No major defect detected in the gene of familial hypo-retinol-binding proteinemia

Retinol-binding protein (RBP), a plasma protein with a molecular weight of 21,000 daltons, binds to retinol with a 1:1 molar ratio and transports it to the peripheral tissues. Familial hypo-retinol-binding proteinemia (hypo-RBPnemia) was detected in a 2-year-old girl who developed keratomalacia, and in her mother and sister. They persistently showed half the normal levels of RBP and retinol. Restriction fragments of leukocyte DNA, created by digestion with BamHI, EcoRI and PstI when human RBP complementary DNA was used as a probe, showed the same patterns in both the affected and unaffected family members. These results indicate that familial hypo-RBPnemia could be attributed to such minor changes as point mutations, rather than large deletion or insertion in the RBP gene.     

Interstitial retinol-binding protein and cellular retinal-binding protein in the mammalian pineal

Antibodies against bovine interstitial retinol-binding protein (IRBP) and cellular retinal-binding protein (CRA1BP) were used in immunochemical and immunocytochemical studies of the pineal glands of cattle, hamsters and rats (RCS and RCS-rdy+). On immunoblots, IRBP (Mr 144,000) was identified in cattle, hamster and rat pineal extracts. The abundance of IRBP in bovine pineals was 33 +/- 6 ng.mg-1 (mean +/- SD, n = 12) soluble protein. RCS (Royal College of Surgeons) rat pineals gave a strong IRBP reaction on immunoblots, even when virtually no IRBP could be found in the eye due to photoreceptor degeneration. In the hamster retina IRBP immunostaining was distributed throughout the entire interphotoreceptor matrix and the outer segment layer. The pineal also showed strong IRBP-like immunostaining scattered uniformly throughout the gland. Other hamster brain regions showed no specific immunostaining; however, an immunoreactive protein with the same Mr as IRBP was detected on Western blots of bovine cerebral cortex, spinal cord and brainstem soluble proteins. Immunoreactive proteins at lower Mr were also detected in these tissues. CRA1BP immunoreactivity (Mr about 32,000) was observed in immunoblots of bovine, hamster and rat pineal proteins. These findings suggest that some mammalian pinealocytes are related to the retinal cells that contain CRA1BP (i.e. pigment epithelium, Muller cells) while others are related to the photoreceptors, which synthesize IRBP.     

The lacrimal gland synthesizes retinol-binding protein.

The rabbit lacrimal gland secretes retinol bound to a 20-21 kDa protein. To test the hypothesis that this protein might be retinol-binding protein (RBP) we probed lacrimal gland for RBP mRNA and lacrimal gland fluid for RBP. A rabbit RBP cDNA clone was used to probe rabbit and rat lacrimal gland RNA using RNase protection analysis. The lacrimal gland contains RBP mRNA at a level 0.1 to 0.03% of that observed in the liver. This RBP mRNA was identical to that observed in the liver based on RNase protection analysis, Northern blot analysis and primer extension analysis. The RBP mRNA levels in the lacrimal gland were not altered by the retinol status of the rabbits. We analysed lacrimal gland fluid for RBP by immunoblotting using a monoclonal antibody that recognizes rat, human and rabbit RBP. A single protein band from the rabbit lacrimal fluid bound the antibody, and this protein comigrated with human RBP which also bound the antibody. We conclude that the lacrimal gland contains RBP mRNA and that the lacrimal gland synthesizes and secretes RBP into the lacrimal gland fluid. This is the first demonstration that an extrahepatic tissue containing RBP mRNA synthesizes and secretes the protein in vivo.     

Interstitial retinol-binding protein (IRBP) in subretinal fluid

Antibodies against bovine interstitial retinol-binding protein (b-IRBP) were used to detect human IRBP (h-IRBP) on immunoblots of eight samples of subretinal fluid (SRF) from patients with retinal detachments of between 2 days' and more than 2 years' duration. Using this sensitive technique, it was found that seven of the samples contained h-IRBP in concentrations estimated to range from below 5% up to 19% of normal human IPM. One of these samples displayed two immunoreactive bands of roughly equal intensity, one at a molecular weight of 135,000 (h-IRBP), the other at 115,000. The latter may have been generated by proteolytic cleavage. No h-IRBP could be detected in an eighth sample from a patient with retrolental fibroplasia. It is concluded that the reduced concentration of h-IRBP in SRF may be due to a number of factors that include dilution, proteolytic degradation, and metabolic inactivation of photoreceptors at the detachment site.     

Electron microscopic immunocytochemistry of interstitial retinol-binding protein in vertebrate retinas

Interstitial retinol binding protein (IRBP) is a soluble glycoprotein found in the interphotoreceptor matrix (IPM) and implicated in shuttling retinol between retina and pigment epithelium (PE) cells. The authors have studied the distribution of IRBP by EM immunocytochemistry. Thin sections of Lowicryl K4M embedded R. pipiens, X. laevis, bovine and human retinas were labeled sequentially with affinity purified rabbit antibovine IRBP, biotinyl-sheep antirabbit F(Ab')2, and avidin-ferritin, or with avidin and biotinyl-ferritin. Antigen was in the interphotoreceptor space and intercalated into the narrow spaces between PE cell microvilli. IRBP penetration between PE cells was delimited abruptly by the PE junctional complexes. IRBP was also observed in small vacuoles in the apical cytoplasm of PE cells and in PE cell phagosomes that contained IRBP surrounding ingested rod tips. IPM was heavily but inhomogeneously labeled. Antigen was usually deposited along the ROS and COS plasma membrane in a confluent layer, but sometimes it was distributed in large (ca. 0.2-micron thick) clumps. In bovine and human retinas, the connecting cilium was ensheathed by antigen at high density but an unlabeled halo surrounded its plasma membrane. The apical plasma membrane of the inner segment aligned along the connecting cilium was also densely coated by antigen. In both frog retinas, the ridges of the periciliary ridge complex (PRC) were coated with antigen. In none of the four species examined was Golgi labeling present. In bovine retinas, labeled vacuoles (granules) in the myoid region were found in very low numbers (15 vacuoles in 358 rod cells). Amphibian retinas also contained only small numbers of myoid vacuoles labeled by anti-IRBP. Absence of antibody binding to intracellular sites of synthesis in any of the cells that abut the interphotoreceptor matrix suggests that the antigen may be masked prior to its release from the synthetic cell(s) or that its level is below limits of detection.     

Some functional characteristics of purified bovine interphotoreceptor retinol-binding protein

Interphotoreceptor retinol-binding protein (IRBP) is a unique retinol carrier: it is a large glycoprotein existing only in the interphotoreceptor matrix (IPM), which is the extracellular material situated between and behind the photoreceptors of the neural retina. IRBP from bovine and human eyes carries endogenous retinol, as evidenced by the protein's fluorescence on gel-filtration (Sephacryl S-300) chromatography and on native (non-SDS) pore-gradient polyacrylamide gel electrophoresis. Bovine IRBP's retinol-binding sites are at most one-third occupied in light-adapted eyes and much less in dark-adapted eyes; this bleach dependence is partially reversible and suggests a role in vitamin A transport during the visual cycle. IRBP can be saturated with exogenous all-trans retinol; one ligand molecule binds per protein molecule. IRBP can be isolated rapidly from IPM by affinity adsorption onto con A-Sepharose; this preparation is 94% pure and yields 0.33 mg or 2.4 nmol of IRBP per bovine eye. An apparently homogeneous preparation can be obtained by subsequent passage through a Sepharose 4B column. IRBP is located only in the IPM, which is harvested by isotonic washing of the retina. (In the absence of this rinse to prepare IPM, the protein is found loosely associated with the retina, although not as a cellular or membrane component.) As the thoroughness of the retinal washing procedure is increased, the yield of IPM proteins (including IRBP) goes up; however, the same set of proteins, in the same ratios, always is retrieved, indicating that retinal-cell components do not contaminate seriously the IPM.     

Human interphotoreceptor matrix contains serum albumin and retinol-binding protein

It is usually assumed that IRBP (interphotoreceptor retinoid-binding protein) is the only protein present in the interphotoreceptor matrix (IPM) capable of shuttling visual-cycle retinoids between photoreceptors and the retinal pigment epithelium. However, this laboratory previously presented qualitative evidence (Western blots) that serum albumin is present in human IPM. Furthermore, Ong and coworkers (1994) found that cultured RPE cells synthesize serum retinol-binding protein (RBP) and secrete it, mainly into the apical culture medium, which would correspond to the IPM in intact eyes. As both of these proteins can bind all- trans -retinol and 11- cis -retinal, it was of interest to quantify the amounts of albumin and RBP in human IPM. We used radial immunodiffusion to accomplish this. The average molar ratio of serum albumin to IRBP in these samples was 1.9; that of RBP to IRBP was 0.015. The presence of a high concentration of serum albumin in the IPM in situ was confirmed by the intense immunohistochemical staining seen in sections of fresh human eyes. The human case is not unique; various concentrations of albumin were found in the IPM of all vertebrate species examined (by gel electrophoresis). These results indicate that both serum albumin, because of its very high concentration in the IPM, and RBP, because of its comparatively tight binding to retinoids, need to be considered, along with IRBP, as proteins that may participate in visual-cycle transport. The accompanying paper addresses this concern.     

Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium

The retinal pigment epithelium (RPE) is responsible for transport of retinol from the choroidal circulation to the photoreceptors. In the intact eye, this process is mediated by membrane receptors for plasma retinol binding protein (RBP) distributed basolaterally on the RPE cells. We have shown that cultured human RPE expresses this receptor. A binding curve exhibiting saturation was generated by incubating enzymatically detached epithelial sheets with increasing concentrations of 125I-labelled RBP. 125I-RBP binding experiments also show that the receptor is expressed at a high level in first passage subcultures, suggesting de novo synthesis, and that basally oriented receptors predominate over those associated with the apical surface, reflecting the polarization characteristic of RPE in vivo. Cultured RPE can internalize 3H-retinol carried by RBP, resulting in synthesis of labelled retinyl palmitate. Production of labelled retinyl ester is competitively inhibited when incubations include an excess of holo-RBP containing non-radioactive retinol. These results indicate that RBP not only binds to the receptor specifically, but also that this interaction is functional, effecting uptake of retinol by the RPE cells. The expression of this property of differentiated RPE favors the use of cultured RPE as a model system for studying vitamin A transport and metabolism.     

The distribution of retinol-binding protein and its mRNA in the rat eye

Although a constant supply of retinol is a critical requirement for the visual cycle, the molecular mechanisms underlying retinol delivery, uptake, storage, and transport in the eye are not well understood. Previously the synthesis of serum retinol-binding protein (RBP) in the mammalian eye was reported. Now the distribution of RBP and RBP mRNA in the rat eye has been studied by immunohistochemical and in situ hybridization techniques has been studied. The RBP mRNA was present only in the cytoplasm of retinal pigment epithelial (RPE) cells, terminating abruptly at the pars plana. On the other hand, RBP immunoreactivity was more widespread. The most intense immunostaining was present in retinal ganglion cells, the corneal endothelium, and under certain conditions of tissue fixation, the corneal epithelium. Consistent but less intense immunoreactivity was detected in the photoreceptors, Müller cells, inner plexiform layer, ciliary epithelium and stroma, iris epithelium, retinal pigment epithelium, lacrimal glandular epithelium, and periorbital soft tissues. These findings suggest that RBP synthesized by the RPE may be secreted to various ocular locations. However, at present, uptake from plasma cannot be excluded as another possible source of ocular RBP. In the plasma, holo-RBP (the retinol-RBP complex) is transported in complex with another plasma protein, transthyretin (TTR). This substance is also synthesized by the RPE and its distribution in the eye is similar to that described for RBP. Taken together, these findings support the proposal that ocular RBP and TTR may function cooperatively in the intraocular translocation of retinol.     

Retinoids bound to interstitial retinol-binding protein during light and dark-adaptation

High-performance liquid chromatography was used to determine the types and amounts of retinoids bound to interstitial retinol-binding protein (IRBP) during light- and dark-adaptation in frogs. IRBP was separated from CRBP and CRA1BP by ion-exchange chromatography and quantitated by determining the amount of Serva Blue R dye bound to it in stained sodium dodecyl sulfate polyacrylamide gels. The amount of IRBP was not significantly different in light- and dark-adapted eyes (0.15 +/- 0.05 nmol/eye compared with 0.18 +/- 0.08 nmol/eye). In the dark-adapted state, IRBP bound mainly 11-cis retinol and 11-cis retinal in quantities that summed to about 1 mol/mol IRBP. After the onset of light-adaptation, all-trans retinol increased from its very low dark-adapted level, peaked at 0.2 mol/mol IRBP and then declined to the dark-adapted level again. Concomitantly, the total retinoid bound to IRBP fell, mainly because there was a drop in the amount of 11-cis retinal. During dark-adaptation, the amount of 11-cis retinal increased. No significant changes were seen in the amount of 11-cis retinol in light and darkness. These findings support the hypothesis that when rhodopsin is bleached IRBP transports all-trans retinol from the retina to the pigment epithelium and that it delivers 11-cis retinal to the rod outer segments for rhodopsin regeneration.     

Synthesis and secretion of interstitial retinol-binding protein by the human retina

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein present between the retina and pigmented epithelium, which may function to shuttle vitamin A derivatives between these tissues. While previous studies have shown that the retina is solely responsible for IRBP synthesis, the specific retinal cell(s) in which this occurs has not been established. Since the carbohydrate moiety of IRBP contains fucose, the authors have analyzed the sites of incorporation of 3H-fucose in the human retina in vitro, using autoradiography. Following a 30-min pulse incubation, all retinal layers exhibited incorporation of label; however, the rod photoreceptor inner segments contained one- to two-fold more radioactivity than was present in any other retinal compartment. In autoradiographs of retinas recovered following a 4 hr chase incubation, all retinal layers retained similar levels of radioactivity with the exception of the rod photoreceptors, cone photoreceptors and cells in the inner nuclear layer, which lost 75, 11, and 14 percent, respectively of the radioactivity present immediately following the 30-min pulse. Proteins present in the chase incubation medium were analyzed by polyacrylamide gel electrophoresis and fluorography. The principal labeled component in the chase medium was identified as IRBP by immunoprecipitation with antibovine-IRBP immunoglobulins. Thus, the major loss of 3H-fucose radioactivity from rod photoreceptors coupled with the appearance of 3H-labeled IRBP in the incubation media suggests that the rod photoreceptors are primarily responsible for the synthesis and secretion of IRBP.     

Vitamin A and interstitial retinol-binding protein in an eye with recessive retinitis pigmentosa

The composition and amount of vitamin A stored in the retinal pigment epithelium and choroid (RPE-Ch) was evaluated in postmortem donor eyes from a patient with retinitis pigmentosa that was probably inherited by an autosomal recessive mode. Additionally, the soluble proteins in the neural retina and RPE-Ch cytosols and interphotoreceptor matrix were examined collectively for the presence of interstitial retinol-binding protein (IRBP). Although there was depletion of the amount of vitamin A stored in the RPE, this was commensurate with the histopathologic findings on the RPE extent and thickness. No evidence was found for an accumulation of free retinol. Nearly all of the vitamin A stored in the RPE was esterified. As in normal eyes, the retinyl esters consisted mainly of palmitate mixed with a small proportion of stearate. Eleven-cis retinyl esters were present, although their proportion was lower than that reported for normals. IRBP could not be detected in stained gels of the soluble proteins, or by autoradiography of these gels after treatment with 125I-concanavalin A. These findings suggest that depletion of stored vitamin A, accumulation of free retinol, or deficiency of 11-cis isomer are unlikely to be causative factors in the retinal degeneration examined here. Although the depletion of IRBP seen at this advanced stage might be secondary to the advanced loss of photoreceptors, the authors cannot rule out the possibility that a relative deficiency or abnormality in this protein at earlier disease stages may contribute to the pathogenesis of retinitis pigmentosa.     

Rhodopsin, vitamin A, and interstitial retinol-binding protein in the rd chicken

In order to determine whether blindness in the rd strain of Rhode Island Red chickens is due to a defect in the vitamin A (visual) cycle, spectroscopy, high performance liquid chromatography, and immunochemical techniques were used to compare the amounts of rhodopsin, interstitial retinol-binding protein, and vitamin A compounds in the dark-adapted eyes of homozygous rd and heterozygous carriers. In both groups of chickens, (up to 6 weeks post-hatching) the distribution of stored vitamin A differed from other vertebrates (mammals, amphibians, fish) in that more than half of the retinyl palmitate/stearate occurred in the neurosensory retina. The 11-cis isomer accounted for nearly 100% of the retinyl palmitate/stearate in the neurosensory retinas of both groups. In the pigmented layers (pigment epithelium and choroid) the 11-cis isomer amounted to 70.1 +/- 4.2% in the carrier, and 65.1 +/- 2.9% in the rd birds. With respect to their content of rhodopsin, IRBP, retinyl palmitate/stearate and unesterified retinol, (both 11-cis and all-trans isomers) no significant difference could be demonstrated between the eyes of rd and carrier chickens (3 days and 28 days post-hatching). These results therefore demonstrate that the ocular tissues of rd chickens do not lack IRBP, the putative extracellular transport protein for vitamin A, that these tissues synthesize and store the 11-cis isomer of vitamin A, and that the 11-cis isomer is used to form rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interstitial retinol-binding protein (IRBP) in the RCS rat: effect of dark-rearing

The effect of light- and dark-rearing on the amounts of rhodopsin and interstitial retinol-binding protein (IRBP) in RCS rats and their congenic controls (RCS-rdy+) was determined. Rhodopsin was measured spectroscopically and IRBP by dot-blot enzyme immunoassay utilizing rabbit antibovine IRBP IgG. After P15-20, dark-reared RCS and RCS-rdy+ rats always had more rhodopsin than their light-reared, age-matched counterparts. The rhodopsin in the light-reared RCS rats peaked at about 2 nmol/eye at P20-25. The rhodopsin in the dark-reared RCS rats peaked at about 4 nmol/eye at P60-70. Maintenance of RCS-rdy+ rats in darkness had no effect on their IRBP content, which continued to increase up to P80-110. In both groups of RCS rats, the amount of IRBP reached a peak at P22. In RCS rats maintained in darkness, the amount of IRBP attained at this peak was about twice that in the corresponding light-reared group and in RCS-rdy+ animals at this age. The decline of IRBP after P22 in RCS rats was slowed in darkness by approximately 10 days. This slowed decline of IRBP is associated with a decreased rate of photoreceptor degeneration, and the results are therefore consistent with the hypothesis that the photoreceptors synthesize and secrete IRBP. The layer of membranous debris would restrict the diffusion of IRBP in the subretinal space and could partially exclude this retinol transport protein from access to the zone adjacent to the apical surface of the retinal pigment epithelium (RPE).     

Transthyretin and retinol-binding protein as discriminators of diabetic retinopathy in type 1 diabetes mellitus

International Ophthalmology - Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus (DM), which is still a major reason for blindness. Transthyretin (TTR) and...     

Interactions of retinol-binding protein with various chromophores and with thyroxine-binding protein. A model for visual pigments

The properties of retinol-binding protein from human serum were compared with those of rhodopsin in an attempt to learn more about the role of protein in visual pigments. In the serum, retinol-binding protein (mol wt. 21 000) tightly binds one molecule of all-trans-retinol and one molecule of the tetrameric protein prealbumin (mol. wt. of tetramer, 54 000). Apo-retinol binding protein was found to be completely dissociated from prealbumin under conditions in which native retinol-retinol-binding protein and reconstituted all-trans-retinol-retinol-binding protein were tightly bound. This difference in binding to prealbumin between apo- and holo-retinol-binding protein is due most probably to different conformations of the apo- and holo-protein. By analogy, it is suggested that the incorporation of the retinal chromophore into apo-visual pigment might act as recognition marker for the incorporation of visual pigment into the photoreceptor disc membrane.Apo-retinol binding protein was shown to form stable 1 : 1 molar complexes with retinols, retinals, retinoic acid, retinyl acetate and retinyl oxime, but not with retinyl palmitate. Only the retinol isomer and retinoic acid-retinol-binding protein complexes were bound to prealbumin at physiological ionic strength, while the other chromophores-retinol·binding protein complexes were not. The various chromophores were all bound to the same site on the retinol-binding chromophore, the binding was noncovalent and irreversible under normal physiological conditions.All the chromophores-retinol-binding protein complexes showed a large induced optical activity of the chromophore absorption band upon binding to the protein. The rotatory strength of the circular dichroism bands of the various chromophore retinol-binding protein complexes was of the same order of magnitude as that of rhodopsin. The binding of retinol and retinal to bovine serum albumin did not produce optical activity in the chromophore absorption band.     

Normal interaction of plasma retinol-binding protein from retinitis pigmentosa with bovine pigment epithelium.

The interaction between retinol-binding protein and normal bovine pigment epithelium has been studied with the use of iodinated retinol-binding protein isolated from the plasma of patients with the recessive form of retinitis pigmentosa and of normal subjects. It is concluded that the capacity of the plasma carrier protein to interact with the retinol-binding protein receptor of bovine pigment epithelium is unimpaired in retinitis pigmentosa with autosomal recessive inheritance.