Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene.

We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

The effect of retinoids and butyrate on the expression of CRX and IRBP in retinoblastoma cells.

We sought to determine whether differentiation agents such as retinoids and butyrate regulate transcript levels of interphotoreceptor retinoid binding protein (IRBP) and cone rod homeobox (CRX), a homeodomain transcription factor that regulates IRBP promoter activity. WERI-Rb1 retinoblastoma cells were treated with all-trans retinol, all-trans retinoic acid, or butyrate. IRBP and CRX mRNA levels were determined by quantitative RT-PCR. Butyrate at low concentrations increased both mRNA levels but suppressed them at higher concentrations. Retinoic acid had minimal effects. Retinol increased CRX mRNA over four fold. IRBP and CRX transcript levels are sensitive to butyrate and CRX expression is sensitive to retinol.

Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells.

PURPOSE: To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity. METHODS: Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT-PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs. RESULTS: Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT-PCR products were obtained for all target sequences. CRX RT-PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected. CONCLUSIONS: The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT-PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.

Experimental autoimmune uveoretinitis in mice (Biozzi ABH and NOD) expressing the autoimmune-associated H-2A(g7) molecule: identification of a uveitogenic epitope.

To determine whether Biozzi ABH (H-2A(g7)) mice were susceptible to chronic experimental autoimmune uveoretinitis (EAU). Biozzi ABH were immunized with the two retinal antigens, interphotoreceptor retinoid binding protein (IRBP) and soluble antigen (S-Ag). Biozzi ABH mice were found to be susceptible to EAU induction with native bovine IRBP. Recombinant protein domains were used to identify IRBP domain 2 (EcR2) as the uveitogenic domain. Histopathological examination indicated that EcR2-induced disease was of a chronic, non-destructive nature in the Biozzi ABH. Using synthetic overlapping peptides corresponding to EcR2, a uveitogenic and immunogenic epitope was identified corresponding to human IRBP511-530. Non-obese diabetic (NOD) mice share the same MHC class II (H-2A(g7)) molecule as the Biozzi ABH, and were also found to be susceptible to EAU induction with EcR2. This study has identified a novel mouse model of EAU, whereby disease is of a chronic, non-destructive nature, which has potential to be used in immune manipulation and neuroprotection studies.

Evidence of a tissue-restricting DNA regulatory element in the mouse IRBP promoter.

The expression of interphotoreceptor retinoid binding protein (IRBP) is limited to photoreceptor cells of the retina and pinealocytes of the pineal gland. We sought to define cis-elements of the mouse IRBP 5' flanking region that are required for this restricted activity. In vitro transient transfections of retinoblastoma and neuroblastoma cells and in vivo experiments with transgenic Xenopus laevis indicate that -1783/+101 and -156/+101 IRBP gene fragments directed expression predominantly to the retina and pineal, with minor neuronal expression elsewhere. In contrast, a -70/+101 fragment was less restrictive in controlling expression, exhibiting activity not only in retina, but also in forebrain, hindbrain, spinal cord, and motor neurons innervating gills.

Extra-hepatic expression of serum albumin mRNA in mouse retina.

PURPOSE: In some mammals, serum albumin protein exists in the interphotoreceptor space (IPS), the space between photoreceptor cells and the retinal pigment epithelium. Serum albumin is synthesized largely in the liver, though low levels of extra-hepatic expression have been documented in several other tissues, including fetal rat kidney, pancreas, lung, and heart. The purpose of this study was to investigate whether serum albumin protein and mRNA are found in mouse retina. METHODS: Using albumin rabbit antibodies and HRP goat anti-(rabbit IgG), we performed immunoassays on mouse IPS washes to detect the presence of serum albumin protein. Protein extracts from IPS washes were subjected to Affigel Blue chromatography. This resin has an affinity for serum albumin. Reverse transcription-polymerase chain reaction (RT-PCR) of retina total RNA was performed to search for albumin mRNA. Also, real-time reverse transcription polymerase chain reaction (RT-RT-PCR) was employed to look at the levels of expression in different age groups. RESULTS: A constituent of the IPS washes specifically bound and eluted from Affigel Blue column, suggesting that the washes contained serum albumin. SDS PAGE revealed that the size of the constituent was 67 kDa, the size of serum albumin. This 67 kDa band reacted with mouse serum antibody. An RT-PCR amplified fragment of serum albumin mRNA from retina displayed the expected size. The sequence of this fragment is identical to authentic serum albumin cDNA sequence. RPE and choroid were negative for serum albumin mRNA. However, rd1(-)/rd1(-) retina was positive, suggesting that at least some serum albumin is synthesized in the inner layers of the retina. RT-RT-PCR showed that serum albumin mRNA levels in whole retina reached a maximum at about postnatal day 15 and gradually decreased to about one-sixth of maximum at 12 months age. CONCLUSIONS: Serum albumin protein and mRNA were found in mouse IPS and retina, suggesting that the protein is synthesized in the retina. The previously demonstrated ability of serum albumin to bind fatty acids and retinoids and its presence in the mouse IPS suggest a role for serum albumin in transporting retinoids in the retina or IPS, especially at young ages when concentrations appear greatest.

Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5' flanking regions in WERI, Y79, chick retina cells, and transgenic mice.

PURPOSE: To determine the sequences of the mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell types using both quantitative and statistical analyses. METHODS: We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region. RESULTS: The 5' flanking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base sequence about 1.25 to 1.50 kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells. CONCLUSIONS: Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.

Prediction of structural and functional relationships of Repeat 1 of human interphotoreceptor retinoid-binding protein (IRBP) with other proteins.

PURPOSE: We compared the structure and function of interphotoreceptor retinoid-binding protein (IRBP) related proteins and predicted domain and secondary structure within each repeat of IRBP and its relatives. We tested whether tail specific protease (Tsp), which bears sequence similarity to IRBP Domain B, binds fatty acids or retinoids, and whether IRBP possessed protease activity resembling Tsp's catalytic function. These tests helped us to learn whether the primary sequence similarities of family members extended to higher order structural and functional levels. METHODS: Predictions derived from multiple sequence alignments among IRBP and Tsp family members and secondary structure computer programs were carried out. The first repeat of human IRBP (EcR1) and Tsp were expressed, purified, and tested for binding properties. Tsp was examined for fluorescence enhancement of retinol or 16-anthroyloxy-palmitic acid (16-AP) to test for ligand binding. IRBP was tested for protease activity. RESULTS: Tsp did not exhibit fluorescence enhancement with retinol or 16-AP. IRBP did not exhibit protease activity. The positions of critical residues needed for the ligand binding properties of retinol were predicted. Primary sequence and three-dimensional similarity was found between Domain A of IRBP Repeat 3 and eglin c. CONCLUSIONS: The sequence similarity of Tsp and IRBP raised the possibility that each might share the function of the other protein: IRBP might possess protease activity or Tsp might possess retinoid or fatty acid binding activity. Our studies do not support such a shared function hypothesis, and suggest that the sequence similarity is the result of maintenance of structure. The finding of similarity to eglin c in Domain A suggests the possibility of a tight interaction between Domain A and Domain B, possibly implying the need for Domain A in retinoid-binding, and suggesting that both Domains should be present in testing mutations. The positions of predicted critical amino acids suggest models in which a large binding pocket holds the retinoid or fatty acid ligand. These predictions are tested in a companion paper.

Effects of dispersed point substitutions in Repeat 1 of human interphotoreceptor retinoid binding protein (IRBP).

PURPOSE: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor retinoid-binding protein (IRBP). First, we predicted important functional amino acids by several computer programs. We also noted the lack of shared functions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues. We analyzed the effects of point substitutions on the retinol and fatty acid binding properties of Repeat 1 of human IRBP at 25 and 50 degrees C. METHODS: To find residues critical to retinol binding that might affect function, a series of thirteen mutations were created by site-specific mutagenesis between positions 140 and 280 in Repeat 1 of human IRBP. These mutants were expressed, purified, and tested for binding properties. The conformations of the proteins were examined by circular dichroism (CD) scans. RESULTS: Seven of the mutations exhibited reduced binding capacity, and five were not expressed at high enough levels to assess binding activity. Four of the mutants were purified, and their CD scans were very similar to those of Repeat 1. Only one of the mutations did not affect binding, folding, or expression when compare to wild type Repeat 1. CONCLUSIONS: Several IRBP mutants containing point mutations retained native structure but lost retinol binding function. The data suggest that retinol binding is affected by many different amino acid substitutions in or near a binding pocket. That even a single point substitution can profoundly affect binding without affecting overall conformation suggests that much of Domain B (from amino acid positions 80 to 300) is involved with ligand binding. This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol can bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat.

G239T mutation in Repeat 1 of human IRBP: possible implications for more than one binding site in a single repeat.

PURPOSE: Interphotoreceptor retinoid-binding protein(IRBP) is a four-repeat protein found in the interphotoreceptor space. Each repeat can bind retinoids and fatty acids. The purpose of this study was to examine the effects of the single amino acid substitution, G239T, versus the wild type sequence of human IRBP Repeat 1, on ligand binding at equilibrium, ligand off rates, and protection of retinol from degradation. METHODS: G239T was created by site-specific mutagenesis, expressed in E. coli, and purified. E. coli expressed wild type Repeat 1 (EcR1) and G239T were subjected to thermal denaturation and analyzed by circular dichroism spectroscopy. We compared the ligand binding properties by fluorescence enhancement of retinol and 16-anthroyloxy-palmitate, tryptophan quenching of the proteins by different ligands, binding competition assays, protection of retinol from degradation, and stopped-flow kinetics to measure transfer of ligands to and from model membranes. RESULTS: Circular dichroism, fluorescence, and absorbance spectroscopy of G239T and EcR1 showed similar wavelength scans. G239T exhibited about three-fold less fluorescence of bound all-trans-retinol or 13-cis-retinol versus EcR1. Retinol quenching of intrinsic protein fluorescence was reduced by 37% in G239T versus EcR1. Other retinoids used as quenchers produced no difference between intrinsic protein fluorescence of either G239T or EcR1; all exhibited saturable high affinity binding to each protein. Docosahexaenoic acid (DHA) served as a competitive inhibitor of retinol fluorescence enhancement with EcR1. However, DHA did not alter retinol fluorescence with G239T. 16-anthroyloxy-palmitate (16-AP) exhibited about 30% higher levels of fluorescence enhancement when bound to G239T versus EcR1. EcR1 prevented oxidative damage of all-trans-retinol, whereas G239T provided much less protection. Each protein could accept 9-cis-retinal from small unilamellar vesicles (SUVs) as measured by stopped flow kinetics. Off rates were the same in comparing G239T and EcR1 as acceptors. CONCLUSIONS: Despite the general similarity in shape between G239T and EcR1 and the nearly identical binding behavior with some ligands, distinct differences exist in the ligand binding properties of G239T and EcR1. Fluorescence enhancement/quenching and retinol protection experiments suggest that retinol binding is reduced by about 50% in G239T versus EcR1. The data suggest that either: (1) EcR1 contains two binding sites for retinol and G239T has lost one site or (2) EcR1 has a single binding site that is altered in G239T to reduce retinol binding. Results of all the experiments were consistent with the first model while some of the data were not consistent with the second model. Thus, it is possible that position 239, found in Domain B2 of IRBP Repeat 1, is located in or near one of two retinol binding sites.

Site-specific DNA hypomethylation permits expression of the IRBP gene.

Interphotoreceptor retinoid binding protein (IRBP), a putative component of the visual cycle, is expressed selectively in the retina and pineal gland. This study examined whether site-specific DNA hypomethylation plays a role in this expression regulation. Southern blotting of HpaII and MspI digests of DNA from various bovine and murine tissues (whole brain, retina, pineal gland, superior colliculus, cortex, thymus, habenular nucleus, cornea, liver, tail, and kidney) revealed that specific CpG dinucleotides in the IRBP gene promoter are hypomethylated in DNA from retinal photoreceptor cells and pineal gland compared to DNA from other tissues. These sites are methylated in DNA from non-photoreceptor retinal cells. Exogenous methylation of these sites diminished DNA:protein binding in electrophoretic mobility shift assays. HpaII methylation of chloramphenicol acetyltransferase reporter constructs suppressed IRBP but not SV40 promoter activity in transiently transfected primary cultures of embryonic chick retinal cells. These data indicate that specific cytosines in the bovine and murine IRBP promoters are unmethylated in photoreceptive cells but methylated in other tissues. This differential DNA methylation may modulate IRBP gene expression since exogenous methylation of the murine sites suppresses reporter gene transcription, apparently by inhibiting DNA:protein binding events.

Transgenic expression of an immunologically privileged retinal antigen extraocularly enhances self tolerance and abrogates susceptibility to autoimmune uveitis.

Interphotoreceptor retinoid-binding protein (IRBP) is an immunologically privileged retinal antigen that can elicit experimental autoimmune uveitis (EAU). The nature and extent of tolerance to immunologically privileged self antigens is poorly understood. To investigate whether transgenic expression of IRBP extraocularly enhances tolerance and protects from EAU we prepared mice that express half of the mouse IRBP gene, containing a potent uveitogenic epitope (residues 161 - 180), under control of MHC class II promoter. Transgene mRNA was detectable in many tissues. Transgenic protein was undetectable by conventional assays, but was detected in thymic tissue by lymphocyte proliferation assay after induction of the promoter. Transgenic mice challenged with p161 - 180 did not develop EAU and had reduced immunological responses, but remained susceptible to EAU induced by whole IRBP, that contains additional uveitogenic epitopes. Disease was also induced by wild type T cells specific to p161 - 180. Thus, extraocular expression of a privileged retinal antigen enhances self tolerance, supporting the notion that sequestration contributes to immune privilege. Exceedingly low levels of transgene expression result in tolerance that is both profound and epitope specific, implying anergy or deletion of the endogenous uveitogenic repertoire. The same level of expression is, however, insufficient to tolerize wild-type effector T cells in the periphery.

Structure-function relationships in the four repeats of human interphotoreceptor retinoid-binding protein (IRBP).

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds hydrophobic ligands in the interphotoreceptor space. Human IRBP consists of 1230 amino acids in four 300 amino acid long repeats. We asked: 1. Whether each of the four repeats can bind retinoids or fatty acids, 2. Whether each repeat can prevent retinol degradation in aqueous solutions, 3. Whether a ligand can stabilize the protein from thermal denaturation, 4. Whether the four repeats can be further classified into two groups. Our rationale was to make each repeat from the human cDNA and then examine structural and functional characteristics. METHODS: Individual repeats were produced in E. coli and the whole protein was expressed in baculovirus. Binding properties with all-trans-retinol were characterized by ligand fluorescence enhancement. The quenching of protein fluorescence by retinol, 9-cis-retinal, all-trans-retinoic acid, beta-ionine, alpha-ionine, trans-parinaric acid, and DHA was also examined. Binding curves were analyzed by nonlinear regression. Prevention of retinol decomposition was measured by absorption spectroscopy. Circular dichroism was examined in the far UV range to study protein secondary structure and the near UV range to study ligand binding effects on the tryptophan environment. RESULTS: Temperature dependent denaturation suggests that EcR1 is the most stable of the four repeats. Each repeat possesses the capability of binding 9-cis-retinal, all-trans-retinol, all-trans retinoic acid, docosahexaenoic acid, alpha- and beta-ionine, and trans-parinaric acid. Protein fluorescence quenching by retinol and retinol fluorescence enhancement assays yielded similar binding parameters for each repeat. Each expressed repeat prevents the degradation of retinol in aqueous solutions. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds both retinoids and analogs, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Together, the results suggest that each repeat retains all functions of the whole protein. However, there are distinguishing characteristics among the repeats in their ligand binding properties, though the four repeats cannot be classified into just two distinctive groups. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.

The 5' flanking regions of IRBP and arrestin have promoter activity in primary embryonic chicken retina cell cultures.

Primary cultures of embryonic chicken cells from various tissues were transiently transfected with plasmid vectors containing reporter genes linked to a 1.8 kb fragment of the mouse interphotoreceptor retinoid-binding protein (IRBP) 5' flanking region, a 1.5 kb fragment of the mouse arrestin 5' flanking region, or a 3.4 kb sequence of the bovine arrestin 5' flanking region. Promoter activity was evident in retina-derived cells, but not in fibroblasts or cells from whole brain. Transfection response also varied with transfection method, plasmid DNA concentration, post-transfection incubation time, and cell density. The data suggest that the primary embryonic chicken retinal cell culture system is a useful tool in studying photoreceptor-specific gene regulation.

A major cis activator of the IRBP gene contains CRX-binding and Ret-1/PCE-I elements.

PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) is expressed exclusively and to high levels in photoreceptive cells. This study was an attempt to delineate the minimal regulated control region of the murine IRBP promoter involved in this expression pattern. METHODS: Fragments of the mouse IRBP 5' flanking region were tested for promoter activity in transient transfections of embryonic chick retina cells in primary culture. Electrophoretic mobility shift assays were used to identify specific cis-acting DNA elements within these fragments. RESULTS: Nested deletion analysis of a 1783 bp fragment of the murine IRBP 5' flanking region shows that high promoter activity is maintained with truncated fragments as short as 70 bp 5' to transcription start, but is lost with truncation to 45 bases. The 1783 bp promoter is active in cultures of retina cells but not brain cells or fibroblasts. The 70 bp fragment is active in retina and brain cells but not fibroblasts. Within retina cell cultures, the 1783 bp fragment is active in photoreceptor-like and amorphous or unidentifiable cells whereas the 70 bp is additionally active in multipolar neuron-like cells. The -70 to -45 interval contains Ret-1/PCE-I (AATTAG in the IRBP gene), a proposed retina-specific consensus sequence cis element, and a same-strand reversed copy of this sequence, GATTAA, the consensus binding element of the photoreceptor-specific trans-acting factor CRX. Mutation of either element suppresses promoter activity. Paralleling promoter tissue-specificity, the -70 to -45 fragment binds a sequence-specific protein complex found in retina and brain extracts but not fibroblasts. Mutation of both or either element inhibits this binding. CONCLUSIONS: These data suggest that a trans-acting complex binds a cis-element in the -70 to -45 sequence. This binding fully activates transcription but confers only partial tissue-specificity to IRBP gene expression.

Structure-function relationships in interphotoreceptor retinoid-binding protein (IRBP).

PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) binds hydrophobic ligands in the retina. The polypeptide consists of 1230 amino acids in four 300 amino acid long repeats. We asked whether each of the four repeats can bind one retinoid or fatty acid analog. Our rationale was to make protein variants from the human cDNA bearing one or more of the repeats and examine binding capacities and dissociation constants. METHODS: Proteins were characterized by SDS-PAGE, western blotting, N-terminal sequencing, and CD spectroscopy. Binding properties with all-trans-retinol and 16-anthryloxy-palmitic acid (16-AP) were characterized by ligand fluorescence enhancement and curve fitting. RESULTS: Binding capacities varied according to the length of each protein. Each repeat possesses the capability of binding retinol and 16-AP. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds a retinoid and fatty acid analog, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.

Expression of retina-specific genes by mouse retinoblastoma cells.

PURPOSE: Two cell lines derived from ocular tumors of a transgenic mouse expressing the SV40 large T antigen have been established as models of human retinoblastoma. One line, TM, originated from a metastasis, and the other, TE, originated from the primary tumor. The authors compared these two lines with the normal adult mouse eye by analysis of the expression of five photoreceptor cell-specific proteins: IRBP, opsin, rod- and cone-specific transducins, and S-antigen. The authors sought to determine which of these proteins was expressed qualitatively and to examine semi-quantitatively for changes in the levels of expression in the cell lines. METHOD: Western blot analysis was used to detect photoreceptor-specific intracellular or secreted proteins. Total RNA was prepared from cultured cells or from mouse adult whole eye. Specific messenger levels in total RNA were determined either by northern hybridization analysis or by a semi-quantitative polymerase chain reaction (PCR), coupled to complementary DNA (cDNA) substrates prepared from total RNA. RESULTS: IRBP was present in the retinoblastoma cell lines and secreted into the medium. Neither S-antigen nor opsin were detectable by immunoblotting. IRBP and cone transducin mRNA were present in both cell lines. In contrast, opsin, rod transducin, and S-Antigen mRNAs were not detectable by PCR. beta-actin was present in the mRNA populations of whole eye and retinoblastoma. SV40 large T antigen mRNA was present only in retinoblastoma cells. CONCLUSIONS: IRBP and cone transducin expression in mouse retinoblastoma cells is independent of signaling provided directly or indirectly through large T antigen or Rb105 regulatory cascades. The pattern of photoreceptor-specific gene expression is similar to that seen in human retinoblastoma cell lines. These murine-derived cell lines may be useful as a tool to study IRBP and cone transducin expression in vitro and to determine early retinoblast expression patterns in the mouse.

Biochemical and biophysical properties of recombinant human interphotoreceptor retinoid binding protein.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds and transports retinoids and fatty acids in the interphotoreceptor space (IPS). To understand the relationship between the protein structure and its functions requires bulk quantities of human IRBP. The authors sought to produce recombinant human IRBP (rhIRBP), a perfect duplicate in amino acid sequence of the authentic human protein. This material could serve as a supply of the protein and later could be used to make mutants of the protein. The goals of the present study were to produce human IRBP in an expression system and to examine some of its biochemical properties. METHODS: A cDNA encoding human IRBP was cloned into the transplacement vector, pVL1392, and the plasmid was recombined with linearized baculovirus on cotransfection into Sf9 cells. Viruses containing the human IRBP cDNA were identified by polymerase chain reaction analysis. IRBP was secreted from virus-infected insect cells. rhIRBP was purified from cell medium and was examined by chromatography, N-terminal protein sequencing, immunologic techniques, and fluorometry. Eyecup and retina washes of human donor eyes provided a source of authentic human IRBP (IPS-IRBP). RESULTS: rhIRBP and IPS-IRBP exhibit similar elution profiles on concanavalin A, ion-exchange, and size exclusion chromatography. rhIRBP contains a five-amino-acid propeptide at the N-terminus as deduced from the cDNA sequence. Retinol binding of rhIRBP has been characterized by fluorometric titration. The dissociation constant is approximately 1.04 microM, close to that reported for bovine IRBP. By scanning fluorometry, the emission and excitation maxima are 479 nm and 339 nm, respectively. CONCLUSIONS: The baculovirus system provides an excellent method to produce and secrete human IRBP. The recombinant protein can be readily purified from cell culture medium. Its behavior in chromatography and in binding studies suggests that the recombinant protein is virtually identical to the authentic protein. This validates its use in place of IRBP from human donor eyes. Small, but significant, differences in biochemical properties in comparing human and bovine material highlight the significance of studying the human protein.

Light deprivation profoundly affects gene expression of interphotoreceptor retinoid-binding protein in the mouse eye.

Ambient light appears to play a role in regulating gene and protein expression of interphotoreceptor retinoid-binding protein (IRBP), a protein that facilitates the transport of retinoids between the neural retina and pigment epithelium in the visual cycle. Pregnant CD-1 mice were placed in the dark approximately 48 hr before parturition, and the pups were reared for 14 days under these conditions. Control animals were reared on a 12 hr light/12 hr dark cycle. Northern blotting of total RNA isolated from whole mouse eyes at post-natal days 7-14 (P7-P14) showed a marked reduction in IRBP message in the light-deprived animals to 10-20% of levels in control animals. Reprobing of the blots for opsin and S-antigen message showed a significant decrease of about 80-90% in opsin message at 5.1 kb but no change in S-antigen message in the eyes of light-deprived mice. Light microscopic examination of the light-deprived mouse retinas showed no apparent abnormalities in morphological development and immunocytochemistry demonstrated normal distribution and levels of IRBP protein. Immunochemical quantitation of IRBP protein confirmed that there was no reduction in light-deprived as compared to normal mouse eyes. Similarly, when adult mice were light-deprived for 14 days, a marked reduction in IRBP message was also observed with no decrease in the amount of IRBP protein. Thus, light deprivation causes a large decrease in IRBP message in the mouse eye, but IRBP protein is not decreased. The dramatic effect of light deprivation on IRBP mRNA and some opsin mRNAs, but not on S-antigen message and the fact that IRBP protein levels are relatively unaffected, suggest a complex pathway of light regulation of photoreceptor function previously not encountered. This may involve regulatory controls at levels including gene transcription, mRNA stability or protein degradation that may make use of a feedback control mechanism involving light- or dark-dependent signal transduction.