Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent M_r is 250 000 by gel-filtration chromatography, and 225 000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent M_r is 140 000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10⁻⁶ M, as measured by fluorimetric titration.     

Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent Mr is 250000 by gel-filtration chromatography, and 225000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent Mr is 140000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10(-6) M, as measured by fluorimetric titration.     

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.     

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.     

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.     

Molecular properties of bovine interphotoreceptor retinol-binding protein

Interphotoreceptor retinol-binding protein (IRBP) is a large retinol-carrying glycoprotein, located only in the interphotoreceptor (or subretinal) space of vertebrate eyes. It has recently been purified to apparent homogeneity. The present report presents its sedimentation, spectroscopic, and binding properties. The molecular weight of bovine IRBP, determined by sedimentation equilibrium, is 133,000. The sedimentation coefficient is 5.8S. The Stokes radius, 56 A, obtained from gel-filtration chromatography, is much larger than that expected for a globular protein of the same molecular weight. These results indicate that IRBP is asymmetric (it can modeled as a prolate ellipsoid of revolution with axial ratio of about 8:1) and explain the overestimates of molecular weight obtained in previous studies based on size-exclusion methods. The molar absorption coefficients for IRBP (at 280 nm) and for bound retinol are both unaffected by ligand dissociation. Fluorescence of the holoprotein displays neither fine structure nor energy transfer from tryptophan to bound retinol. Circular dichroism suggests a secondary structure containing approximately 15% alpha-helix and approximately 20% beta-structure, unchanged by the presence of ligand. The binding of retinol creates a positive, extrinsic Cotton effect at 330 nm, proportional to the amount of retinol bound. The apparent dissociation constant for all-trans-retinol is 1.3 X 10(-6) M. This relatively loose binding implies that, if required during the visual cycle, IRBP should be able to transfer its ligand to other binding proteins in the neural retina and retinal pigment epithelium.     

Crystal structure of the functional unit of interphotoreceptor retinoid binding protein.

Interphotoreceptor retinoid binding protein (IRBP), the major soluble component of the interphotoreceptor matrix, is critical to the function, integrity, and development of the vertebrate retina. Although its role is poorly understood, IRBP has been thought to protect 11-cis retinal and all-trans retinol while facilitating their exchange between the photoreceptors and retinal-pigmented epithelium. We determined the X-ray structure of one of the functional units, or modules, of Xenopus laevis IRBP to 1.8 A resolution by multiwavelength anomalous dispersion. The monomeric protein consists of two domains separated by a hydrophobic ligand binding site. A structural homology to the recently solved photosystem II D1 C-terminal-processing protease and the enoyl-CoA isomerase/hydratase family suggests the utility of a common fold used in diverse settings, ranging from proteolysis to fatty acid isomerization to retinoid transport.     

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-[15-³H] 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 μm 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-[15-³H] 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 [³H]leucine or [³H]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.     

The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides

The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.     

Goldfish cones secrete a two-repeat interphotoreceptor retinoid-binding protein

Vitamin A and fatty acids are critical to photoreceptor structure, function, and development. The transport of these nutrients between the pigment epithelium and neural retina is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP, a 133-kDa (human) glycolipoprotein, is the major protein component of the extracellular matrix separating these two cell layers. In amphibians and mammals, IRBP consists of four homologous repeats of about 300 amino acids which form two retinol and four fatty acid-binding sites. Here we show that IRBP in teleosts is a simpler protein composed of only two repeats. Western blot analysis shows that goldfish IRBP is half the size (70 kDa) of IRBP in higher vertebrates. Metabolic labeling studies employing Brefeldin A taken together with in situ hybridization studies and the presence of a signal peptide show that goldfish IRBP is secreted by the cone photoreceptors. The translated amino acid sequence has a calculated molecular weight of 66.7 kDa. The primary structure consists of only two homologous repeats with a similarity score of 52.5%. The last repeats of human and goldfish IRBPs are 69.1% similar with hydrophobic regions being the most similar. These data suggest that two repeats were lost during the evolution of the ray-finned fish (Actinopterygii), or that the IRBP gene duplicated between the emergence of bony fish (Osteichthyes) and amphibians. Acquisition of a multirepeat structure may reflect evolutionary pressure to efficiently transport higher levels of hydrophobic molecules within a finite space. Quadruplication of an ancestral IRBP gene may have been an important event in the evolution of photoreceptors in higher vertebrates. Correspondence to: F. Gonzalez-Fernandez     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.     

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.     

Purification and characterization of laccase from Monocillium indicum Saxena

Summary An ascomycete Monocillium indicum Saxena producing extracellular laccase was isolated. The culture filtrate on native polyacrylamide gel electrophoresis (PAGE) revealed four bands of activity, one of which was a major one. The major laccase band, a glycoprotein, was purified and characterized. Gel filtration chromatography showed that the relative molecular weight (M_r) of laccase was 100 000. On sodium dodecyl sulphate (SDS)-PAGE the major laccase band further resolved into three proteins of M_r 72 000, 56 000 and 24 000. The enzyme had a pH optimum of 3.0 and was active on a number of o-phenols and aromatic acids. The 72 000 M_r protein was found to share common immunological properties with laccases of Coriolus versicolor, Agaricus bisporus and lignin peroxidase of Phanerochaete chrysosporium. Correspondence to: K. Koteswara Rao     

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.     

Cellular retinol-binding protein (type two) is abundant in human small intestine

Human small intestine was found to contain a retinol-binding protein similar to the gut-specific cellular retinol-binding protein, type two [CRBP (II)], described in the rat. This newly detected human protein was immunochemically distinct from human cellular retinol binding protein previously described but immunochemically similar to rat CRBP (II). The partially purified protein bound retinol and exhibited fluorescence excitation and emission spectra distinct from those spectra for retinol bound to pure human CRBP but similar to the spectra for retinol bound to rat CRBP (II). Human CRBP (II) could be localized to the villus-associated enterocytes by immunohistochemistry, using antiserum against rat CRBP (II). The protein was abundant representing 0.4% of the total soluble protein in a jejunum mucosal extract. This protein may play an important role in the absorption and necessary intestinal metabolism of vitamin A.     

Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein.

This study investigates whether the interphotoreceptor retinoid-binding protein (IRBP) is necessary for the release of 11-cis-retinaldehyde (RAL) or if the retinoid is constitutively released from the retinal pigment epithelium (RPE) following synthesis. The strategic location of IRBP in the interphotoreceptor matrix (IPM) and its retinoid-binding ability make it a candidate for a role in 11-cis-RAL release. Fetal bovine RPE cells were grown in permeable chambers, and their apical surfaces were incubated with medium containing either apo-IRBP, the apo form of cellular retinaldehyde-binding protein (CRALBP), the apo form of serum retinol-binding protein (RBP), or bovine serum albumin (BSA) or with medium devoid of binding proteins. [3H]-all-trans-Retinol (ROL) was delivered to the basal surface of the cells by RBP. High-performance liquid chromatography demonstrated that [3H]-11-cis-RAL was optimally released into the apical medium when apo-IRBP was present. The most surprising result was the diminished level of [3H]-11-cis-RAL when apo-CRALBP was in the apical medium. Circular dichroism demonstrated that CRALBP had not been denatured by the photobleaching required for endogenous ligand removal. Therefore, apo-CRALBP should have been able to bind [3H]-11-cis-RAL if it was constitutively released into the apical medium. In addition, when proteins other than apo-IRBP were present, or if the cells were incubated with medium alone, the observed decrease in apical [3H]-11-cis-RAL was concomitant with a buildup of intracellular [3H]-all-trans-retinyl palmitate and [3H]-all-trans-ROL in the basal culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Monoclonal antibodies to a Mr 68000 pore complex glycoprotein interfere with nuclear protein uptake in Xenopus oocytes

Using a monoclonal antibody (PI1) raised against mouse lymphocyte nuclear matrix fractions we have identified a N-acetylglucosamine (G1cNAc)-containing glycoprotein of M_r 68000 as a component of the nuclear pore complexes of Xenopus laevis oocytes. The antigenic determinant recognized by antibody PI1 comprises both the sugar moiety and protein sequences since, on the one hand, added G1cNAc competed effectively for antibody binding and, on the other hand, the antibody reacted in immunoblots with only one member of the G1cNAc-containing pore complex glycoprotein family. By using immunogold-electron microscopy we could demonstrate that the M_r 68000 glycoprotein was located preferentially to the cytoplasmic side of the pore complex channel. When radiolabeled soluble nuclear proteins were injected into the cytoplasm of Xenopus oocytes, their reentry into the nucleus was almost completely inhibited in the presence of antibody PI1 as shown by two-dimensional gel electrophoresis. The results indicate that the evolutionarily conserved M_r 68000 glycoprotein is involved in transport processes of karyophilic proteins from the cytoplasm into the nucleus.     

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.     

Purification and immunochemical characterization of human adrenal tyrosine hydroxylase

Tyrosine hydroxylase (TH) was purified from the soluble fraction of human adrenal glands. The enzyme in human adrenal glands that was purified to apparent homogeneity had an apparent M_r of about 280,000. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a single band with a M_r of 60,000 similar to the M_r of bovine adrenal enzyme. The enzyme is considered to be composed of four identical subunits. The specific activity of the final preparation was approximately 310 nmol 3,4-dihydroxyphenylalanine (DOPA) formed/min/mg protein. The use of the “Western Blot” method showed that human adrenal TH did not aggregate as rapidly as bovine adrenal TH.