retinaldehyde and 3-hydroxyretinal

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Topics: Animals; Apoproteins; Drosophila melanogaster; Drosophila Proteins; Mechanoreceptors; Opsins; Retinaldehyde

(R)-all-trans-3-hydroxyretinal 1, (S)-all-trans-4-hydroxyretinal and (R)-all-trans-4-hydroxyretinal have been synthesized stereoselectively by Horner-Wadsworth-Emmons and Stille cross-coupling as bond-forming reactions. The CBS method of ketone reduction was used in the enantioface-differentiation step to provide the precursors for the synthesis of the 4-hydroxyretinal enantiomers. The kinetic constants of Xenopus laevis ADH8 with these retinoids have been determined.

Topics: Alcohol Dehydrogenase; Diterpenes; Hydrogenation; Ketones; Kinetics; Retinaldehyde; Xenopus Proteins

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.

Topics: Animals; Drosophila; Drosophila Proteins; Hydroxylation; Isomerism; Oxidoreductases; Retinaldehyde; Spectrometry, Mass, Electrospray Ionization; Vitamin A

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaG(P330) mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaG(P330). These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal.

Topics: Animals; Chromatography, High Pressure Liquid; Drosophila; Drosophila Proteins; Phenotype; Retina; Retinaldehyde; Rhodopsin

A dietary source of retinoid or carotenoid has been shown to be necessary for the biosynthesis of functional visual pigment in flies. In the present study, the larvae or adults of Drosophila melanogaster were administered specific carotenoid-containing diets and high performance liquid chromatography was used to identify and quantify the carotenoids in extracts of wild type and ninaD visual mutant flies. When beta-carotene was fed to larvae, wild type flies were shown to hydroxylate this molecule and to accumulate zeaxanthin and a small amount of beta-cryptoxanthin. Zeaxanthin content was found to increase throughout development and was a major carotenoid peak detected in the adult fly. Carotenoids were twice as effective at mediating zeaxanthin accumulation when provided to larvae versus adults. In the ninaD mutant, zeaxanthin content was shown to be specifically and significantly altered compared to wild type, and was ineffective at mediating visual pigment synthesis when provided to both larval and adult mutant flies. It is proposed that zeaxanthin is the larval storage form for subsequent visual pigment chromophore biosynthesis during pupation, that zeaxanthin or beta-crytoxanthin is the immediate precursor for light-independent chromophore synthesis in the adult, and that the ninaD mutant is defective in this pathway.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Chromatography, High Pressure Liquid; Cryptoxanthins; Diet; Drosophila melanogaster; Larva; Mutation; Retinal Pigments; Retinaldehyde; Xanthophylls; Zeaxanthins

In the class Insecta, retinal and 3-hydroxyretinal are used as chromophores of visual pigments, but the absolute structure of the 3-hydroxyretinal chromophore has yet to be clarified. This study investigates the chirality of 3-hydroxyretinal in the compound eyes of five representative orders of insects. In the orders Odonata, Hemiptera, Neuroptera and Lepidoptera, and suborders Nematocera and Brachycera of the Diptera, only (3R)-3-hydroxyretinal isomers were detected, but dipterans of the suborder Cyclorrhapha (higher flies) had the (3S)-11-cis enantiomer and a mixture of (3R)-all-trans and (3S)-all-trans 3-hydroxyretinal enantiomers; the ratio of the (3R) enantiomer to the sum of both enantiomers of the all-trans isomer was in the range 9-32%. Despite differences in feeding habits, including one species that is a butterfly parasite, all higher flies analysed to date share the same pattern of 3-hydroxyretinal enantiomers, making them a unique group with regard to the nature of the visual pigment chromophore.

Topics: Animals; Chromatography, High Pressure Liquid; Diptera; Eye; Hemiptera; Insecta; Lepidoptera; Retinal Pigments; Retinaldehyde; Stereoisomerism

The sequence encoding opsin from the mantid Sphodromantis sp. has been determined by dideoxynucleotide sequencing of PCR products from a cDNA derived from eye cup tissue. The 376-amino-acid (aa) residues show approx. 56% identity and 85% similarity to known insect opsins (Drosophila melanogaster and Calliphora erythrocephala). The predicted protein structure, based on the hydropathy profile and placement of key aa residues, reveals a seven-transmembrane structure typical of a rhodopsin. Unlike the previously characterised insect visual pigments which have 3-hydroxy retinal in their binding sites, mantid rhodopsin contains 11-cis retinal. Comparison of transmembrane sequences from the opsin family was performed in order to identify any specific aa substitutions which are able to account for the selection of retinal or its 3-hydroxy derivative by insect opsins.

Topics: Amino Acid Sequence; Animals; Base Sequence; DNA, Complementary; Hydrogen Bonding; Molecular Sequence Data; Orthoptera; Polymerase Chain Reaction; Protein Structure, Secondary; Retinaldehyde; Rod Opsins; Sequence Homology, Amino Acid

Opsin expression is extremely suppressed by carotenoid deprivation in Drosophila. Carotenoid replacement in deprived flies promotes the recovery of visual pigment with an increase in opsin, as well as the chromophore 11-cis-3-hydroxyretinal. Here, we show that opsin mRNA and opsin peptide in an intermediate step of posttranslational processing were present in carotenoid-deprived flies. By supplementing chromophore to photoreceptor cells, intermediate opsin was made mature. During this process, opsin peptide underwent multiple modifications involving glycosylation. Based on these results, we present a novel mechanism of protein regulatory expression; that is, chromophore posttranslationally controls the expression of apoprotein by promoting its maturation.

Topics: Animals; Drosophila melanogaster; Gene Expression; Glycosylation; Immunoblotting; Photoreceptor Cells; Protein Processing, Post-Translational; Retinaldehyde; Rhodopsin; RNA, Messenger; Rod Opsins; Transcription, Genetic

The metabolism of 3-hydroxyretinoids in the cytosol of the compound eyes of a species of butterfly, Papilio xuthus, was investigated. The cytosol was found to contain 25-30% of the total 3-hydroxyretinal and 70-82% of the total 3-hydroxyretinol in the eye. These percentages of 3-hydroxyretinoids in the cytosol were found to be constant regardless of whether the eyes are light-adapted or dark-adapted. 3-Hydroxyretinal can be newly synthesized in the cytosol of light-adapted eyes. Blue light specifically increases the amount of 11-cis and all-trans 3-hydroxyretinal ca 2.5 and 1.8 times respectively, compared to pre-irradiation. When 3-hydroxyretinal was synthesized, 3-hydroxyretinol was decreased or disappeared in the cytosol. When retinol (non-native chemical) was added to the cytosol, it was converted into retinal. This result indicates that an oxidative system exists in the compound eye which can convert 3-hydroxyretinol to 3-hydroxyretinal.

Topics: Adaptation, Ocular; Animals; Butterflies; Cytosol; Eye; Female; Light; Male; Retinaldehyde; Time Factors

Racemic all-trans-3-hydroxyretinal (3-OH-RAL) (1) was converted by a reaction with (-)-camphanic acid chloride (CpCl) into a diastereomixture of camphanates (2a) and (2b) which was separated by preparative high-performance liquid chromatography (HPLC) to give two esters (2a) and (2b) in pure state, respectively. Saponification of (2a) and (2b) independently afforded optically active (3S)- and (3R)-3-OH-RALs (3a) and (3b), respectively, whose absolute structures were determined by circular dichroism (CD) spectra. Racemic 3-OH-RAL was separated to two peaks by HPLC using chiral column (ChiraSpher, Merck). Cochromatography with authentic specimens (3a) and (3b) showed that the peak with a short retention time corresponded to (3R)-isomer and the other to (3S).

Topics: Chromatography, High Pressure Liquid; Retinaldehyde

The quantum yields of bleaching for two artificial pigments, bovine opsin combined with (3R)-3-hydroxy retinal or (3R,S)-3-methoxy retinal, were determined in comparison to the value for regenerated bovine rhodopsin. Regeneration of the visual pigments was performed by incubation of 3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1- propanesulfonate (CHAPSO)-solubilized opsin with the 11-cis isomers of retinal and the respective retinal derivatives. The extinction coefficients of the pigments in CHAPSO were determined to 35,000 M-1 cm-1 (native rhodopsin), 35,300 M-1 cm-1 (regenerated rhodopsin) and 34,500 M-1 cm-1 (3-OH retinal opsin). With respect to rhodopsin (lambda max: 500 nm), the pigments carrying the substituted chromophores exhibit blue shifted absorbance maxima (3-hydroxy and 3-methoxy retinal opsin: 488 nm). In parallel experiments under absolutely identical conditions we find related to the value of CHAPSO solubilized rhodopsin (identical to 1) a quantum efficiency of bleaching for the 3-hydroxy pigment of 1.2.

Topics: Animals; Cattle; Cholic Acids; Detergents; Eye Proteins; Quantum Theory; Retinaldehyde; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Solubility; Spectrophotometry

Larvae of the tobacco hornworm moth Manduca sexta were reared on either a carotenoid-supplemented or a carotenoid-deficient diet. The former yields fortified adults with normal visual function, whereas visual sensitivity and rhodopsin content are reduced by 2-4 log units in the compound eyes of the deprived moths reared on the latter. We characterized the retinoids of fortified retinas and investigated the recovery of visual function in deprived moths that were provided with retinaldehyde as a source of photopigment chromophore. Retinoids were identified and measured by high-performance liquid chromatography (HPLC). Fortified retinas contained mainly 3-hydroxyretinaldehyde (R3); 11-cis R3 predominated in dark-adaptation, all-trans in light-adaptation, indicating that R3 is the photopigment chromophore. No retinoids could be measured in deprived eyes. Retinaldehyde (R1) was delivered to the retinas of deprived moths by "painting" solutions of 11-cis or all-trans R1 in dimethylsulfoxide (DMSO) on the corneal surfaces of the compound eyes or on the head capsule between the eyes. 11-cis R1 induced rapid recovery: during 3 days, sensitivity rose to within a log unit of that measured from fortified animals. By 7 days, sensitivity was close to normal. Although rhodopsin and P-face particle densities of photoreceptor membranes increased, neither rose to the levels found in fortified animals. All-trans R1 induced only a slight increase in sensitivity that could have resulted from some nonspecific isomerization of the all-trans to the 11-cis isomer; we found no evidence for a retinal isomerase that functions in darkness. Small amounts of R3 were measured in recovering retinas, indicating some conversion of R1 to R3.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Chromatography, High Pressure Liquid; Cornea; Dark Adaptation; Isomerism; Light; Moths; Photoreceptor Cells; Retina; Retinaldehyde; Retinoids; Vision, Ocular; Vitamin A Deficiency

The distribution of 3-hydroxyretinal (R3), a recently discovered retinoid used as the visual pigment chromophore in some insects, was investigated in the class Insecta using HPLC technology. We studied 138 species in 24 orders, sampling from a wide range of taxonomic groups as well as varied habitats. In addition to groups already known to have R3, we find this retinoid in Hemiptera (suborder Heteroptera), Plecoptera, Megaloptera, and Hymenoptera. We also find retinal (R1) in Hemiptera (suborder Homoptera), Mecoptera, and Trichoptera, groups previously thought to have only R3. The pattern of R3 occurrence indicates that this retinoid cannot be considered a phylogenetic marker, having a scattered distribution in the class Insecta as well as within some orders of insects. Several environmental factors that might influence the selection of chromophore have been considered, but none correlates with its distribution. The evolutionary reasons for the pattern of occurrence of R3 therefore remain unknown.

Topics: Animals; Insecta; Phylogeny; Retinaldehyde; Retinoids; Species Specificity

Retinoids in the compound eyes of nymphs and adult dragonflies in 11 families of the 3 suborders were extracted by the oxime method, and analysed by high performance liquid chromatography. Almost all of the species examined contained both retinal and 3-hydroxyretinal in the compound eye. The ratio of 11-cis 3-hydroxyretinal to 11-cis retinal (3-OH ratio) was calculated as an index of the retinoid composition. The 3-OH ratios of the whole eye of nymphs in all the suborders and of adults of the suborder Zygoptera were very high, 2.2 at the minimum, but in Anisozygoptera and Anisoptera most of the ratios were distributed between 1 and 2.7. In the family Gomphidae, exceptionally low 3-OH ratios, less than 1, were observed in several species. The regional distributions of the retinals in the adult compound eyes were also examined. In the Zygopteran compound eye, both retinals were distributed evenly all over the eye, while in the compound eye of the other two suborders, the 3-OH ratios in the dorsal area of the eye were extremely low. In several species of Gomphidae and Libellulidae the ratios in the dorsal areas were zero. From the correspondence of these results and the compartment of the compound eye, it appeared that the large ommatidia in the dorsal area contained only retinal. This was confirmed when the large facet region in the dorsal part of the compound eye of an Anax was excised and examined, and only retinal was detected. However, the ventral area of the true dragonflies' compound eye which did not include the large ommatidia contained both retinals, and the 3-OH ratio was more than ten. The biological significance of using both retinals as chromophores of visual pigments in the dragonfly eye is discussed in relation to the structure of the ommatidia and to the vision of dragonflies.

Topics: Animals; Chromatography, High Pressure Liquid; Eye; Insecta; Nymph; Retinaldehyde; Retinoids; Species Specificity

When the fruitfly, Drosophila melanogaster, was reared on media deficient in carotenoids and retinoids, the level of 3-hydroxyretinal (the chromophore of fly rhodopsin) in the retina decreased to less than 1% compared with normal flies. The level of 3-hydroxyretinal increased markedly in flies that were given a diet supplemented with retinoids or carotenoids. The retinas of flies fed on all-trans retinoids and maintained in the dark predominantly contained the all-trans form of 3-hydroxyretinal, and showed no increase in the level of either the 11-cis isomer or the visual pigment. Subsequent illumination of the flies converted substantial amounts of all-trans 3-hydroxyretinal to its 11-cis isomer. The action spectrum of the conversion by illumination showed the optimum wavelength to be approximately 420 nm, which is significantly greater than the absorption maximum of free, all-trans 3-hydroxyretinal. Flies that were fed on carotenoids showed a rapid increase of the levels of 11-cis 3-hydroxyretinal and of visual pigment in the absence of light.

Topics: Animals; Carotenoids; Drosophila melanogaster; Light; Photochemistry; Pigment Epithelium of Eye; Retinaldehyde; Retinoids

The bioluminescent squid, Watasenia scintillans has three visual pigments. The major pigment, based on retinal (lambda max 484 nm), is distributed over the whole retina. Another pigment based on 3-dehydroretinal (lambda max approximately 500 nm) and the third pigment (lambda max approximately 470 nm) are localized in the specific area of the ventral retina just receiving the downwelling light. Visual pigment was extracted and purified from the dissected retina. The chromophores were then extracted and analyzed with HPLC, NMR, infrared and mass spectroscopy, being compared with the synthetic 4-hydroxyretinal. A new retinal derivative, 11-cis-4-hydroxyretinal, is identified as the chromophore of the third visual pigment of the squid.

Topics: Animals; Chromatography, High Pressure Liquid; Decapodiformes; Diterpenes; Luminescent Measurements; Magnetic Resonance Spectroscopy; Retina; Retinaldehyde; Retinoids; Spectrophotometry, Ultraviolet

All-trans and 11-cis 3-hydroxyretinals were synthesized and the presence of these substances in the head of Drosophila melanogaster was shown by using high performance liquid chromatography. Even when the head extract was prepared in the dark from the flies reared successively in the dark, both of the 3-hydroxyretinal isomers were detected. In the culture medium, they were not present. D. melanogaster must have an 11-cis 3-hydroxyretinal forming-system that does not need light.

Topics: Animals; Chromatography, High Pressure Liquid; Darkness; Drosophila melanogaster; Retinaldehyde; Retinoids