: (1) Quantitative extraction of IRBP from the IPM for biochemical analysis. (2) Routes of delivery of radiolabeled precursor to achieve a pulse label in vivo. (3) Selection of labeled precursor in order to minimize reutilization of radiolabel. Using Western blot analysis, immunoprecipitation and immuno-electron microscopy, we found that IRBP can be quantitatively extracted from the IPM by a simple saline wash. IRBP was radiolabeled by systemic or intravitreal injection of either [35S]methionine or carboxyl-terminal labeled [1-14C]leucine. The specific activity of matrix IRBP was determined by either phosphorimaging or fluorography of Coomassie blue-stained SDS-polyacrylamide gels. Intravitreal injection of tracer was more effective than systemic delivery in achieving a pulse of radiolabel to the retina. This may be due to intravitreal injection allowing the body to act as a 'sink' for radiolabeled amino acid. When radiolabeled precursor was delivered by intravitreal injection, the calculated half-life of matrix IRBP using [35S]methionine was 25. 6+/-0.82 hr; in contrast, it was 10.7+/-2.9 hr using [1-14C]leucine. The faster apparent IRBP turnover using [1-14C]leucine is interpreted in context of the early decarboxylation of leucine during its degradation. Our results demonstrate rapid turnover of IRBP in the Xenopus IPM in vivo and suggest that the IPM is a dynamic structure undergoing continuous renewal.     

Differential distribution of interphotoreceptor retinoid-binding protein (IRBP) around retinal rod and cone photoreceptors

Light microscopic immunogold cytochemistry was used to examine the distribution of interphotoreceptor retinoid-binding protein (IRBP) in the interphotoreceptor matrix (IPM) surrounding rod and cone photoreceptors. Silver enhancement of retinas reacted with anti-IRBP antibodies using the two stage labeling procedure showed dense staining of the IPM around rod photoreceptor outer segments and the apical region of the RPE. However, the IPM around cone photoreceptors was lightly labeled for IRBP. This region of light labeling extended from the RPE to the distal one-fourth to one-third of the cone inner segment. Although most of the area surrounding cone outer segments was lightly labeled, a dense band of label was seen along the margins of cone outer segments. This study confirms that heterogeneity exists in the distribution of IRBP in the IPM and provides new evidence that IRBP is not homogeneously distributed around rod and cone photoreceptors.     

Photosensitized light-induced damage of IRBP (interphotoreceptor retinoid-binding protein): effects on binding properties

PURPOSE: To determine if IRBP (interphotoreceptor retinoid-binding protein) is damaged following irradiation by visible light in the presence of bound all-trans retinal. METHODS: Following irradiation of the IRBP-all-trans retinal complex, the retinal was removed and damage to IRBP measured as loss of titratable thiol groups, loss of tryptophan fluorescence, and changes in retinol-binding-induced fluorescence. RESULTS: IRBP irradiated by itself showed only minimal loss of tryptophan fluorescence; this loss was substantially increased by irradiation in the presence of all-trans retinal. Thiol groups and retinol-binding activity were also shown to be reduced. The damage to IRBP seemed to involve photosensitization by the all-trans retinal, which was in turn protected from bleaching by the IRBP. The binding affinity was shown to be reduced ten-fold following irradiation. CONCLUSION: In the eye, IRBP can stabilise vitamin A and debatably may be responsible for transport of different forms of vitamin A between the photoreceptor cells and pigment epithelium. If this is the case, it would play a key role in rhodopsin regeneration after bleaching. IRBP also appears to be necessary to sustain photoreceptor cells. Light was shown to cause photosensitized damage to IRBP, and thus might impair the regeneration process and photoreceptor viability.     

mRNA for interphotoreceptor retinoid-binding protein (IRBP): distribution and size diversity in vertebrate species

Northern blots of total retinal RNA from a number of different vertebrate species were probed with a cDNA fragment corresponding to translated portions of bovine IRBP mRNA. A hybridizing band was detected in normal human retina (4.6 kb), Y-79 human retinoblastoma cells (4.4 kb), and in retinas of monkey (4.6 kb), guinea-pig (4.9 kb), mouse (6.1 kb), rat (two bands at 5.4 kb and 6.6 kb), rabbit (6.3 kb), cow (6.5 kb), and hamster (7.6 kb). Thus, the IRBP gene is expressed in the retinas of a wide variety of mammalian species. The mRNAs could be readily detected in about 20 micrograms of total RNA, suggesting that, in these species, IRBP message is relatively abundant. In contrast, only very weak hybridization was detected on northern blots of the three bird species examined (chicken, duck, quail). IRBP mRNA is thus relatively well expressed in mammals, but not in birds.     

Photoreceptor-specific activity of the human interphotoreceptor retinoid-binding protein (IRBP) promoter in transgenic mice.

In order to define the cellular specificity of the interphotoreceptor retinoid-binding protein (IRBP) promoter in the retina, we linked the human IRBP promoter to the beta-galactosidase (lacZ) gene and made five lines of transgenic mice. In three of the five transgenic mouse lines, retinas showed positive staining upon incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Mice from one line (OVE278B) showed positive X-gal staining throughout the retina except for the most peripheral regions. Interestingly, the staining was heterogeneous throughout the retina. Heavily stained regions were interspersed with lightly stained areas. Mice in two other lines showed highly mosaic X-gal staining patterns. Histological examination demonstrated that staining was confined to photoreceptor cells in all three expressing families. Furthermore, electron microscopy showed that the promoter is active in both rod and cone cells. Our results demonstrate that the human IRBP promoter can be used to obtain photoreceptor-specific gene expression in transgenic mice.     

The functional unit of interphotoreceptor retinoid-binding protein (IRBP)--purification, characterization and preliminary crystallographic analysis.

To define the relationship between the structure and function of interphotoreceptor retinoid-binding protein (IRBP) we seek to prepare crystals of IRBP suitable for X-ray crystallographic analysis. As recent studies suggest that each of IRBPs four homologous regions or modules possess ligand-binding activity, we here explore the feasibility of preparing crystals from an individual module. Xenopus laevis IRBP, which has a similar four-module structure as that of mammalian and avian IRBPs, was selected for these studies in view of the advantage of the Xenopus retina for cellular and transgenic approaches. In the present study we focused on the second module of Xenopus IRBP. This module was expressed as a thioredoxin/histidine-patch fusion protein to promote its soluble expression in Escherichia coli and subsequent purification. The ligand-binding properties of the fusion protein were determined by fluorescence spectroscopy. For the preparation of crystals, the module was enzymatically separated from the fusion tag. Crystals of the native and selenomethionine derivatized module were prepared by vapor diffusion in hanging drops. Module II of IRBP binds 1.57 +/- 0.041 and 1.49 +/- 0.15 equivalents of at all- trans retinol and 9-(9-anthroyloxy) stearic acid, respectively, with KDs in the 0.1 microM range. Crystals of this module had an elongated rectangular beam-like morphology. A complete dataset of a frozen selenomethionine crystal extending to 1.85 A resolution was collected. Focusing on the individual modules will likely provide an effective strategy to correlate biochemical and structural data to define the functional domains of IRBP. The quality and resolution of the data obtained suggests that it will be possible in the near future to solve the X-ray crystal structure of the IRBP modules.     

Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina.

Differentiation and maturation of the photoreceptor outer segments are key steps in the development of the visual system. Morphological studies presented here show that the cow and human are nearly identical in the timing of outer segment appearance during fetal development, implying that the bovine retina is a good model system for the final stages of human photoreceptor development. To study photoreceptor maturation, rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) were quantified by ELISA in a developmentally staged series of fetal bovine retinas. In addition, their localization within these retinas was determined by immunogold electron microscopy. Rhodopsin, as detected by antibodies directed against either the N- or C-terminal portions of the molecule, is first found at about 5.5 months gestation. It is first detected on the plasma membrane of the immature cilia and on the earliest emergent outer segment membrane, even before organized disk membranes are apparent. In contrast, whereas rhodopsin levels and outer segments are nearly undetectable before 5 months gestation, IRBP accumulates to a significant level (4-5% of the adult) as early as 3 months gestation. Immunogold electron microscopy confirmed this finding, with localization of IRBP predominantly in the subretinal space.     

Suppression of interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis by pretreatment with anti-idiotypic monoclonal antibody]

In an experimental model of interphotoreceptor retinoid binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) in Lewis rats, EAU onset was suppressed by pretreatment with anti-idiotypic antibody (TRD3). In the pretreated group, the titers of anti-IRBP antibody and idiotypic antibody were lower and the titer of anti-idiotypic antibody was higher than in the control group, and the skin test (DTH) against IRBP was also suppressed. Histopathological findings of the control group showed destruction of retinal outer granular layer, loss of the outer segment and lymphocytic infiltration, although no definite inflammatory signs appeared in the pretreated group. We suppose that the mechanism of idiotypic suppression is via induction of idiotype-specific or idiotype-related suppressor T cells.     

Coordination between production and turnover of interphotoreceptor retinoid-binding protein in zebrafish

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), which is secreted by the photoreceptors of most vertebrates, is the major soluble protein component of the interphotoreceptor matrix (IPM). Recent studies suggest that IRBP is short lived in the IPM (half-life, approximately 11 hours). The mechanisms coordinating the production and removal of IRBP are not known. Zebrafish provide a useful system to study the regulation of these two processes, because its IRBP mRNA levels are under circadian regulation. In the present study, the relationship between the quantity of IRBP, the rate of its turnover, and the expression of its mRNA in the zebrafish retina were examined. METHODS: Full-length zebrafish IRBP was expressed in Escherichia coli and an antiserum generated against purified recombinant IRBP. Western and protein dot blot analyses and indirect immunofluorescence were used to define the temporal and spatial patterns of IRBP expression in the adult zebrafish. In vivo and in vitro metabolic labeling experiments were used to examine the regulation of IRBP turnover by both environmental light and the light-dark cycle. RESULTS: Despite the known rhythmicity in IRBP mRNA expression, neither the amount of IRBP nor its localization changes significantly during the light-dark cycle. IRBP is rapidly removed from the zebrafish eye (half life, approximately 7 hours). This rapid turnover is independent of environmental lighting conditions during subjective day and is more rapid during the day than at night. CONCLUSIONS: Because the amount of IRBP remains constant throughout the day, the enhanced daytime IRBP mRNA expression may function to compensate for an increased turnover of the protein during the day. These findings suggest that the processes of IRBP production and removal are coordinately regulated.     

Proteoglycans in the mouse interphotoreceptor matrix. III. Changes during photoreceptor development and degeneration in the rds mutant

The distribution of sulfated proteoglycans in the interphotoreceptor matrix (IPM) was examined during development and degeneration of photoreceptors in the rds mouse with electron microscopy after staining with the cationic dye Cupromeronic Blue (CmB). Three distinct CmB-positive filaments types were observed: type A (45-55 nm long and around 5 nm in diameter), type B (up to 0.5 micron long and 5-10 nm in diameter), and type C filaments (up to 1 micron long and 15-25 nm in diameter. During early postnatal development, before degenerative changes occur in photoreceptors, CmB-positive filaments were virtually identical in morphology and pattern of development as those recently reported for the normal mouse IPM (Tawara, Varner and Hollyfield, 1989, Exp. Eye Res. 48, 815-39). From 10 days to 1 year of age, during the period of progressive degeneration and loss of photoreceptor cells, numerous type B and type C filaments were present in the IPM. Type B filaments were distributed throughout the IPM, whereas type C were predominantly located around the apical termination of photoreceptor inner segments and between the pigment epithelial microvilli. Type A filaments were located principally in the apical cytoplasm of the pigment epithelial cells and in the proximal IPM. In the 20-month-old rds mouse, a time when virtually no photoreceptor cells remain, only minimal CmB staining was evident at the interface between the pigment epithelium and retina. Pretreatment with chondroitinase AC eliminated most CmB-positive filaments from the 18-day-old and 20-month-old mouse IPM. These findings suggest that there are no major differences in structural type or early postnatal development of chondroitin sulfate-type proteoglycans in the IPM between rds and normal mice. Any differences in distribution of chondroitin sulfate-type proteoglycans between rds and normal mice can be accounted for by the absence of photoreceptor outer segments and progressive loss of photoreceptor cells in this mutant. The disappearance of these IPM proteoglycans following photoreceptor degeneration suggests that photoreceptors may be critically involved in the maintenance of these matrix components.     

Interphotoreceptor retinoid-binding protein (IRBP) in progressive rod-cone degeneration (prcd)--biochemical, immunocytochemical and immunologic studies

Interphotoreceptor retinoid-binding protein (IRBP) is synthesized and secreted by photoreceptor cells and is thought to facilitate the transport of retinoids during the visual cycle as well as fatty acids essential to the maintenance of normal outer segment membranes. Proteins such as IRBP, which are unique to the photoreceptor cells in the retina, are prime candidates in the consideration of biochemical defects which could contribute to photoreceptor cell degeneration in man and animals. In this study, the association between IRBP and retinal degeneration was examined using the progressive rod-cone degeneration (prcd) mutant retina in dogs as an animal model. This study shows that loss of IRBP is not an early occurrence in prcd. IRBP is present in relatively normal amounts and distribution even at 1.7 years of age, a time when there is extensive visual cell disease and degeneration. By 2.7-3.0 years of age, IRBP loss correlates with the severity of the disease and concomitant loss of photoreceptor cells. IRBP immunoreactivity was present in the interphotoreceptor matrix (IPM) as long as inner segments were present to a significant degree. The late loss of IRBP immunoreactivity seems to be, therefore, the result of advanced degeneration and end-stage atrophy of the retina. In addition, immunological studies were carried out in order to examine the possible role of an autoimmune response against IRBP in the disease cascade. Normal, heterozygote and prcd-affected dogs had measurable antibody titers to IRBP, but there was no correlation between disease state and antibody levels.     

Immunocytochemical localization of interphotoreceptor retinoid-binding protein in developing normal and RCS rat retinas

Interphotoreceptor retinoid-binding protein (IRBP) was localized immunocytochemically in developing normal and RCS rat retinas. IRBP was present in normal and RCS neural retinas on the day after birth (postnatal day 2, P2) to P8 in the space between the neuroblastic layer and the retinal pigment epithelium (RPE). The presence of IRBP prior to the development of outer segments (OS) suggests that OS formation is not linked temporally with IRBP secretion. On P10, staining was confined to the interphotoreceptor space with an intense band of label adjacent to the RPE. This staining pattern persisted in normal rats throughout development and until P18 in RCS rats. On P18, anti-IRBP staining in the RCS was spread evenly throughout the OS layer with no intense band of label adjacent to the RPE and after P18, there was decreased staining with anti-IRBP. On P45 and later, no staining of the RCS retina was found with anti-IRBP. Immunoblots of normal and RCS retinas corroborated the results from immunocytochemical staining. These findings suggest that IRBP may be synthesized in the photoreceptors, but is not abnormal in amount or distribution prior to onset of retinal degeneration in the RCS rat.