Robert A. Landers

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.

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.

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.

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.

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.

Increased Retinal Synthesis of Heparan Sulfate Proteoglycan and HNK‐1 Glycoproteins Following Photoreceptor Degeneration

Abstract: 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 35SO42− 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 35SO42−‐labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na235SO4 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 HCI/detergent extracts of the retinas by ion‐exchange chromatography. The 35SO42−‐proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of 35SO42−‐HSPG found in control incubations. The RCS and light‐damaged rats also showed increased synthesis of 35SO42−‐chondroitin sulfate proteoglycan relative to the control, though the increase was of lesser magnitude than the HSPG effect. 35SO42−‐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.

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.

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.

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)

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 retinolbinding 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.