Evidence of retinoid metabolism within cells of inner retina.
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Aldehyde Dehydrogenase↗
Biomedical subjects
J C Saari
Publications and source records attributed to J C Saari.
Rod outer segment retinol dehydrogenase: substrate specificity and role in phototransduction.
The reaction catalyzed by all-trans-retinol dehydrogenase of rod outer segments completes the quenching of photoactivated rhodopsin and initiates the cycle of reactions leading to regeneration of visual pigment. The goal of this study was to determine the kinetic parameters of the dehydrogenase at physiological levels of bleaching, to investigate its specificity, and to determine its possible role in modulating phototransduction. Reduction of all-trans-retinal could be measured after bleaching < 0.15% rhodopsin. Kinetic parameters for the forward reaction determined with endogenous all-trans-retinal were Km = 1.1 microM; Vmax = 7 nmol/min/mg rhodopsin. The low enzymatic activity suggests that at high bleach rates, all-trans-retinal could accumulate, increasing the steady state level of bleaching intermediates or promoting formation of pseudophotoproducts. Active pseudophotoproducts, which stimulate Gt activation and opsin phosphorylation by rhodopsin kinase, are formed with opsin and all-trans-retinal as well as retinal analogues lacking the 13 methyl or the terminal two carbons of the polyene chain. Addition of all-trans-retinol, NADP, and [32P]ATP to rod outer segments increased rhodopsin phosphorylation. Kinetic parameters for the reverse reaction determined with exogenous all-trans-retinol were Km = 10 microM; Vmax = 11 nmol/min/mg rhodopsin. Our results support the hypothesis that all-trans-retinol dehydrogenase could influence the phototransduction cascade, including activities of Gt, rhodopsin kinase, and binding of arrestin, by impeding the recycling of rhodopsin at high bleach levels.
Alcohol Oxidoreductases↗
Control of substrate flow at a branch in the visual cycle.
Photoisomerization of rhodopsin's chromophore, 11-cis-retinaldehyde, and subsequent regeneration of the 11-cis configuration are accomplished in vertebrates by a series of reactions known as the visual cycle. At one point in the cycle, 11-cis-retinol can either be enzymatically oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration or be enzymatically esterified and stored. Partition of substrate at this branch was examined in this study and found to be influenced by cellular retinaldehyde-binding protein (CRALBP), a retinoid-binding protein found in retina. Esterification was reduced to about 10% and oxidation stimulated 2-3-fold in the presence of this protein. Other experiments confirmed that "free" 11-cis-retinol was esterified more rapidly than 11-cis-retinol complexed with CRALBP and that CRALBP.11-cis-retinol was not an inhibitor of the esterification. Following oxidation of CRALBP.11-cis-retinol, the reaction product, 11-cis-retinaldehyde, was found associated with the binding protein. 11-cis-Retinaldehyde is not available for reaction with carbonyl reagents when the retinoid is bound to CRALBP. However, enzymatic oxidation of CRALBP.11-cis-retinol in the presence of O-ethylhydroxylamine produced ca. 30% retinaldehyde O-ethyloxime and 70% free 11-cis-retinaldehyde, suggesting that about one-third of the retinol oxidized had dissociated from the binding protein. Neither oxidation nor esterification of CRALBP.11-cis-retinol was inhibited by including CRALBP.11-cis-retinaldehyde in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Interactions of all-trans-retinol and long-chain fatty acids with interphotoreceptor retinoid-binding protein.
Interphotoreceptor retinoid-binding protein (IRBP), a predominant protein in the interphotoreceptor matrix of the retina, has been implicated in transfer of retinoids between retinal pigment epithelium and photoreceptor cells. The interactions of IRBP with all-trans-retinol have been studied by three fluorescence-based methods and by measurements of binding of 3[H]-labeled all-trans-retinol to this protein. It was found that IRBP contains two sites with similar but not identical affinities for all-trans-retinol. The dissociation constant of the all-trans-retinol-IRBP complex at the first site was 0.1 microM, which is about 10-fold lower than previously reported values. The second site had about 2.5-fold lower affinity for all-trans-retinol as compared to the first site. Long-chain fatty acids were found in this study to displace all-trans-retinol from the stronger retinol-binding site on IRBP. Displacement of all-trans-retinol was used to study the interactions of fatty acids with this protein. It was found that docosahexaenoic acid (DHA C22:6n-3), an essential fatty acid which plays an important role in vision, had the highest apparent affinity for the site probed on IRBP of all the fatty acids studied.
Animals↗
Assays of retinoid dehydrogenases by phase partition.
Two modifications of an extraction assay for retinoid dehydrogenases are described. The first method involves the transfer of tritium from carbon-15 of [15-3H]-retinol or [15-3H]retinaldehyde to NAD, whereas in the second method, tritium from [3H]NADPH is transferred to all-trans-retinaldehyde. Since both versions of the assay involve the interconversion of water-soluble and -insoluble tritium-labeled compounds, a simple phase partition is sufficient to separate labeled products from labeled reactants. The assays are shown to provide reliable estimations of the reaction progress for three retinoid dehydrogenases of the visual system when compared to HPLC analysis of retinoid products or gel filtration analysis of pyridine nucleotide. The assays will be useful in studying retinoid dehydrogenases from other tissues and in principle can be modified for other dehydrogenase reactions with water-insoluble substrates.
Alcohol Oxidoreductases↗
Retinol esterification in bovine retinal pigment epithelium: reversibility of lecithin:retinol acyltransferase.
Esterification of all-trans-retinol is a key reaction of the vertebrate visual cycle, since it produces an insoluble, relatively non-toxic, form of the vitamin for storage and supplies substrate for the isomerization reaction. CoA-dependent and -independent pathways have been described for retinol esterification in retinal pigment epithelium (RPE). The CoA-independent reaction, catalysed by lecithin:retinol acyltransferase (LRAT) was examined in more detail in this study. Addition of retinol to RPE microsomes results in a burst of retinyl ester synthesis, followed by a rapid apparent cessation of the reaction. However, [3H]retinol, added when retinyl ester synthesis has apparently ceased, is rapidly incorporated into retinyl ester without a net increase in the amount of ester. The specific radioactivities of [3H]retinol and [3H]retinyl ester reach the same value. [14C]Palmitate from palmitoyl-CoA is incorporated into preexisting retinyl ester in the absence of net ester synthesis, too. These exchange reactions suggest that the reaction has reached equilibrium at the plateau of the progress curve and that only the accumulation of retinyl ester, and not its synthesis, has stopped during this phase of the reaction. Studies with geometrical isomers of retinol revealed that the rate of exchange of all-trans-retinol with all-trans-retinyl esters was about 6 times more rapid than exchange of 11-cis-retinol with 11-cis-retinyl ester. This is the first demonstration of the reversibility of LRAT and the first example of stereospecificity of retinyl ester synthesis in the visual system. Reversal of the LRAT reaction could contribute to the mobilization of 11-cis-retinol from 11-cis-retinyl ester pools.
Acyltransferases↗
Autoantibodies against retinal bipolar cells in cutaneous melanoma-associated retinopathy.
PURPOSE: This study's goal was to determine the pathophysiology of the retinopathy that occurs in patients with metastatic cutaneous melanoma and sudden onset of night blindness, the so-called melanoma-associated retinopathy (MAR) syndrome. We tested the hypothesis that sera from two MAR patients contained autoantibodies that reacted with "on" bipolar cells of the human retina. METHODS: Immunofluorescence was performed on cryostat sections of unfixed normal human retinas. Sera and IgG fractions were tested from the two MAR patients and 38 control subjects (28 patients with metastatic melanoma, but no visual symptoms; two patients with non-MAR retinopathy; and eight normal subjects). RESULTS: The sera and IgG fractions from both MAR patients but from none of the control subjects produced heavy immunostaining of bipolar cells, which were identified as rod bipolars by a double labeling procedure using anti-protein kinase C. CONCLUSIONS: We hypothesize that MAR patients generate autoantibodies against a melanoma antigen that cross react with bipolar cells of the retina. These antibodies, by an unknown mechanism, may cause abnormalities of the rod and cone systems that are characteristic of MAR.
Adult↗
Assignment of the gene (RLBP1) for cellular retinaldehyde-binding protein (CRALBP) to human chromosome 15q26 and mouse chromosome 7.
Cellular retinaldehyde-binding protein (CRALBP) has properties that suggest that it is involved in the visual process and, therefore, potentially with retinal diseases. A human cDNA probe has been used to map this gene to human chromosome 15q26 (somatic cell hybrids and in situ hybridization) and to mouse chromosome 7 by somatic cell hybrids.
Animals↗
Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.
Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.
Amino Acid Sequence↗
Immunolocalization of cellular retinoic acid binding protein to Müller cells and/or a subpopulation of GABA-positive amacrine cells in retinas of different species.
Retinoic acid (RA) and its specific binding protein, cellular RA binding protein (CRABP), are found in relative abundance in bovine and rat retinas. Since RA does not participate in the visual cycle, the presence of RA and its binding protein in retina suggests that they may be involved in other aspects of retinoid action. As an initial step in identifying the role of RA and its binding protein in retina, monoclonal antibodies were prepared against CRABP purified from bovine retina and used to localize this antigen by immunocytochemistry in retinas of different species. Human and monkey retinas showed specific cytoplasmic labeling of Müller cells. Cat, bovine, rabbit, rat, turtle, and chick retinas showed specific cytoplasmic labeling of some somata in the inner nuclear and ganglion cell layers and characteristic strata in the inner plexiform layer. Cat and bovine retinas also showed cytoplasmic labeling of Müller cells. Immunoreactivity in these species was absent with nonimmune serum or abolished when the antibodies were preabsorbed with purified antigen. Chameleon, goldfish, and frog retinas were nonreactive. We used double-labeling immunofluorescence experiments to determine if the CRABP-positive cells were also positive for known neurotransmitters or associated enzymes. CRABP-positive amacrine cells of cat, cow, rabbit, rat, and chick represented a subset of the more numerous gamma-aminobutyric acid (GABA)-positive amacrine cells. However, turtle CRABP-positive amacrine cells were negative for GABA despite the fact that turtle retina contains many GABA positive cells. CRABP-positive amacrine cells in rat retinas were not immunoreactive for glycine, choline acetyltransferase, somatostatin, or tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Immunolocalization of cellular retinol-, retinaldehyde- and retinoic acid-binding proteins in rat retina during pre- and postnatal development.
Cellular retinol-, retinaldehyde- and retinoic acid-binding proteins were localized in rat retina during pre- and postnatal development by indirect immunofluorescence. Cryostat tissue sections were prepared daily from embryonic day 11 until the day of birth (E11-22) and from postnatal days 1-32 (P1-32). Cellular retinaldehyde- and retinol-binding proteins were first detected in retinal pigment epithelium on E13 and E18, respectively, and in Müller cells at P1 and P15. Parallel studies showed that in adult retina cellular retinoic acid-binding protein is present in a subpopulation of GABAergic amacrine cells. During retinal differentiation, cellular retinoic acid-binding protein was first detected at E18 in cells sclerad to the developing inner plexiform layer, suggesting that this binding protein is expressed in amacrine cells very early during differentiation. During early ocular morphogenesis, cellular retinoic acid-binding protein was present in mesenchymal cells enveloping the eye (E12-15), in the neuroblastic layer of the retina (E13-15), in the nerve fibre layer (E14-15), and the developing optic nerve (E15). Our results suggest that retinoic acid, the natural ligand of cellular retinoic acid-binding protein, may be involved in neuronal differentiation in the inner retina. The studies further support a role for cellular retinoic acid-binding protein in mediating the effects of retinoic acid on developing neural crest cells and raise new questions about the role of cellular retinaldehyde-binding protein in the visual cycle and during development.
Aging↗
Localization of cellular retinoic acid-binding protein to amacrine cells of rat retina.
Monoclonal antibodies to performic acid-oxidized cellular retinoic acid-binding protein (CRABP) from bovine retina were prepared by fusion of spleen cells from immunized mice with mouse myeloma cells. Five antibodies were studied in detail. It was established by ELISA that the antibodies react with CRABP and oxidized CRABP, but not with other oxidized or unmodified retinoid-binding proteins. Competitive ELISA demonstrated that the antibodies react with heat-denatured antigen but not with native protein. Western blotting and immunostaining, following sodium dodecyl sulfate gel electrophoresis, provided evidence for recognition of a single component in retinal supernatants whose staining is prevented by preabsorption of the antibody with heat-denatured CRABP. The insoluble fraction from a retinal homogenate contains residual CRABP and two weakly-reacting components, whose staining is not affected by preabsorption of the antibody with antigen. Each antibody produces the same staining pattern on cryostat sections of rat retina by indirect immunofluorescence. Amacrine somata on both sides of the inner plexiform layer are labeled, as well as processes forming laminae within this layer. These results suggest that retinoic acid may play a functional role in the inner retina.
Animals↗
Acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase activities of bovine retinal pigment epithelial microsomes.
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Acyltransferases↗
Lecithin:retinol acyltransferase in retinal pigment epithelial microsomes.
Microsomal preparations from retinal pigment epithelium carry out phosphatidylcholine synthesis upon incubation with 1-palmitoyllysophosphatidylcholine and fatty acyl-CoA. Phosphatidylcholine synthesized in situ in this manner is an acyl donor for retinyl ester synthesis, demonstrating the existence of lecithin:retinol acyltransferase. Although acyl transfer to retinol is from the 1-position of phosphatidylcholine, the fatty acid in the 2-position is important in substrate recognition. The finding of this novel enzyme activity in retinal pigment epithelial microsomes suggests that phosphatidylcholine is the endogenous acyl donor in CoA-independent retinol esterification observed in these preparations.
Acyltransferases↗
The complete primary structure of the cellular retinaldehyde-binding protein from bovine retina.
Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The complete amino acid sequence of CRALBP from bovine retina has been determined by direct microanalysis of the protein. Bovine CRALBP contains 316 residues in a single amino-terminal-blocked chain corresponding to a molecular weight of 36,421, inclusive of the blocking group. Overlapping peptides were generated by cleavage of lysyl, arginyl, methionyl, glutamyl, and one tryptophanyl bond and sequenced by gas-phase Edman degradation. Analysis of amino-terminal arginyl and methionyl peptides by fast atom bombardment mass spectrometry identified the N alpha-blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 9 residues. Comparison of CRALBP with other known protein sequences reveals no significant structural relatedness. The present results provide a basis for relating CRALBP domains with physiological function and for the future development of a more detailed three-dimensional model of the interaction of 11-cis-retinaldehyde with protein.
Amino Acid Sequence↗
Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures.
A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.
Animals↗
CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium.
Washed, buffered microsomes from bovine retinal pigment epithelium catalyze retinyl ester synthesis from retinol in the absence of an exogenous acyl donor. A plot of retinyl ester synthesis versus time reaches a plateau at 123 +/- 26 nmol of retinyl ester mg-1 microsomal protein, providing a minimum value of the concentration of the endogenous acyl donor. Fatty acyl-CoA analysis by three different methods employing high performance liquid chromatography resulted in the detection of less than 1 nmol mg-1 protein of acyl-CoA, indicating that fatty acyl-CoA is not the endogenous acyl donor. Stimulation of the rate of retinyl ester synthesis by palmitoyl-CoA or ATP, CoA, and palmitate is observed following its addition at the beginning of the reaction or after the endogenous acyl source has been exhausted by 20 min of reaction with retinol. Palmitate from [14C]palmitoyl-CoA is incorporated into retinyl ester at a rate similar to that for the incorporation of [3H] retinol, demonstrating the presence of an apparent acyl-CoA:retinol acyl transferase activity. The acyl group from palmitoyl-CoA can be transferred initially to a component of the microsomes and subsequently to retinol. The product of retinyl ester synthesis from all-trans-retinol and palmitoyl-CoA is all-trans-retinyl palmitate, indicating that the stereochemical configuration is retained during esterification. The kinetic parameters for the esterification of 11-cis-retinol and all-trans-retinol are similar.
Acyl Coenzyme A↗
Purification of cellular retinaldehyde-binding protein from bovine retina and retinal pigment epithelium.
Cellular retinaldehyde-binding protein (CRALP) has been purified from extracts of bovine retina or retinal pigment epithelium by a procedure employing an initial, high-capacity anion exchange chromatographic step and anion exchange HPLC for removal of a persistent contaminant. The procedure also yields fractions containing three other retinoid-binding proteins present in retina (cellular retinol-, cellular retinoic acid- and interphotoreceptor retinol-binding proteins; CRBP, CRABP and IRBP, respectively). Procedures are described for labeling CRALBP with 9-cis-retinaldehyde, 11-cis-retinaldehyde, or 11-cis-retinol. There are approx. 3 nmol of CRALBP per adult bovine eye and the binding protein is ca. 0.5% of the soluble protein of a retinal supernatant.
Animals↗