Phosphorylation of interphotoreceptor retinoid-binding protein (IRBP)

The phosphorylation of interphotoreceptor retinoid-binding protein (IRBP), the major soluble (glycolipo) protein of the interphotoreceptor matrix (IPM) and a putative intercellular retinoid-transport vechicle, has been examined in a crude bovine IPM wash using [γ-³²P]ATP. SDS-polyacrylamide gel electrophoresis and autoradiography, size-exclusion high-performance liquid chromatography (HPLC) and ion-exchange HPLC all showed IRBP to be phosphorylated in this system. The phosphorylation probably is of serine and/or threonine residues rather than of tyrosine. Interestingly, phosphorylated IRBP was bound tightly to concanvalin A (Con A)-Sepharose and was not eluted by 50 mM α-methyl-d-mannoside indicating a marked alteration in binding characteristics upon phosphorylation.     

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP)

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.     

Butyrate enhances the synthesis of interphotoreceptor retinoid-binding protein (IRBP) by Y-79 human retinoblastoma cells

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) from Y-79 human retinoblastoma cells was investigated using immunocytochemistry and SDS-polyacrylamide gel electrophoresis. Indirect immunofluorescence of cells growing in monolayer culture for 11 and 13 days showed no significant IRBP staining although by SDS-polyacrylamide gel electrophoresis, a small amount of IRBP was detected in the culture medium, suggesting synthesis and extracellular secretion. Butyrate (2mM) treatment of cells starting on the eighth day of culture resulted in a dramatic increase of IRBP fluorescence 3-5 days after treatment. Treatment of cells in all conditions with 1 microM monensin for 3 h showed concentration of IRBP in the Golgi apparatus of about 10-20% of cells as proved by a double immunofluorescent technique, employing anti-IRBP antibody and wheat-germ agglutinin. Incubation of cells with either radiolabeled amino acids or glucosamine followed by analysis of cell cytosol and culture medium by SDS-polyacrylamide gel electrophoresis also confirmed that 1) IRBP is synthesized by the Y-79 cells and secreted into the medium and 2) its production is markedly increased by butyrate treatment. The enhancement of IRBP synthesis by butyrate suggests biochemical differentiation of Y-79 cells possibly into photoreceptor-like cells and offers a new system for studying the properties of this unique retinoid-binding protein and of factors that control its synthesis and secretion.     

Interphotoreceptor retinoid-binding protein (IRBP)

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retinal pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.     

Carotenoids as possible interphotoreceptor retinoid-binding protein (IRBP) ligands: A surface plasmon resonance (SPR) based study

Uptake, transport and stabilization of xanthophylls in the human retina are important components of a complex multistep process that culminates in a non-uniform distribution of these important nutrients in the retina. The process is far from understood; here, we consider the potential role of interphotoreceptor retinoid-binding protein (IRBP) in this process. IRBP is thought to facilitate the exchange of 11-cis-retinal, 11-cis-retinol and all-trans-retinol between the retinal pigment epithelium (RPE), photoreceptors and Müller cells in the visual cycle. Structural and biochemical studies suggest that IRBP has a variety of nonequivalent ligand binding sites that function in this process. IRBP is multifunctional, being able to bind a variety of physiologically significant molecules including fatty acids in the subretinal space. This wide range of binding activities is of particular interest because it is unknown whether the lutein and zeaxanthin found in the macula originate from the choroidal or retinal circulations. If from the choroidal circulation, then IRBP is a likely mediator for their transport across the interphotoreceptor matrix. In this report, we explore the binding interactions of retinoids, fatty acids, and carotenoids with IRBP using surface plasmon resonance (SPR)-based biosensors. IRBP showed similar affinity toward retinoids and carotenoids (1–2 μM), while fatty acids had approximately 10 times less affinity. These results suggest that further studies should be carried out to evaluate whether IRBP has a physiologically relevant role in binding lutein and zeaxanthin in the interphotoreceptor matrix.     

Synthesis of interphotoreceptor retinoid-binding protein (IRBP) by monkey retina in organ culture: effect of monensin

Whole monkey retinas were incubated in short-term organ culture with either radiolabeled amino acids or glucosamine. Soluble retinal proteins and proteins in the culture medium were analyzed by SDS-poly-acrylamide gel electrophoresis. Fluorography showed that the interphotoreceptor retinoid-binding protein (IRBP), a 146,000 Mr glycoprotein localized in the extracellular matrix, is synthesized by the neural retina and rapidly secreted into the medium. Secretion is blocked by 10-5M monensin. No significant IRBP synthesis was observed in the pigment-epithelium-choroid complex. IRBP is thus the major component synthesized and secreted by the neural retina into the interphotoreceptor space. This, and its affinity for retinoid makes it a prime candidate for an extracellular retinoid transport vehicle.     

Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures

A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.     

Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.     

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) by isolated normal and rd mouse retinal photoreceptor neurons in culture

Cultures of dissociated retinal neurons and photoreceptors from homozygous wild-type, heterozygous rd/+ and homozygous rd/rd retinas have been used to investigate the capacity of isolated photoreceptor cells to synthesize and secrete the interphotoreceptor retinoid-binding protein (IRBP). Retinal cells were dissociated on postnatal day 2 and grown in chemically defined medium in the absence of glial and pigmented epithelial cells. Expression of IRBP immunoreactive materials in these cultures was cell type-specific and developmentally regulated. Thus increasing numbers of rod photoreceptor cells showed immunoreactivity during the first week in culture, whereas nonphotoreceptor cell types remained consistently negative. Photoreceptor immunoreactivity could be detected in permeated (detergent-treated) as well as in unpermeated preparations, the latter suggesting that some IRBP is associated with the photoreceptor cell surface. These materials appeared to be loosely bound to the photoreceptors, since they disappeared when the cultures were exposed for 6 hr to IRBP-free medium but not when they were exposed to IRBP-containing medium. IRBP synthesis and secretion could be demonstrated by analyzing either cell extracts or conditioned medium by "slot blot" and Western blot techniques using affinity purified antibodies against bovine IRBP as well as by fluorographic analysis after metabolic labeling of the cultures with 35S-methionine. Comparisons of cultures from the different genotypes showed many similarities, including the abundance of IRBP-immunoreactive photoreceptors in 7 day cultures. However, immunochemical analysis showed lower conditioned medium/cell extract IRBP ratios in rd/rd cultures, an observation consistent with previous reports suggesting that IRBP secretion may be deficient in rd/rd photoreceptor cells.     

Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promotor and first exon is linked to expression of the gene.

The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene.     

Reduced level of interphotoreceptor retinoid-binding protein (IRBP), a possible cause for retinal degeneration in the Abyssinian cat

Summary Retinae of Abyssinian cats homozygous for a retinal degeneration gene, and normal controls, have been investigated using antibodies directed against opsin, transducin (TD-), S-antigen (48K protein), interphotoreceptor retinoid-binding protein (IRBP), and cone outer segments. IRBP-immunoreactivity (IR) is much reduced at stage 2 of the disease in affected retinae; later massive photoreceptor cell death occurs. In cats, at a late stage of the disease, the retina exhibits few S-antigen-IR cells in the peripheral part of the retina whereas, in the central part, some patches of cells exhibiting opsin-IR, TD--IR, and S-antigen-IR are present in remnants of the outer nuclear layer (ONL). No IRBP-IR is detectable at this stage. The form and size of the majority of these remaining cells, however, does not resemble that of normal photoreceptors. No, or only rudimentary, inner and outer segments are present; long bifurcating basal protrusions often occur. These cells, which could be remains of cone elements, are S-antigen immunoreactive. Double labelling for different retina-specific proteins reveals a co-localization of opsin, TD- and S-antigen in some, but not all, remaining photoreceptor elements. Cells exhibiting opsin-IR also show TD--IR and S-antigen-IR located within the entire cell and its protrusions. In control retinae and retinae at early stages of the disease, immunoreactions are comparable with all antibodies used. However, TD--IR is less intensive in the photoreceptor terminals. S-antigen-IR cones are most frequently present in the peripheral retina. Reduction of IRBP at an early stage of the disease could be one of the factors leading to photoreceptor cell death at later stages.     

Repeated determinants within the retinal interphotoreceptor retinoid-binding protein (IRBP): immunological properties of the repeats of an immunodominant determinant.

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes in the retina and pineal gland, induces inflammatory changes in these organs (EAU and EAP, respectively) when injected into various mammals. We have previously identified a determinant (residues 1169-1191) in bovine IRBP which is immunodominant and highly immunogenic and immunopathogenic in Lewis rats. IRBP exhibits a fourfold repeat structure and we report here on the comparison between the active sequence 1179-1191 and its three repeat peptides. Only one of the repeats, 271-283, cross-reacted with 1179-1191 and exhibited immunodominance, albeit of a low level. Peptide 271-283 was also immunogenic and immunopathogenic in Lewis rats, but with a minimal dose approximately 100 times higher than that of 1179-1191. Peptide 880-892, a nondominant determinant, resembled 271-283 in its immunogenicity, but was markedly less immunopathogenic. No immunological activity was detected in the fourth repeat peptide, 579-591. Peptide 1179-1191 was superior to the other repeats also in its antigenicity, i.e., the capacity to stimulate presensitized lymphocytes in culture: the minimal stimulatory concentrations of 1179-1191 was greater than 1000 times lower than those of 271-283 or 880-892. Furthermore, 1179-1191 was stimulatory at concentrations lower than those of 271-283 even when tested with lymphocytes sensitized against 271-283. A correlation was also found between the immunological activities of the repeat peptides and their amphipathicity. This study thus identifies two new immunopathogenic determinants of IRBP and provides additional data to show the association between immunodominance of peptides and their various immunological activities.     

The presence of a soluble interphotoreceptor retinol-binding protein (IRBP) in the retinal interphotoreceptor space

A new, gentle technique has been developed for washing of the retinal interphotoreceptor space (IPS) to obtain soluble components of the extracellular matrix (ECM). Using this method, we have determined that the major soluble coustituent of monkey IPS is a 146,000 M_r glycoprotein, which binds [³H]retinol, sediments on sucrose gradients at 7S and has an R_f of 0.42 on native gel electrophoresis. Using size-exclusion high performance liquid chromatography, the apparent molecular weight of the native protein was calculated to be 250,000 daltons. In contrast to previous studies, no 15,000-dalton cellular retinol-binding protein (CRBP) or 33,000-dalton cellular retinaldehydebinding protein (CRALBP) was observed in the IPS wash, indicating that these proteins are probably not involved in retinol transport between retina and pigment epithelium (PE). In the supernatant fraction of retinal homogenates that contains soluble intracellular proteins as well as extracellular constituents, the 146,000 M_r protein was closely associated with a 93,000 M_r protein that could be separated on SDS-gel electrophoresis; the 93,000 M_r protein was not found in the IPS wash. The 146,000 M_r interphotoreceptor retinol-binding protein (IRBP) may function in extracellular retinol transport in the IPS.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Size and shape of bovine interphotoreceptor retinoid-binding protein by electron microscopy and hydrodynamic analysis

Individual molecules of interphotoreceptor retinoid-binding protein (IRBP), a protein likely to be important in the visual cycle, were visualized by means of electron microscopy. IRBP was coated with a very thin layer of tungsten and photographed by dark-field imaging. IRBP is seen to be a flexible, elongated molecule about 24 nm in length by 3-4 nm in width (statistical modes). These dimensions agree very well with those calculated from the frictional ratio obtained from sedimentation data. Approximately half of these rod-shaped IRBP molecules are straight, and half are bent in the middle, usually with an angle of 60-90 degrees between the two arms. A representation of IRBP as a bendable string of beads yields calculations of dimensions and of hydrodynamic parameters consistent with the electron microscopic and sedimentation data; the sedimentation coefficients derived from this representation are nearly insensitive to molecular bending. When IRBP is bound to saturating amounts of its endogenous ligands, all-trans- or 11-cis-retinol, its sedimentation behavior is unchanged, and the same types of particles are visualized by electron microscopy as with the free protein; however, a greater proportion of the molecules are bent. Deglycosylation of IRBP (with peptide:N-glycosidase F) results in a somewhat smaller molecule that retains its rod-like shape, as shown by gel filtration and sedimentation data. The results indicate that IRBP is an elongated molecule and suggest that a structural change may occur upon ligand binding.     

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.     

Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone

We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.     

Uveitogenic potential of lymphocytes sensitized to interphotoreceptor retinoid-binding protein

In our previous study rats immunized with bovine retinal interphotoreceptor retinoid-binding protein (IRBP) were found to develop inflammation in the eye and the pineal gland. This inflammatory disease was distinct in several aspects from experimental autoimmune uveoretinitis (EAU) induced by the retinal S-antigen (S-Ag). The current study examined the adoptive transfer of IRBP-induced EAU. We established that lymphocytes from IRBP immunized donor rats were capable of transferring EAU after in vitro stimulation with either IRBP (lymph node or spleen cells) or concanavalin A (spleen cells only). Recipients of these cells developed uveoretinitis and pinealitis identical to the actively induced disease. As compared with the S-Ag system, recipients of IRBP sensitized cells developed disease earlier, and smaller numbers of cells were needed to transfer EAU. Development of inflammation was directly related to a cellular response to the specific retinal antigen used for sensitization. Moreover, the unique nature of ocular inflammation was reestablished in the IRBP system: high proportions of polymorphonuclear leukocytes were found in the inflamed tissue of certain recipients despite a lack of a humoral response to the specific antigen. In contrast to the eye, only mononuclear leukocytes comprised the inflammation in the pineal gland.