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J. Biol. Chem., Vol. 279, Issue 48, 50230-50242, November 26, 2004
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From the
Departments of Ophthalmology, ¶Pharmacology, and ||Chemistry, University of Washington, Seattle, Washington 98195
Received for publication, August 10, 2004 , and in revised form, September 7, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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max = 290 nm, compared with the parent compound, all-trans-retinol (max = 325 nm), and its MS analysis (m/z = 288) indicates saturation of a double bond. The product was further identified as all-trans-13,14-dihydroretinol, since its characteristics were identical to those of a synthetic standard. Mouse RetSat is membrane-associated and expressed in many tissues, with the highest levels in liver, kidney, and intestine. All-trans-13,14-dihydroretinol was also detected in several tissues of animals maintained on a normal diet. Thus, saturation of all-trans-retinol to all-trans-13,14-dihydroretinol by RetSat produces a new metabolite of yet unknown biological function. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to dietary sources, retinoids are derived from the cleavage of C40 provitamin A carotenoids such as 
- and 
-carotene and cryptoxanthin to produce retinal, which can be converted to all-trans-retinol. Provitamin A carotenoids also represent a major storage form of retinoids in tissues, serum, and the vertebrate egg yolk. Only two enzymes involved in the metabolism of -carotene in animals have been identified: ,-carotene-15,15'-monooxygenase (BCO-I) (20), which carries out the symmetric cleavage of -carotene to produce retinal, and ,-carotene-9',10'-oxygenase (BCO-II), which carries out the asymmetric cleavage to generate -ionone and -apo-10'-carotenal (21). BCO-I and -II have sequence similarity to VP14, the 9-cis-neoxanthin cleavage enzyme from Zea mais, and other carotenoid cleavage enzymes from plants (reviewed in Ref. 22). BCO-I was first identified in flies based on its similarity to VP14 (20) and later cloned from mice (23) and humans (24). Other, more limited dietary sources of retinoids are all-trans-retinyl esters and free all-trans-retinol. In addition to -carotene, retinal, and retinoic acid, animal tissues also retain considerable amounts of nonprovitamin A carotenoids such as lutein and zeaxanthin in the primate macula and lycopene in serum and most tissues. Nonprovitamin A carotenoids as well as uncleaved -carotene have been implicated in the prevention of cancer, macular degeneration, and heart disease (reviewed in Refs. 25 and 26). Despite this interest, the enzymes involved in the metabolism and physiology of carotenoids in animals await molecular identification.
Powerful genetic approaches and readily identifiable phenotypes aided in the discovery of the biochemical pathways of carotenoid synthesis in plants and bacteria. These enzymes could serve as a model to uncover carotenoid or retinoid enzymes in vertebrates. Recently, the enzymes responsible for the isomerization of (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene (also known as prolycopene) to all-trans-lycopene in plants (27, 28) and cyanobacterium Synechocystis (29, 30) (Scheme 1) have been characterized (reviewed in Ref. 31). Tomato CRTISO mutants are known for their tangerine phenotype due to accumulation of (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene (32). The protein sequences of CRTISO enzymes are similar to CrtI from nonphotosynthetic bacteria, an enzyme that catalyzes the direct conversion of phytoene into all-trans-lycopene (33). It also bears resemblance to phytoene desaturase and 
-carotene desaturase from plants, which each introduce two double bonds during the four desaturation steps from phytoene to (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene (trans-H elimination at 11,11' by phytoene desaturase and cis-H elimination at 7,7' by -carotene desaturase) (reviewed in Ref. 34).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inducible Expression of LRAT Protein in HEKK Cells—Mouse LRAT cDNA was cloned as described elsewhere (2). For expression, LRAT coding region was amplified using the primers 5'-GCCACCATGAAGAACCCAATGCTGGAAGCT-3' and ACATACACGTTGACCTGTGGACTG. The PCR product was ligated into the pCR-Blunt II-TOPO vector (Invitrogen) and then subcloned into the EcoRI site of pcDNA4/TO. TetR-expressing HEKK cells were transfected with the pcDNA4/TO-LRAT construct and selected with zeocin. Stable clones were verified for expression of LRAT protein using the anti-LRAT monoclonal antibody described elsewhere (2).
Purification of a Bacterially Expressed His-tagged Mouse RetSat Fragment, Polyclonal and Monoclonal Antibody Production—The NcoI fragment of RetSat cDNA, corresponding to nucleotides 440–1391 of mouse RetSat cDNA (coding for the 148MASPF... MTALVPM465 polypeptide fragment) was cloned into the NcoI site of the inducible bacterial expression vector pET30B (Invitrogen). This resulted in a recombinant protein tagged at both amino and carboxyl termini with hexahistidine tags. The plasmid was transformed into BL-21RP cells (Stratagene), and expression was induced with isopropyl-1-thio--D-galactopyranoside. The double His6-tagged fragment of the mouse RetSat protein (40 kDa) was purified by Ni2+-nitrilotriacetic acid affinity using the manufacturer's protocol (Qiagen, Valencia, CA). The purified protein was examined by gel electrophoresis. Following in-gel trypsin digestion, the eluted tryptic peptides were examined by microsequencing by liquid chromatography/mass spectrometry to verify the identity of the recombinant RetSat fragment (not shown). The purified protein was used to immunize mice as described before (35), and the monoclonal antibody was produced by established methods (36). Rabbit polyclonal antiserum was raised in collaboration with Cocalico Biologicals Inc. (Reamstown, PA). The sera and monoclonal antibody were tested for their specificity by immunocytochemistry and immunoblotting of RetSat-transfected versus untransfected cells. Anti-RetSat IgG was purified from the ascitic supernatant of RetSat-producing hybridoma cells using a HiTRAP protein G HP (Amersham Biosciences), using the manufacturer's protocol. The purified antibody was coupled with fluorophore using the Alexa Fluor 488 monoclonal antibody coupling kit (Invitrogen) following the manufacturer's protocol.
Northern Blot Analysis of Mouse RetSat Transcripts—Northern blot analysis was performed using a commercially available premade blot containing 2 µg of poly(A) RNA from various mouse tissues per lane (FirstChoice Northern blot Mouse Blot I; Ambion) following the manufacturer's protocol. The -32P-radiolabeled probe was constructed by run-off PCR of mouse RetSat cDNA using the 5'-TCTGGCTCTTCTCTGAACGGACTACATC-3' reverse primer and the Strip-EZ probe synthesis kit from Ambion following the manufacturer's protocol. Alternatively, a radiolabeled antisense mouse beta-actin probe was constructed using the T7 primer and the pTRIamp18 -actin template (Ambion).
Immunoblotting and Immunohistochemistry Analysis of Mouse RetSat—To establish the membrane association of RetSat, mouse liver was homogenized in 50 mM Tris-HCl, pH 8.0, containing 250 mM sucrose, 5 mM dithiothreitol, and 1x protease inhibitor mixture (Sigma) using a Dounce homogenizer. The nuclei and extracellular matrix were pelleted by centrifugation for 30 min at 20,000 x g and discarded. The high speed cytosolic supernatant and postnuclear membranes were separated by centrifugation at 145,000 x g for 90 min. Postnuclear membranes were homogenized in 10 mM Tris, pH 8.0, containing 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 10 µM phenylmethylsulfonyl fluoride. The protein concentration was measured in whole cell lysate, high speed cytosolic supernatant, and postnuclear membrane fraction using the Bradford assay (37). Equal amounts of protein were resolved on SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and stained with a 1:1000 dilution of anti-RetSat monoclonal antibody and 1:104 goat anti-mouse IgG (Fc) (Promega, Madison, WI). To examine tissue-specific expression of RetSat, various mouse tissues were dissected and homogenized with 10 mM Tris, pH 8.0, containing 10 mM 2-mercaptoethanol and 10 µM phenylmethylsulfonyl fluoride, with the aid of a Dounce homogenizer. The membranes were pelleted by centrifugation at 12,000 x g for 30 min. The protein concentration was measured using the Bradford assay (37). Equal amounts of protein (10 µg) from the membrane fraction of each tissue were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and stained by immunoblotting with a 1:1000 dilution of rabbit anti-RetSat polyclonal antiserum and alkaline phosphatase-coupled 1/104 goat anti-rabbit IgG (Fc) (Promega) secondary antibody. The mouse monoclonal anti-RetSat showed the same reactivity in the examined tissues as the polyclonal antiserum. Untransfected HEKK or HEKK-RetSat cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) on glass bottom microwell dishes (MatTek Corp., Ashland, MA). Expression of RetSat was induced by the addition of 1 µg/ml tetracycline. Cells were harvested after 48 h and fixed with 4% paraformaldehyde (Fisher, Hampton, NH) in PBS (136 mM NaCl, 11.4 mM sodium phosphate, pH 7.4) for 10 min and washed by PBS. To block nonspecific labeling, the cells were incubated in 1.5% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) in PBST (136 mM NaCl, 11.4 mM sodium phosphate, 0.1% Triton X-100, pH 7.4) for 15 min at room temperature. The cells were incubated overnight at 4 °C in Alexa 488-coupled anti-RetSat monoclonal IgG diluted with PBST. The sections were rinsed in PBST and mounted in 50 µl of 2% 1,4-diazabicyclo-[2.2.2]octane (Sigma) in 90% glycerol to retard photobleaching. For confocal imaging, the cells were analyzed on a Zeiss LSM510 laser-scanning microscope.
Retinol Isomer Purification and HPLC Analysis of Retinoids—All procedures involving retinoids were performed under dim red light unless otherwise specified. Retinoids were stored in N,N-dimethylformamide under argon at -80°C. All retinol and retinal substrates were purified by normal phase HPLC (Beckman Ultrasphere-Si, 5 µm, 4.6 x 250 mm) with 10% ethyl acetate, 90% hexane at a flow rate of 1.4 ml/min using an HP1100 high performance liquid chromatograph with a diode-array detector and HP Chemstation A 08.03 software. For all-trans-13,14-dihydroretinol, we used an extinction coefficient 
= 16,500 at 290 nm. The following extinction coefficients were used for retinoids (in M-1 cm-1): all-trans-retinol, = 51,770 at 325 nm; 9-cis-retinol, = 42,300 at 323 nm; 11-cis-retinol, = 34,320 at 318 nm; 13-cis-retinol, = 48,305 at 328 nm; all-trans-retinal, = 48,000 at 368 nm in hexane (38); and all-trans-retinoic acid, = 45,300 at 350 nm in ethanol. Retinoic acid was dissolved in ethanol and examined by a reverse-phase HPLC System II (Zorbax ODS, 5 µm, 4.6 x 250 mm; Agilent, Foster City, CA) with an isocratic mobile phase of 70% acetonitrile, 29% water, 1% glacial acetic acid and flow rate of 1.4 ml/min.
(7Z,9Z,9'Z,7'Z)-Tetra-cis-lycopene Purification and Reverse-phase HPLC Analysis of Carotenoids—Carotenoid extraction and analysis were performed under dim red light. One g from the fruit of a tangerine tomato was freeze-thawed three times and extracted with 2 ml of PBS, 2 ml of ethanol, and 6 ml of hexane with the aid of a Dounce homogenizer. The organic phase was dried and resuspended in ethanol/tetrahydrofuran (9:1) and examined by reverse-phase HPLC System I (Prontosil, 200-3-C30, 3 µm, 4.6 x 250 mm; Bischoff Chromatography, Leonberg, Germany) with a mobile phase of 75% tert-butyl methyl ether, 25% methanol and a flow rate of 1 ml/min. More than 90% of the extract consisted of (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene, which was identified based on its published UV-visible absorption spectrum in hexane (37) with the following characteristics: shoulder at = 417 nm, = 90,000 M-1 cm-1, max = 437 nm, = 105,000 M-1 cm-1, shoulder at = 461 nm, = 70,000 M-1 cm-1.
Enzyme Assays of RetSat- and CRTISO-catalyzed Reactions—For enzyme assay, cells were seeded in 6-well plates, and expression of RetSat or CRTISO was induced with 1 µg/ml tetracycline 48 h prior to analysis. Substrate preparation and addition were conducted under dim red light. Retinoid substrates were purified by HPLC as described above and dissolved in N,N-dimethylformamide to a final concentration of 4 mM. Organic extract of tangerine tomatoes was dried under a stream of argon and resuspended in N,N-dimethylformamide. The substrates were diluted in 300 µl of complete medium (tetracycline, 1 µg/ml) to a 40 µM final concentration, overlaid on cells, and incubated overnight in the dark at 37 °C in 5% CO2 and 100% humidity. Media and cells were collected by scraping and mixed with an equal volume of methanol. For retinol and dihydroretinol analysis, the methanol/water mixture was extracted with two volumes of hexane, and then the organic phase was dried, resuspended in hexane, and analyzed by normal phase HPLC. Retinal and dihydroretinal analysis was performed by treatment of the methanol/water mixture with 12.5 mM hydroxylamine followed by organic extraction and normal phase HPLC. For carotenoid analysis, the organic phase was dried and resuspended in ethanol/tetrahydrofuran (9:1) and examined by reverse-phase HPLC System I. For retinoic acid analysis, the 1:1 methanol/water mixture was acidified with 0.1 volumes of 12 N HCl and extracted with 1 volume of chloroform, dried, and resuspended in ethanol and examined by reverse-phase HPLC System II.
Chemical Synthesis of All-trans-13,14-dihydroretinol—All reagents were purchased from Sigma or Fluka and were used without additional purification. Solvents were dried under standard procedures prior to use. All operations with retinoids were performed under dim red light unless otherwise specified. -Ionone was condensed with triethyl phosphonoacetate in anhydrous tetrahydrofuran in the presence of NaH to give ethyl trans--ionylideneacetate. This ester was then reduced with LiAlH4 to alcohol and reacted overnight with triphenylphosphine hydrobromide to give Wittig salt. Ethyl 4-oxo-3-methylcrotonate was hydrogenated in methanol with H2 using 10% palladium on carbon as a catalyst to yield ethyl 4-oxo-3-methylbutyrate, which was then reacted with Wittig salt using t-BuOK as a base in anhydrous CH2Cl2 in the presence of 18-crown-6. The obtained mixture of ethyl 11-cis- and all-trans-13,14-dihydroretinoates was reduced with LiAlH4 to 13,14-dihydroretinols, and all-trans-isomer was separated from 11-cis-dihydroretinol by flash chromatography of silica gel using 5% ethyl acetate in hexane. NMR data were recorded on a Bruker 500-MHz spectrometer using CDCl3 as an internal standard. 1H NMR analysis of synthetic all-trans-13,14-dihydroretinol: NMR (CDCl3, 
, ppm) 6.41 (dd, 1H, H-12, J = 11.3, 14.75 Hz), 6.10–6.12 (m, 3H, H-7, H-8, H-10, J = 15.7 Hz), 5.6 (dd, 1H, H-11, J = 8.34, 14.95 Hz), 3.67 (m, 2H, CH2-15), 2.41 (m, 1H, H-13, J = 6.7 Hz), 1.96 (m, 2H, CH2-14), 1.90 (s, 3H, CH3-9), 1.69 (s, 3H, CH3-5), 1.46 (m, 2H, CH2-2), 1.6 (m, 4H, CH2-3, CH2-4), 1.06 (d, 3H, CH3-13 J = 6.7 Hz), 1.00 (s, 6H, 2x CH3-1).
Isomerization and Electron Impact Mass Spectrometry Analysis of All-trans-13,14-dihydroretinol—Equal amounts (by UV absorbance) of the synthetic and biosynthetic compounds were resuspended in ethanol and exposed to sunlight for 30 min, followed by the addition of an equal volume of water and two volumes of hexane. The compounds were extracted, the organic phase was dried and resuspended in hexane, and the isomeric mixture was examined by normal phase HPLC. For MS analysis, the unknown biosynthetic metabolite and the chemically synthesized all-trans-13,14-dihydroretinol were purified by normal phase HPLC and analyzed by electron impact mass spectrometry analysis using a JEOL HX-110 direct probe mass spectrometer. Some of the ions in the fragmentation patterns of both samples were as follows: 288 [M]+, 273 [M-CH3]+, 243 [M-CH2CH2OH]+, 215 [M-CH(Me)(CH2)2OH]+, 202, 187, 159. The spectra are shown without manipulation.
Enzymatic Assays for Saturase Activity in Homogenized Cells—Cells were homogenized with 15 mM Tris-HCl, pH 8.0, containing 10 mM dithiothreitol and 0.32 M sucrose. One aliquot of cells was boiled for 10 min at 95 °C as a negative control. Cell aliquots of 200 µl were supplemented with 1 mM ATP and 40 µM all-trans-retinol final concentrations. Some aliquots were also supplemented with 0.4 mM NADH or 0.4 mM NADPH to regenerate the redox state of the reaction. The cell homogenate was incubated with retinol substrate with shaking at 37 °C for 1 h in the dark. This was followed by the extraction of retinoids with one volume of methanol and two volumes of hexane. The organic phase was dried and resuspended in hexane and then examined by normal phase HPLC.
Esterification Assay of All-trans-retinol and All-trans-13,14-dihydroretinol—HEKK-LRAT cells were homogenized in 250 mM sucrose, 10 mM Tris-HCl with the aid of a Dounce homogenizer. RPE microsomes were prepared as previously described (39). A substrate solution, 2 µl of 1 mM stock in N,N-dimethylformamide, was added to a 1.5-ml Eppendorf tube containing 20 µl of 10% bovine serum albumin and 20 µl of UV-treated RPE microsomes or 100 µl of membrane homogenate of HEKK-LRAT cells and 10 mM BTP (pH 7.5) buffer to a total volume of 200 µl. The reactions were incubated at 37 °C for 10 min. Retinoids were extracted with 300 µl of methanol and 300 µl of hexane. Then 100 µl of the hexane extract was analyzed by normal phase HPLC, using first 0.5% ethyl acetate in hexane for 10 min to separate retinyl esters and then 20% ethyl acetate in hexane for an additional 10 min to separate retinols. Elution was monitored at 290 and 325 nm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cDNA for mouse and macaque monkey RetSat orthologous proteins was cloned from reverse transcribed RNA from the retina and RPE. Sequencing of several independent clones ensured that the sequence was verified. The sequence of the submitted mouse RetSat cDNA (AY704159 [GenBank] ) has five bases that are different from the sequence available in the data base (gi18483252), two of which result in amino acid changes. In a previous study, rat RetSat expression and other gene products were identified as down-regulated in rat mammary adenocarcinomas, and the rat cDNA was tentatively designated rat mammary tumor-7 (43). No further biochemical characterization of the enzyme was carried out. The sequence we deposited to GenBankTM for mouse RetSat (AY704159 [GenBank] ) corresponds perfectly to multiple expressed sequence tag sequences, and it is more similar to human RetSat than the sequence currently available in the data base. Monkey RetSat protein (GenBankTM submission AY707524 [GenBank] ) has 97% conserved substitutions, including 94% identical residues with the human protein sequence available in the data base (gi46329587).
Characterization of the Tissue Distribution and Subcellular Localization of Mouse RetSat Protein—Mouse RetSat expression was examined by Northern blot analysis using a radiolabeled antisense RetSat probe and a commercially available premade blot containing equal amounts of RNA from various tissues. RetSat mRNA appears as a 2200-bp transcript expressed predominantly in the liver and kidney among the tissues examined (Fig. 1D, a, top panel). We also examined the expression of the 2100-bp nonmuscle -actin mRNA in the same tissues to verify the quality of the RNA on the blot (44) (Fig. 1D, a, bottom panel). Greater amounts of RNA from spleen and lung tissues are present, based on the level of actin detected. Despite this, RetSat mRNA cannot be detected in the corresponding lanes of spleen and lung in the top panel of Fig. 1D (a), whereas it is clearly present in the kidney and liver at the same exposure of the blot (30 min). Very low levels of RetSat were detectable only after much longer exposure of the blot (5 h) in other tissues besides kidney and liver. This was confirmed by reverse transcription-PCR, indicating that RetSat is expressed predominantly in the kidney and liver and at very low levels in many other tissues examined (not shown). A rabbit polyclonal antiserum and a monoclonal antibody were prepared against recombinant mouse RetSat. For both mice and rabbit immunogens, a bacterially expressed fragment of the mouse RetSat protein was used as antigen. The recombinant protein fragment was chosen to eliminate the putative dinucleotide-binding domain that may result in cross-reaction with related proteins. Glycosylation-deficient HEK cells obtained from Dr. Khorana (45), HEKK, were transfected with the tetR gene and mouse RetSat cDNA under the control of the tetracycline (Tet)-inducible promoter. Stable clones of transfected cells were selected, pooled, and used for further analysis. These cells were designated HEKK-RetSat. Both polyclonal (Fig. 1D, b) and monoclonal antibodies (Fig. 2D) reacted with a specific protein of 70 kDa, similar to the predicted mass of mouse RetSat protein and identical to the mass of the protein detected in Tet-induced HEKK-RetSat cells. Equal amounts of protein from several tissues were analyzed by SDS-PAGE and immunoblotting with anti-RetSat polyclonal antibody. RetSat protein was detected in many tissues, with the highest expression in liver, kidney, and intestine (Fig. 1D, b). This expression pattern was also confirmed by immunoblotting with the monoclonal anti-RetSat antibody (not shown).
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Tomato CRTISO and Mouse RetSat Exhibit Different Enzyme Activities—Tomato CRTISO was cloned from RNA isolated from the skin and pulp of a fresh red tomato fruit. Tomato CRTISO was expressed in HEKK cells under the control of an inducible promoter. The natural substrate of tomato CRTISO, (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene, was isolated by organic extraction of a tangerine tomato, which accumulates (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene (32). The tangerine tomato extract consisted mostly of (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene (greater than 90%) as determined by reverse-phase HPLC analysis and the UV absorbance spectrum of the main peak, which matched published spectra (peak S in Fig. 3A, a). The (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene exhibits a shifted absorbance maxima max of 440 nm compared with all-trans-lycopene max of 475 nm and has a distinct UV absorbance spectrum (46). Untransfected HEKK, RetSat- and CRTISO-expressing cells were incubated in the presence of (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene in the dark (Fig. 3A). The products of the reaction were analyzed by a reverse-phase HPLC System I. There was no difference in the profile of eluted carotenoids from either untransfected cells or RetSat-expressing cells (Fig. 3A, a and b). As expected, CRTISO-expressing cells converted the substrate (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene (peak S in Fig. 3A, c) into all-trans-lycopene, with a max of 475 nm (P in Fig. 3A, c). All-trans-lycopene was identified based on its absorbance spectrum and co-elution with an available standard obtained from Dr. Kurt Bernhard (CaroteNature GmbH, Lupsingen, Switzerland) and Dr. Regina Goralczyk (Roche Vitamins Ltd., Basel, Switzerland). CRTISO also catalyzed the conversion of (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene into a compound labeled 1 in Fig. 3A, which we tentatively identified as a 7,9-di-cis-lycopene isomer based on its absorbance spectrum (46). This observation would suggest a two-step reaction mechanism for CRTISO, in which both cis-bonds of first one end and then the other of the carotenoid are isomerized. Thermally induced or light-induced isomerization can convert (7Z,9Z,9'Z,7'Z)-tetra-cis-lycopene into the (7Z,9Z)-di-cis-isomer at a slower rate, since this compound was also present in the original tomato extract (peak 1 in Fig. 3A, a and b) and reported by other investigators (47).
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max was 290 nm (peak P in Fig. 3B, b). The peak was absent in untransfected and CRTISO-expressing cells. cis-Isomers of retinol (peaks 2–4 in Fig. 3B) generated during the overnight incubation were present in all cells regardless of background. Based on the hypsochromic-shifted UV absorbance maximum of 290 nm, we deduced that the new compound has one fewer double bond as compared with the parent compound retinol exhibiting a max of 325 nm. A survey of the literature indicates that all-trans-13,14-dihydroretinal exhibits a maximum absorption at 289 nm (48). To prove the hypothesis that the unknown compound is all-trans-13,14-dihydroretinol, it was chemically synthesized as depicted in Scheme 2, purified by HPLC, and characterized by 1H NMR spectrum. The unknown compound produced by RetSat-expressing cells was purified by collecting the appropriate fraction from a normal phase HPLC. The purity of the unknown biosynthetic compound and synthetic all-trans-13,14-dihydroretinol was verified by normal phase HPLC (Fig. 4A, a and b). The amount of the purified unknown compound precluded us from conducting its 1H NMR analysis. However, both all-trans-13,14-dihydroretinol and the unknown compound exhibit the same chromatographic properties on normal phase HPLC, since they co-eluted as one peak when combined (Fig. 4A, c). The two compounds have identical UV absorbance spectra (Fig. 4B), and light-induced isomerization of equal amounts of the two compounds generates a series of cis-isomers identical in both elution profile and intensity (Fig. 4C). Acetylation of the synthetic all-trans-13,14-dihydroretinol and the extracted compounds produced ester compounds that co-eluted on a normal phase HPLC (data not shown). More importantly, MS analysis revealed that the biosynthetic compound has an m/z mass of 288, an increase of 2 daltons from the mass of the parent compound, all-trans-retinol (m/z = 286) (Fig. 4D, a, inset). This observed mass is the same as the mass of synthetic all-trans-13,14-dihydroretinol (Fig. 4D, b, inset). The MS fragmentation pattern of the both synthetic and biosynthetic compounds is identical (Fig. 4D, a and b). Since C13 becomes a chiral center in 13,14-dihydroretinol, further NMR analysis will be necessary to establish the absolute configuration of the biosynthetic compound. These findings lead us to propose that RetSat catalyzed the saturation reaction of the 13–14 double bond of all-trans-retinol as depicted in Scheme 3.
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max = 290 nm) were examined on the same HPLC system to establish product elution conditions (Fig. 6B and inset spectra). However, no dihydroretinal-oximes were detected in RetSat-expressing cells incubated with retinal (6–8-min elution time) (Fig. 6A). It was not possible to conclusively establish whether retinal is a substrate for RetSat given the rapid conversion of retinal to retinol in cultured cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vertebrate (13,14)-All-trans-retinol Saturase: An Ancient Enzyme—In addition to RetSat, enzymes involved in retinoid processing such as retinal dehydrogenase and CYP26 and one retinoic acid receptor can be found in the translation of the draft genomic sequence of the primitive chordates, the ascidians C. intestinalis and C. savignyi (51). The ascidian tadpole-larva contains a notochord and a dorsal tubular nerve cord much like a vertebrate tadpole and is considered a good approximation of the chordate ancestor. The acquisition of the anterioposterior organized body plan in chordates coincides with the innovation of RA and its nuclear receptor to control development. No retinoic acid receptors have so far been found in nonchordate species (52). Identification of a putative ascidian RetSat underscores the potential importance of the pathway that starts with the saturation of the 13–14 double bond of retinol. As retinoid metabolism evolved, chordate metabolism modified an existing enzyme, possibly an ancient phytoene desaturase, in order to create new metabolites with novel functions.
Carotenoid- and retinoid-modifying enzymes share many features determined by the highly related nature of their substrates. The 9-cis-neoxanthin cleavage enzyme from plants, VP14, is similar to ,-carotene-oxygenases BCO-I and -II from flies, ninaB, and vertebrates (22). Another vertebrate protein related to carotenoid cleavage enzymes is RPE65, which is essential for the production of 11-cis-retinol, a key step of the visual cycle (53). The function of RPE65 is not clear, since it was shown to bind retinyl esters (54), yet no catalytic role has been ascribed to it. In vertebrates, P450 enzymes CYP26A1 and -B1 convert retinoic acid (a diterpenoid) to hydroxylated metabolites (15, 17). Closely related P450 enzymes from plants hydroxylate abscisic acid, a sesquiterpene hormone that controls the plant life cycle, and taxol, a plant diterpenoid (55, 56). Based on its activity, RetSat is a retinoid-saturating enzyme related to carotenoid desaturases (phytoene desaturase, -carotene desaturase, and CrtI), whereas the primary amino acid sequence relates to CRTISO.
Structural Analysis of the RetSat Enzyme—Sequence analysis of the vertebrate RetSat family proteins reveals a dinucleotide-binding motif (40, 57), U4G(G/A)GUXGLX2(A/S)X2L(X6–12)UX(L/V)UE(X4)UGG(X9–13)(G/V)X3(D/E)XG, where U is a hydrophobic residue and X is any residue. Many proteins with this motif, including monoamine oxidases, protoporphyrinogen oxidases, and many phytoene desaturases, have been shown to be stimulated by FAD (58, 59), and others have been shown to be stimulated by NAD or NADP (60). The presence of a putative dinucleotide-binding motif in the sequence of RetSat argues that saturation of the double bond occurs through the transfer of a hydride (H-) ion from a reduced cofactor (NAD(P)H or FADH2) and a proton from the solution. This may explain the labile nature of RetSat in homogenized cells (i.e. cells in which the redox state has been altered).
We show that mouse RetSat is membrane-associated and appears to localize to the ER compartment of transfected cells. A cleavable signal sequence can be readily identified at the amino terminus of the protein, indicating that the protein is targeted to the ER membrane. In addition, a stretch of hydrophobic amino acids from residue 568 to 588 is a strong candidate for a transmembrane domain.
13,14-Dihydroretinols in Biological Systems—Retinoids containing saturated 13–14 double bonds such as 9-cis-13,14-dihydroretinoic acid and its taurine conjugate (61), 9-cis-4-oxo-13,14-dihydroretinoic acid (62), were previously identified in animals supplemented with 9-cis-retinoic acid or retinyl palmitate, respectively. Another saturated all-trans-13,14-dihydroxy-retinol was detected in retinol-treated lymphoblastoma 5/2 cells and was shown to support the proliferation of lymphocytes (63). The RetSat-catalyzed saturation reaction prefers all-trans-retinol as a substrate, which leads to specific synthesis of all-trans-13,14-dihydroretinol. Here we show that all-trans-13,14-dihydroretinol is detectable in unsupplemented animals (Fig. 9). It is preferable to demonstrate the existence of metabolite in vivo in animals maintained on a normal diet or receiving physiological levels of labeled precursor. This is the first report of this retinoid in vivo. Future studies will examine whether all-trans-13,14-dihydroretinol has biological activity or is metabolized to other active compounds. Although it is possible that all-trans-13,14-dihydroretinol is a breakdown product of all-trans-retinol, we find this unlikely, since retinol and retinoic acid are degraded through oxidation to polar catabolites. The precise role of all-trans-13,14-dihydroretinol in vivo remains to be established.
Is All-trans-13,14-dihydroretinol on the Pathway for the Degradation or Formation of All-trans-13,14-dihydroretinoic Acid?— The 13,14-dihydroretinoic acid and its catabolic product 4-oxo-13,14-dihydroretinoic acid have been previously identified in supplemented animals (61, 62). The 13,14-dihydroretinoic acid can be formed by oxidation of 13,14-dihydroretinol or by saturation of the 13–14 double bond of retinoic acid. In our assay system, RetSat cannot convert all-trans-retinoic acid into all-trans-13,14-dihydroretinoic acid, which implies that that the previously observed metabolites are downstream products of the oxidation of all-trans-13,14-dihydroretinol. The oxidation of all-trans-13,14-dihydroretinol most likely employs the same enzymes as all-trans-retinol, members of the short-chain alcohol dehydrogenase family or of the medium-chain alcohol dehydrogenase family, to generate all-trans-13,14-dihydroretinal and retinal dehydrogenase 1, 2, or 3 to further generate all-trans-13,14-dihydroretinoic acid. Given that previously observed metabolites were in 9-cis conformation, it is unclear at what stage 9–10 bond isomerization occurs and whether this reaction is enzymatic.
Relationship between Plant and Vertebrate Enzymes: A Productive Pathway of Discovery—Carotenoids and retinoids play essential roles in biology. Their unique light-absorbing properties allow carotenoids to mediate photosynthesis and photoprotection and allow retinoids to form the visual chromophore. Through metabolites they can also regulate gene expression as seen for abscisic and retinoic acid. The only natural source of carotenoids, and hence retinoids, are plants and photosynthetic bacteria. Although vertebrates do not synthesize carotenoids or retinoids, they are able to transform them to generate a unique series of metabolites. Vertebrate enzymes involved in carotenoid and retinoid processing probably evolved by changing the substrate of an existing terpenoid modifying enzyme or by reactivating an ancestral gene inherited from a common ancestor of animals, plants, and photosynthetic bacteria. Studying the relationship between plant and vertebrate enzymes is a productive pathway of discovery. Both carotenoid and retinoid biochemistry can gain a new level of understanding through cross-fertilization of the two fields.
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To whom correspondence may be addressed: Dept. of Ophthalmology, University of Washington, Box 356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-221-6784; E-mail: moise{at}u.washington.edu. ** To whom correspondence may be addressed: Dept. of Ophthalmology, University of Washington, Box 356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-221-6784; E-mail: palczews{at}u.washington.edu.
