Binding of 11-cis-retinaldehyde to cellular retinaldehyde-binding protein from pigment epithelium.
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Animals↗
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Identification of human liver aldehyde dehydrogenases that catalyze the oxidation of aldophosphamide and retinaldehyde.
Biotransformation of the biologically and pharmacologically important aldehydes, retinaldehyde and aldophosphamide, is mediated, in part, by NAD(P)-dependent aldehyde dehydrogenases catalyze the oxidation of the aldehydes to their respective acids, retinoic acid and carboxyphosphamide. Not known at the onset of this investigation was which of the several known human aldehyde dehydrogenases (ALDHs) catalyze these reactions. Thus, human liver aldehyde dehydrogenases were chromatographically resolved and the ability of each to catalyze the oxidation of retinaldehyde and aldophosphamide was assessed. Only one, namely ALDH-1, catalyzed the oxidation of retinaldehyde; the Km value was 0.3 microM. Three, namely ALDH-1, ALDH-2 and succinic semialdehyde dehydrogenase, catalyzed the oxidation of aldophosphamide; Km values were 52, 1193, and 560 microM, respectively. ALDH-4, ALDH-5 and betaine aldehyde dehydrogenase did not catalyze the oxidation of either aldophosphamide or retinaldehyde. ALDH-1 and succinic semialdehyde dehydrogenase accounted for 64 and 30%, respectively, of the total hepatic aldehyde dehydrogenase-catalyzed aldophosphamide (160 microM) oxidation. ALDH-1-catalyzed oxidation of aldophosphamide was noncompetitively inhibited by chloral hydrate; the Ki value was 13 microM. ALDH-2- and succinic semialdehyde dehydrogenase-catalyzed oxidation of aldophosphamide was relatively insensitive to inhibition by chloral hydrate. These observations strongly suggest an important in vivo role for ALDH-1 in the catalysis of retinaldehyde and aldophosphamide biotransformation. Succinic semialdehyde dehydrogenase-catalyzed biotransformation of aldophosphamide may also be of some in vivo importance.
Adult↗
Identification of mouse liver aldehyde dehydrogenases that catalyze the oxidation of retinaldehyde to retinoic acid.
NAD(P)-linked aldehyde dehydrogenases catalyze the oxidation of a wide variety of aldehydes. Thirteen of these enzymes have been identified in mouse tissues; eleven are found in the liver. Some are substrate-nonspecific; others are relatively substrate-specific. The present investigation sought to determine which of these enzymes are operative in catalyzing the oxidation of retinaldehyde to retinoic acid, a metabolite of vitamin A that promotes the differentiation of epithelial and other cells. Spectrophotometric and HPLC assays were used for this purpose. Enzyme-catalyzed oxidation of retinaldehyde (25 microM) was restricted to the cytosol (105,000 g supernatant fraction) and occurred at a rate of 211 nmol/min/g liver; oxidation of acetaldehyde (4 mM) by this fraction proceeds about ten times faster. At least 90% of this activity was NAD dependent. Of the approximately 10% that was apparently NAD independent, two-thirds was inhibited by 1 mM pyridoxal, a known inhibitor of aldehyde oxidase. Of the six cytosolic aldehyde dehydrogenases, only two, viz. AHD-2 and AHD-7, catalyzed the oxidation of retinaldehyde to retinoic acid. An additional NAD-dependent enzyme, viz. xanthine oxidase (dehydrogenase form), also catalyzed the reaction. Catalysis by AHD-2 accounted for more than 90% of the total NAD-dependent activity. Km values were 0.7, 0.6 and 0.9 microM, respectively, for the AHD-2-, AHD-7- and xanthine oxidase (dehydrogenase form)-catalyzed reaction. AHD-4, an aldehyde dehydrogenase found in the cytosol of mouse stomach epithelium and cornea, did not catalyze the reaction.
Aldehyde Dehydrogenase↗
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↗
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↗
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↗
Analysis of a soluble lipid-protein complex carrying endogenous 11-cis retinaldehyde from bovine retinal pigment epithelium.
A soluble lipid-protein complex in bovine retinal pigment epithelium is shown to carry endogenous 11-cis retinaldehyde, in the extent of 15% of the total 11-cis retinaldehyde found in this tissue. The complex, analyzed with respect to its chemical composition, exhibits a lipid composition close resembling the lipid composition of the rod outer segment membrane; the SDS-PAGE evidences the presence of a number of protein bands, two of which of 34 and 27 kDa appear glycoproteins. Finally, the lipid-protein complex exhibits a discrete level of a Cathepsin D-like protease activity. From the above, the possibility is discussed that the soluble lipid-protein complex could represent some phagolysosomal inclusion occurring in the pigment epithelial cells upon rod outer segment phagocytosis.
Animals↗
[Detection of retinaldehyde in mouse liver].
Vitamin A metabolites were studied in the liver of white mice, hamsters, guinea-pigs and rabbits after a single administration of retinyl acetate per os. In addition to retinol and retinyl palmitate, the mouse showed retinaldehyde which accumulated in the liver in the maximum concentration 3 hours later after the vitamin administration. Retinaldehyde isolated on aluminium oxide by means of TCA was examined with respect to absorption spectra in the UV-light, NaBH4 reduction with subsequent isolation of anhydrovitamin A from the reduced product as well as to absorption peaks in reactions with SbCl3 and thiobarbituric acid.
Administration, Oral↗
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↗
Analysis of the DNA of patients with retinitis pigmentosa with a cellular retinaldehyde binding protein cDNA.
We used a cDNA fragment corresponding to the human cellular retinaldehyde binding protein (CRALBP) gene to search for mutations at this locus in patients with autosomal dominant, autosomal recessive, or isolate retinitis pigmentosa, and Usher's syndrome, type I. No gene deletions or rearrangements could be detected in any patient by Southern blotting. We identified a Pvu II restriction fragment length polymorphism (RFLP) defining two alleles at the CRALBP locus in the normal population. We used this RFLP to analyze the genomic DNA of large sets of unrelated patients with autosomal dominant, autosomal recessive, or isolate retinitis pigmentosa. Within each of these groups, RFLP alleles at the CRALBP locus showed no linkage disequilibrium (departure from Hardy-Weinberg equilibrium). In addition, two autosomal dominant, two autosomal recessive, and three Usher's syndrome, type I pedigrees each showed no cosegregation of the CRALBP locus and the disease locus. We could find no evidence that mutations of the CRALBP gene are associated with the common forms of retinitis pigmentosa or Usher's syndrome, type I.
Alleles↗
A water-soluble polylysine-retinaldehyde Schiff base. Stability in aqueous and nonaqueous environments.
In order to improve the existing models of retinal-protein Schiff bases, a water-soluble polylysine-retinaldehyde imine has been synthesized and its stability assessed under a variety of conditions through changes in the visible absorption spectrum. The compound absorbs at 342 nm and consists of a 90-kDa poly-L-lysine containing a retinal Schiff base in about 2% of the lysyl epsilon-amino ends. Retinal is mostly in the all-trans form; under no conditions is more than 15% of the 13-cis isomer detected. The absorption maximum exhibits a pH-dependent reversible shift to 402 nm, with an apparent pKa approximately 3.4. In the presence of the anionic surfactant sodium dodecyl sulfate, this pKa is shifted to approximately 8.9, probably because of electric neutralization of lysyl epsilon-amino groups. Other detergents (cetyltrimethylammonium bromide, Triton X-100) do not modify the Schiff base pKa, but rather promote its hydrolysis; in this case detergents act in the same way as certain solvent mixtures, by providing an amphiphillic environment to the imine that in turn stabilizes the products of hydrolysis. Our results suggest that once the surfactant reaches the Schiff base, preferential partition of retinal into detergent micelles is the main factor facilitating imine bond breakdown. The response of our synthetic Schiff base to changes in pH or solvent polarity point together to an important role of the supporting polypeptide in providing a suitable environment to the chromophore.
Hydrogen-Ion Concentration↗
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↗
Cloning and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to retinaldehyde-binding protein and yeast SEC14p.
Protein tyrosine phosphorylation is important in the regulation of cell growth, the cell cycle, and malignant transformation. We have cloned a cDNA that encodes a cytosolic protein-tyrosine-phosphatase (PTPase), MEG2, from MEG-01 cell and human umbilical vein endothelial cell cDNA libraries. The 4-kilobase cDNA sequence of PTPase MEG2 corresponds in length to the mRNA transcript detected by Northern blotting. The predicted open reading frame encodes a 68-kDa protein composed of 593 amino acids and has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C-terminal region has a PTPase catalytic domain that has 30-40% amino acid identity to other known PTPases. The N-terminal region has 254 amino acids that are 28% identical to cellular retinaldehyde-binding protein and 24% identical to yeast SEC14p, a protein that has phosphatidylinositol transfer activity and is required for protein secretion through the Golgi complex in yeast. Recombinant PTPase MEG2 expressed in Escherichia coli possesses PTPase activity. PTPase MEG2 mRNA was detected in 12 cell lines tested, which suggests that this phosphatase is widely expressed. The structure of PTPase MEG2 implies that a tyrosine phosphatase could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus.
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↗
An improved HPLC method for the separation of retinaldehyde isomers from visual pigments.
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Animals↗
Retinaldehyde-protein pigments.
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Animals↗
Synthesis of retinoids by human retinal epithelium and transfer to rod outer segments.
The synthesis and release of 11-cis-retinoids by primary cultures of human retinal pigment epithelium (RPE) and the transfer of these retinoids to co-incubated human rod outer segments (ROS) were studied. Monolayers of 2-3-week-old cultured RPE incorporate tritiated all-trans-retinol, esterify it to the corresponding retinyl palmitate, form 11-cis-retinol and 11-cis-retinaldehyde and release retinaldehyde into the culture medium. The ratio of 11-cis to all-trans isomers of retinol, retinyl palmitate and retinaldehyde formed in the cells along with retinaldehyde released and incorporated into the ROS progressively increases, indicating a progressive increase in the concentration of 11-cis isomer from the time it is formed in RPE cells until its transfer to ROS. Incorporation of 11-cis-retinaldehyde into the ROS is directly related to the amount of albumin present in the media, suggesting the transfer of retinoids from RPE to photoreceptor to be a protein-mediated process. Events leading to isomerization, esterification, oxidation and release of retinoids by human RPE and incorporation of retinoids into ROS can therefore be examined in vitro.
Adult↗
Affinity labeling of bovine opsin by trans-retinoyl chloromethane.
All-trans-retinoyl chloromethane is a potent irreversible inactivator of bovine opsin in retinal rod outer segments. The inactivation appears to be due to specific modification of the apoprotein at the 11-cis-retinaldehyde binding site. The reaction follows pseudo first order kinetics at 37 degrees C. A moderate dissociation constant for the initial reversible complex of 6.1 mM could be derived with a first order rate constant of 1.8x10(-1) min-1 for the conversion to the irreversible inactivated form. Native rhodopsin or rhodopsin regenerated from opsin by addition of 11-cis-retinaldehyde is completely protected against inactivation by trans-retinoyl chloromethane. All-trans-retinaldehyde does not provide this protection for the irreversible inactivation.
Affinity Labels↗