An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis.

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.

N-terminal sequence homologies in interstitial retinol-binding proteins from 10 vertebrate species.

We report here the first comprehensive comparative NH2-terminal sequence studies of interstitial retinol-binding protein (IRBPs) from nine mammals (including cattle) and one amphibian. This study has revealed that in many species the N-terminus of IRBP includes a 3-6 amino acid extension. IRBP possessing this leader sequence is sometimes mixed with IRBP from which this sequence has been excised.

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.

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.

Interstitial retinol-binding protein (IRBP) in retinoblastoma.

Interstitial retinol-binding protein (IRBP) is an extracellular glycoprotein that appears to be synthesized by the photoreceptors in the normal retina and may be involved in shuttling retinoids through the interphotoreceptor matrix between the retina and pigment epithelium. The present work demonstrated immunochemically that IRBP of the same molecular weight as normal human IRBP (135,000) was present in three retinoblastomas, irrespective of their degree of differentiation. Tumor 1 was classified as differentiated; Tumors 2 and 3 were classified as poorly differentiated. The level of IRBP in the soluble proteins of Tumor 2 was about 8 times that in Tumor 1 and about one-half that in the soluble proteins (including adhering interphotoreceptor matrix) from a pair of normal retinas from a 31-year-old donor. IRBP occurred in the interstitial space of Tumor 3. Most of the IRBP in this tumor was recovered from the medium in which the undifferentiated cells were dispersed. Incubation of the isolated cells from Tumor 3 in medium containing [(3)H]leucine demonstrated that [(3)H]IRBP was secreted into the medium. The [(3)H]IRBP was immunoreactive with rabbit antibovine IRBP antibodies. It was inferred that the [(3)H]IRBP was glycosylated because it was bound by immobilized concanavalin A and could be displaced with ?-methylmannopyranoside. Since IRBP is not normally found in retinas until the time of photoreceptor differentiation, regulation of its gene may be defective in this malignant neuroectodermal neoplasm. The present findings are relevant to the possible role of retinoids and their binding proteins in neoplastic cells, because they demonstrate for the first time the presence of an extracellular retinoid-binding protein in tumor tissue.

An extracellular retinol-binding glycoprotein in the rat eye-characterization, localization and biosynthesis.

We have identified and partially purified interstitial retinol-binding protein (IRBP) from the subretinal space of the rat. It appeared to be glycosylated. Its apparent mol. wt was 270,000 by gel-filtration and 144,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rat IRBP cross-reacted with anti-bovine IRBP sheep and rabbit sera, bound all-trans-[15-(3)H] retinol and was bound by concanavalin A. IRBP was not detected in the cytosols of the neural retina or retinal pigment epithelium and choroid. This distribution was confirmed by immunocytochemistry using a fluorescence-labeled second antibody. Immunospecific fluorescence was most intense in the interphotoreceptor matrix in a 6.5 ?m band adjacent to the retinal pigment epithelium. It was less intense over the remainder of the rod outer segment layer and was comparatively faint over the inner segment region. Its occurrence in the interstitial space between the photoreceptors and retinal pigment epithelium coupled with the fact it bound all-trans-[15-(3)H] retinol supports the concept that it may be implicated in the transport of retinoids between the retina and the retinal pigment epithelium during the visual cycle. When incubated with [(3)H]leucine or [(3)H]glucosamine, isolated retinas (but not retinal pigment epithelium and choroid) secreted labeled IRBP into the medium. This suggests that the retina plays a role in regulating the amount of IRBP in the subretinal space.

Varieties of rhodopsin in frog rod outer segment membranes: analysis by isoelectric focusing.

Purified ROS membranes from cattle, R. pipiens and R. catesbeiana adults and tadpoles were investigated by analytical and preparative isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The rhodopsin from single specimens of R. pipiens displayed two closely-spaced bands with Mr at 34.7 and 37.0 k on SDS polyacrylamide gels. Both were found to be phosphorylated when prepared from retinas incubated with 32Pi and then exposed to light. When purified ROS membranes were solubilized in octyl glucoside and examined by isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis, the low-Mr component focused in two bands (I, IIa) at pH 8.8 and 8.1. Band I and a trace amount of band IIa were observed if band I was eluted and refocused. The high-Mr component focused in one band (IIb) at pH 8.0. Identical isoelectric focusing patterns were observed with ROS membranes from R. catesbeiana tadpoles and the dorsal and ventral retina areas from R. catesbeiana adults. Bovine rhodopsin, on the other hand, had a single Mr component and focused in a major band at pH 6.2.

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.

Bovine interstitial retinol-binding protein (IRBP)--isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA.

Three clones for b-IRBP were isolated by anti b-IRBP screening of two bovine retina libraries in the expression vector lambda gt11. The cDNA inserts were then used as hybridization probes to screen and isolate three more clones in a bovine retina library in the non-expression vector lambda gt10. The six overlapping clones generated a b-IRBP cDNA sequence of 3400 nucleotides. An open reading frame encoded the complete amino acid sequences of 8 of the 35 b-IRBP tryptic peptides purified in the present study. One tentative glycosylation site was identified. The coding region was followed by TAG translation terminating codon and an untranslated stretch of about 1700 nucleotides that ended in a sequence containing a presumptive AATAAA polyadenylation signal that was 18 nucleotides upstream from a 10 nucleotide oligo(A) tract. The coding region for b-IRBP would be expected to be 3300 bp long, but Northern blot hybridization experiments performed with bovine retina polyadenylated RNA and probes containing part of the coding region established that the mRNA for b-IRBP consisted of a major species of about 6300 bp, and a minor species of 5200 bp. In vitro translation of bovine retina polyadenylated RNA in a rabbit reticulocyte lysate system yielded an immunoreactive protein that was comparable in size with nonglycosylated, mature IRBP, showing that it is not synthesized from a large precursor, and supporting our finding that the mRNA contains an extensive non-coding region.

Development of the interphotoreceptor matrix in Xenopus laevis.

Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.

Proteoglycans in the mouse interphotoreceptor matrix. VI. Evidence for photoreceptor synthesis of chondroitin sulfate proteoglycan using genetically fractionated retinas.

To determine the role of photoreceptors in the synthesis of chondroitin sulfate proteoglycan (CS-PG) present in the interphotoreceptor matrix (IPM), 35SO4(2-) was used as a tracer for comparison of proteoglycans synthesized in vitro in the absence of the pigment epithelium by normal retinas and retinas from retinal degeneration (rd) mice at stages before and after photoreceptor degeneration. Isolated retinas from 10 day post-partum (P-10) pups, adult normal mice (C57BL/6J ++/++) and retinal degeneration mice (C57BL/6J rdle/rdle) were incubated for 7 hr with 35SO4(2-) to label newly synthesized sulfated proteoglycans. At P-10, rd retinas have not undergone extensive photoreceptor degeneration, whereas in the adult retinas from this strain, only a few cone photoreceptors remain. At the termination of the labeling period, proteoglycans in the incubation medium and those remaining in guanidine hydrochloride (GuHCl) extracts of the retina were analysed separately and identified by their susceptibility to enzymatic or nitrous acid depolymerization. At P-10, no significant differences were observed in the types or sizes of newly synthesized proteoglycan in normal and rd retinas. Medium samples from P-10 retinas contained near equal amounts of 35S-labeled CS-PG and heparan sulfate proteoglycan (HS-PG), while in GuHCl extracts, approximately 90% of the 35SO4(2-) was incorporated into HS-PG, with the remainder found in CS-PG. Comparisons of adult tissue revealed a divergence of proteoglycan synthesis profiles. Retinas from normal adults label predominantly CS-PG. [35S]proteoglycan from normal retina incubation medium was approximately 96% CS-PG, and GuHCl extracts were about 73% CS-PG. From adult rd retinas these values were 18 and 10%, respectively. Per retina, this shows the rd retinas labeling less than 4% of the medium CS-PG, and about 50% of the GuHCl extractable CS-PG compared to normal retinas. Labeled HS-PG comprised about 28% of the normal retina GuHCl extracts, but was not detected in the incubation medium. In contrast, HS-PG synthesis accounted for about 76% of the medium proteoglycan label, and about 85% of the extracted proteoglycan in the adult rd retina. In fact, 35SO4(2-) labeling of HS-PG in the rd retina GuHCl extracts exceeded by 1000% the level observed in normal retina extracts on a per retina basis. Retinas from both strains incorporate significant amounts of 35SO4(2-) into proteins with rd achieving higher specific activity. IRBP was identified as a 35SO4(2-) labeled protein by immunoadsorption from aliquots of the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)

A serum-free defined medium for retinal pigment epithelial cells.

Human and bovine RPE cells underwent changes in morphology and culture doubling times when passaged in serum-supplemented medium (CM). Furthermore, late passage human RPE cells subcultured in CM medium increased synthesis of three acidic, 43 000-63 000 D proteins. In order to provide a controlled environment for the study of RPE cells in vitro, we have developed a method for growing human and bovine RPE in a serum-free defined medium (DM). RPE cells grown in DM required a 24 h pretreatment with CM to allow the cells to attach and spread on the substrate. Cells grown in DM retained an epithelioid morphology, a stable culture doubling time, and similar 2-D PAGE patterns through several subculturings.

The carbohydrate of bovine interstitial retinol-binding protein.

Pronase digestion of IRBP yielded one major glycopeptide (IRBP-GP1) of approximate Mr 2,500 IRBP-GP1 was heterogeneous by anion exchange chromatography, an observation that was attributed to the presence of varying numbers of sialic acid residues. Asialo-IRBP-GP1 was also heterogeneous by gel-filtration chromatography, possibly because of varying degrees of fucosylation. A minimum of four (possibly five) concanavalin A-binding glycopeptides was generated by cyanogen bromide cleavage of IRBP. These findings, in conjunction with earlier observations, suggest that bovine IRBP contains 4-5 N-linked oligosaccharide chains. These chains appear to have the same basic complex biantennary structure. They contain galactose, mannose and N-acetylglucosamine, in addition to sialic acid and fucose. Nearly all of the IRBP secreted by bovine retinas incubated with (3H)-leucine in the presence of tunicamycin was nonglycosylated and did not bind to concanavalin A. On SDS polyacrylamide gels non-glycosylated IRBP had an Mr that was 5,000-6,000 below that of the glycosylated protein. Non-glycosylated IRBP had an Mr of 250,000 on gel-filtration columns, a value that was identical with that of glycosylated IRBP. It is concluded that the oligosaccharide has little influence on the molecular conformation of IRBP, which is believed to be responsible for the anomalously high Mr seen on these columns. When mixed, the glycosylated and nonglycosylated forms of IRBP appeared to associate as stable aggregates. Bovine retinas incubated with (14C)-leucine and (3H)-fucose in the presence of castanospermine, an inhibitor of glucosidase I, secreted IRBP that appeared to have a reduced number of fucose residues. Swainsonine, an inhibitor of mannosidase II, effected only a marginal reduction in the degree of fucosylation of secreted IRBP.

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties.

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.

Proteoglycans in the mouse interphotoreceptor matrix. IV. Retinal synthesis of chondroitin sulfate proteoglycan.

To determine whether the mouse retina contributes chondroitin sulfate proteoglycan (CS-PG) to the interphotoreceptor matrix (IPM), 35SO4(2-) was used as a tracer for newly synthesized proteoglycan by retinas maintained in vitro in the absence of pigment epithelium. Following incubation with the tracer for 3 hr, the 35S-labeled proteoglycans present in the incubation medium and associated with isolated photoreceptor outer segments were analyzed separately. Proteoglycan was extracted with 4 M guanidine, and then separated on a G-25 column followed by DEAE ion exchange chromatography in the presence of 8 M urea. The proteoglycan fraction was eluted with a linear NaCl gradient of 0.15-1.0 M. Eluted 35S-labeled macromolecules were susceptible to chondroitinase AC and ABC degradation, indicating that virtually all the 35S-labeled proteoglycan synthesized by the mouse retina and secreted into the incubation media is of the chondroitin sulfate type. In parallel autoradiographic analysis of retinas following 35SO4(2-) incubation, silver grains were present over all retinal compartments, with 41-48% associated with the photoreceptor layer. Quantitative autoradiography of retinas following chondroitinase AC digestion of fixed retinas revealed significant (P less than 0.025) reduction in silver grains associated with the photoreceptor outer segment layer as compared to controls. These combined biochemical and autoradiographic studies indicate that the retina, possibly the photoreceptors, synthesize at least a portion of the CS-PG present in the IPM of the mouse.

Increased retinal synthesis of heparan sulfate proteoglycan and HNK-1 glycoproteins following photoreceptor degeneration.

In an earlier analysis of the retinal biosynthesis of proteoglycan, we noted that, following photoreceptor degeneration in the rd (retinal degeneration) mouse, the remaining inner retina exhibited a marked elevation in synthesis of heparan sulfate proteoglycan (HSPG), well above the level observed in the normal (nondegenerate) retina, as well as a pronounced increase in sulfation of protein substrates. Biochemical and autoradiographic results of 35S-amino acid utilization reported here confirm that the 35SO4(2-) differences seen previously are accompanied by increased protein synthesis in the rd retina. An intact photoreceptor cell layer is neither a barrier to nor a sink for the amino acid precursor. Further, we have examined sulfate utilization in four other rodent strains with photoreceptor degenerations. In each of the models examined, an increase in retinal synthesis of 35SO4(2-)-labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na2(35)SO4 isolated retinal cultures from the following: (a) mice with light-induced photoreceptor degeneration; (b) rd mice; (c) transgenic mice with photoreceptor degeneration; (d) RCS rats; and (e) rats with light-induced photoreceptor degeneration. Comparisons were made with concurrent cultures of control nondegenerate retinal tissues. Protein and proteoglycan-enriched fractions were prepared from the incubation media and guanidine HCl/detergent extracts of the retinas by ion-exchange chromatography. The 35SO4(2-)-proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of 35SO4(2-)-HSPG found in control incubations. The RCS and light-damaged rats also showed increased synthesis of 35SO4(2-)-chondroitin sulfate proteoglycan relative to the control, through the increase was of lesser magnitude than the HSPG effect. 35SO4(2-)-protein in degenerate and light-damaged retinas always contained at least twice the radioactivity found in comparable control preparations. The bulk of the increased radiolabeling was found in N-linked oligosaccharides, including several recognized by the HNK-1 antibody. These data suggest that a sustained increase in HSPG and HNK-1 glycoprotein synthesis is a consistent response of inner retinal cells following loss of photoreceptors and is independent of the cause of photoreceptor degeneration.

Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina.

A retinol-binding glycoprotein ( IRBP ) was purified in milligram quantities from the extracellular matrix ( interphotoreceptor matrix) that occupies the subretinal space in bovine eyes. IRBP binds 2.2 molecules of all-trans retinol with a KD of approximately 10(-6) M. The holoprotein has lambda max at 280 nm (E1%1 cm = 10.99) and at 330 nm (E1%1 cm = 7.88). When freshly isolated from light-exposed eyes, IRBP contains up to 0.6 molecule of all-trans retinol, together with small amounts of the 11-cis and 13-cis isomers. IRBP also binds exogenous cholesterol, alpha-tocopherol, and all-trans retinoic acid, all of which are completely displaced by all trans retinol. The affinity of alpha-tocopherol for IRBP was at least several orders of magnitude less than that of all-trans retinol. IRBP contains 8.4% by weight of carbohydrate, which consists of sialic acid, neutral hexoses, and glucosamine in the molar ratio of approximately 1:3:2. No galactosamine was detected. Observations on the binding of 125I-labeled lectins to IRBP in sodium dodecyl sulfate-polyacrylamide gels before and after desialosylation suggest that at least one oligosaccharide chain is of the sialated biantennary complex type and contains fucose. The Mr of IRBP on calibrated size-exclusion columns averaged 249,000; on sodium dodecyl sulfate-polyacrylamide gels (with or without dithiothreitol) the apparent Mr was 144,000. IRBP exists in at least four isoelectric forms that bind concanavalin A and have pI values ranging from 4.4 to 4.8. Rabbit anti-bovine IRBP antiserum gave a single precipitin line against purified bovine IRBP , which showed a line of complete identity with crude bovine interphotoreceptor matrix and a line of partial identity with human interphotoreceptor matrix. The human material contains a prominent protein with lectin-binding properties similar to bovine IRBP but with a somewhat faster electrophoretic mobility. When isolated bovine neural retinas were incubated with 3H-labeled fucose, glucosamine, or leucine, a solitary labeled protein identified as IRBP was secreted into the medium. Labeled IRBP could not be detected in the medium when retinal pigment epithelium was incubated with these precursors under the same conditions. Neural retinas incubated with 3H-labeled leucine in the presence of tunicamycin secreted a form of IRBP that did not bind concanavalin A and had an Mr reduced by approximately 5,000.

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates.

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.

Photoreceptor outer segment development in Xenopus laevis: influence of the pigment epithelium.

Opsin gene expression, synthesis, and photoreceptor outer segment morphology were evaluated during retinal development in Xenopus laevis. Retinal rudiments were harvested during in vivo development from embryonic stages 31 through 46 or were allowed to develop in vitro after removal from stage 33/34 embryos for 1, 2, or 3 days either with or without an investing pigment epithelium. Opsin mRNA was detected at stage 33/34 and the transcript level increased until stage 40 and remained at this level through stage 46. Opsin was first detected at stage 37/38 and progressively increased through stage 46. Rudimentary photoreceptor outer segment membranes occasionally appeared as early as stage 33/34 and they gradually increased in length, forming well-defined stacks of collapsed membranous saccules (discs) during in vivo development. The maturation of eye rudiments in culture was followed to determine how closely in vivo and in vitro development compare and to examine the ability of photoreceptors to differentiate when maintained in the absence of an overlying pigment epithelium (PE) layer. With the PE present, opsin mRNA as well as opsin content steadily increased over the entire culture period. After 1 day of culture, short cilia with minimal amounts of outer segment membranous material were present. By Day 3, the degree of outer segment differentiation corresponded morphologically to approximately stage 43 of in vivo development. When cultured in the absence of an investing PE, the opsin mRNA level increased minimally during the 3 days in culture. Opsin content increased, yet the relative amount was approximately 50% less than that present in retinas developing in the presence of the PE. Membranous material was elaborated; however, the outer segments appeared to be highly disorganized and formed whorl-like structures rather than the normal stacked disc morphology. These results suggest that the PE may be involved in regulating opsin at the transcriptional and/or translational levels and also participates in the organization of rod outer segment membranes.