Identification of all-trans-retinol:all-trans-13,14-dihydroretinol saturase

Abstract

Retinoids carry out essential functions in vertebrate development and vision. Many of the retinoid processing enzymes remain to be identified at the molecular level. To expand the knowledge of retinoid biochemistry in vertebrates, we studied the enzymes involved in plant metabolism of carotenoids, a related group of compounds. We identified a family of vertebrate enzymes that share significant similarity and a putative phytoene desaturase domain with a recently described plant carotenoid isomerase (CRTISO), which isomerizes prolycopene to all-trans-lycopene. Comparison of heterologously expressed mouse and plant enzymes indicates that unlike plant CRTISO, the CRTISO-related mouse enzyme is inactive toward prolycopene. Instead, the CRTISO-related mouse enzyme is a retinol saturase carrying out the saturation of the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol. The product of mouse retinol saturase (RetSat) has a shifted UV absorbance maximum, lambda(max) = 290 nm, compared with the parent compound, all-trans-retinol (lambda(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.

FIG. 1. Identification of vertebrate proteins with similarity to plant and cyanobacteria CRTISO

A, sequence comparison of human RetSat (RetSat Hom-gi46329587), macaque-monkey RetSat (RetSat Maq-AY707524 submitted sequence), mouse RetSat (RetSat Mus-AY704159 submitted sequence), and rat RetSat (RetSat Rat-gi34855900) with tomato CRTISO (CRTISO Lyc-gi19550437), Arabidopsis CRTISO (CRTISO Ara-gi42561764), and cyanobacterial CRTISO (CRTISO Syn-gi16331999). White letters on a black background represent identical residues. White letters on a gray background represent conserved substitutions in all but one of the species examined, whereas black letters on a light gray background indicate substitutions conserved in four of the seven species examined. The dashed lines represent gaps introduced to maximize the alignment. The alignment was built using the program T-Coffee and the matrix BLOSUM62 (64) with gap penalties: existence-11, extension-1. Sequence-based predictions such as the signal peptide and a putative dinucleotide binding motif are indicated. A phylogenetic tree of CRTISO-like enzymes was built using the ClustalW neighbor-joining distance algorithm with numbers indicating evolutionary distances (65) (B). The percentage similarity to human RetSat is indicated in parentheses beside the gene name. C, gene structure of human RetSat as it is found on the minus strand of chromosome 2 from 85,556,195 to 85,543,754. The numbered black boxes indicate exons, white boxes indicate untranslated regions, and lines represent introns. The length of each intron is indicated in kbp. The start (ATG) and stop of translation are also indicated. D, tissue distribution of mouse RetSat. a, Northern blot analysis of mouse RetSat expression in various mouse tissues (top panel) indicates that mouse RetSat is expressed predominantly in the liver and kidney among the tissues examined. Control hybridization was performed by stripping and reprobing of the same blot using an antisense probe to nonmuscle β-actin (bottom panel). The size of detected transcripts is shown at the right side of the panels. Lysates of various mouse tissues containing 10 µg of protein per lane were subjected to immunoblotting using rabbit polyclonal anti-mouse RetSat serum (b). The lane labeled HEKK-RetSat shows the immunoreactivity of the mouse RetSat protein from the lysate of Tet-induced, HEKK-RetSat cells corresponding to 1 µg of total loaded protein. There is no immunoreactive band in the lysate of untransfected cells immunoblotted with either rabbit polyclonal or mouse monoclonal antibody (not shown). The apparent molecular mass of mouse RetSat is 70 kDa and is indicated to the right of the panel.

FIG. 2. Subcellular localization of mouse RetSat in transfected cells

The anti-mouse RetSat monoclonal antibody (red) was used to stain Tet-induced HEKK-RetSat-transfected cells (A) and untransfected cells (B). HEKK-RetSat cells stained with the anti-RetSat monoclonal antibody examined under higher magnification show the perinuclear and reticular membrane localization of RetSat in transfected cells (C). Scale bar, 20µm. D, subcellular analysis of RetSat protein in mouse liver cells. Immunoblotting of equal amounts of protein from the cytosolic supernatant, postnuclear membrane fraction, and whole cell lysate of mouse liver cells indicates that the RetSat protein is membrane-associated. An immunoreactive band of a protein with apparent molecular mass of 70 kDa was identified as the mouse RetSat protein, confirmed by its presence in the lysate of Tet-induced HEKK-RetSat cells. The blots were probed with the anti-mouse RetSat monoclonal antibody.

FIG. 3. Enzyme activities of tomato CRTISO and mouse RetSat in transfected cells

A, analysis of the effect of tomato CRTISO and mouse RetSat on the conversion of (7Z,9Z,9′Z,7′Z)-tetra-cis-lycopene into all-trans-lycopene. Cells were incubated with (7Z,9Z,9′Z,7′Z)-tetra-cis-lycopene substrate (S), extracted, and examined by reverse-phase HPLC System I for the conversion of S into all-trans-lycopene product (P). The analysis indicates that the conversion occurs in cells expressing tomato CRTISO (c) but not in untransfected (a) or RetSat-expressing cells (b). A compound whose absorbance spectrum corresponds to 7,9-di-cis-lycopene was observed in all cells and more intensely in CRTISO-expressing cells (indicated by 1). B, analysis of the effect of tomato CRTISO and mouse RetSat on the conversion of all-trans-retinol into a new product. Cells were incubated with all-trans-retinol substrate (S), extracted, and examined by normal phase HPLC for the conversion of S into a novel product (P) whose maximum absorbance peak is 290 nm. The analysis indicates that the conversion occurs in cells expressing mouse RetSat (b) but not in untransfected (a) or CRTISO-expressing cells (c). Additional peaks with absorbance spectra corresponding to 13-cis-retinol (2), 9,13-di-cis-retinol (3), and 9-cis-retinol (4) were observed in all cells regardless of background and are most likely the result of thermal isomerization. The experiment was performed in duplicate samples and repeated. mAU, milliabsorbance units.

FIG. 4. Identification of the biosynthetic product of the conversion of all-trans-retinol by mouse RetSat

The HPLC-purified biosynthetic product of the RetSat reaction was compared with 13,14-dihydroretinol for its elution characteristics on normal phase HPLC (A). The retention times for both all-trans-13,14-dihydroretinol (a) and the biosynthetic product (b) are identical, and when mixed, the two compounds co-elute as a single peak (c). The absorbance spectrum for the two compounds is identical with a maximum absorbance at 290 nm (B). Both all-trans-13,14-dihydroretinol and the biosynthetic compound generate the same pattern of isomers following light-induced isomerization (C). Electron impact MS analysis of the biosynthetic product (a) and all-trans-13,14-dihydroretinol (b) shows that they have the same mass of 288 m/z, corresponding to retinol plus 2H (D). The base peak is shown in the inset. The MS fragmentation patterns of biosynthetic compound (a) and all-trans-13,14-dihydroretinol compound (b) show that they generate ions of the same mass and relative intensity. mAU, milliabsorbance units.

FIG. 5. Isomeric form of the substrate of mouse RetSat

Tet-induced HEKK-RetSat cells were incubated overnight with pure isomers of retinol (>95% pure by HPLC, assayed before incubation). Following incubation, retinoids were extracted and analyzed by normal phase HPLC. The appearance of 13,14-dihydroretinol isomers was monitored at 290 nm, since the absorbance maxima of most isomers of 13,14-dihydroretinol differ by less than 5 nm from 290 nm, the λ_max of all-trans-13,14-dihydroretinol (spectra not shown). In each panel an arrow indicates the substrate investigated to distinguish it from the additional retinol isomers that were generated by thermal isomerization during overnight incubation in tissue culture. The numbers indicate the identity of eluted peaks based on absorbance spectra and comparison with pure standards, specifically 13-cis-retinol (1), all-trans-13,14-dihydroretinol (2), 9-cis-retinol (3), all-trans-retinol (4), 9,13-di-cis-retinol (5), and 11-cis-retinol (6). No isomers of 13,14-dihydroretinol were detected other than the all-trans isomer. The retention times in the bottom right panel are slightly longer due to variations in the solvent system. The experiment was performed in triplicate and repeated.

FIG. 6. RetSat activity toward all-trans-retinal

A, analysis of retinal conversion in RetSat-expressing cells. Tet-induced HEKK-Ret-Sat or untransfected cells were incubated overnight with pure all-trans-retinal (>99% pure by HPLC, assayed before incubation). Following incubation, retinals were derivatized with hydroxylamine, extracted, and analyzed by normal phase HPLC. The appearance of syn- and anti-oximes of 13,14-dihydroretinal was monitored at 290 nm (expected 6–8 min after injection, as indicated). The peak numbers represent 13-cis-retinol (1), all-trans-13,14-dihydroretinol (2), and all-trans-retinol (3). B, synthetic standards of 13,14-dihydroretinal derivatized with hydroxylamine were examined by normal phase HPLC in order to establish product elution profile. The inset shows the spectra of the different isomers of 13,14-dihydroretinal-oximes.

FIG. 7. RetSat activity toward all-trans-retinoic acid

A, analysis of retinoic acid conversion in RetSat-expressing cells. Tet-induced HEKK-RetSat or untransfected cells were incubated overnight with pure all-trans-retinoic acid (>90% pure by HPLC, assayed before incubation). Following incubation, retinoic acid was extracted and analyzed by reverse- phase HPLC System II. The appearance of 13,14-dihydroretinoic acid isomers was monitored at 290 nm (expected 25–30 min after injection). The peak numbers represent 13-cis-retinoic acid (1), 9,13-di-cis-retinoic acid (2), 9-cis-retinoic acid (3), and all-trans-retinoic acid (4). B, mixture of isomers of synthetic standards of 13,14-dihydroretinoic acid were examined by reverse-phase HPLC System II in order to establish the product elution profile. The inset shows the spectra of the different isomers of 13,14-dihydroretinoic acid. *, an unrelated compound. The experiment was performed in triplicate samples and repeated.

FIG. 8. RetSat activity in homogenized cells

Untransfected cells (solid gray trace) or Tet-induced HEKK-RetSat cells (solid black trace) were homogenized and incubated with all-trans-retinol substrate, followed by retinoid extraction and normal phase HPLC analysis. The elution profile was monitored at 290 nm for the appearance of all-trans-13,14-dihydroretinol. In control samples (short dashed black trace) cell homogenate from HEKK-RetSat cells was boiled 10 min at 95 °C prior to incubation with substrate. The addition of 0.4 mM NADH or NADPH had no effect on the yield of all-trans-13,14-dihydroretinol. The experiment was performed in duplicate. The peak numbers represent 13-cis-retinol (1), all-trans-13,14-dihydroretinol (2), 9,13-di-cis-retinol (3), 9-cis-retinol (4), and all-trans-retinol (5). mAU, milliabsorbance units.

FIG. 9. Identification of all-trans-13,14-dihydroretinol in various tissues

Retinoids were extracted from mouse liver (0.3 g, top left panel), kidney (0.2 g, top right panel), bovine retina (0.2 g, bottom left panel), and RPE (0.2 g, bottom right panel) and examined by normal phase HPLC. The elution of 13,14-dihydroretinol was monitored at 290 nm. Based on its retention time and absorbance spectrum, a peak corresponding to all-trans-13,14-dihydroretinol was identified in all tissues examined; it elutes on normal phase HPLC between 13-cis-retinol (1) and 9,13-di-cis-retinol (2). Other peaks corresponding to all-trans-retinol (3) and 11-cis-retinol (4; bovine retina and RPE) were also identified. The experiment was performed in duplicate from tissues of different animals. The yield of all-trans-13,14-dihydroretinol was slightly higher (<10%) by saponification of the extract before HPLC analysis. mAU, milliabsorbance units.

FIG. 10. LRAT activity

Two nmol of retinols were incubated with RPE microsomes and with homogenized HEKKLRAT cells for 10 min. The production of esters was monitored by HPLC measuring absorbance at 325 nm for all-trans-retinol (black bars) and 290 nm for all-trans-14,13-dihydroretinol (gray bars). Protein concentrations were not equalized. No activity was observed in controls with protein boiled for 10 min at 95 °C. Experiments were performed in triplicate.

SCHEME 1. Reaction catalyzed by plant and cyanobacterial CRTISO

SCHEME 2. Synthesis of all-trans-13,14-dihydroretinol

a, (EtO)₂P(O)-CH₂COOEt. NaH, tetrahydrofuran, room temperature, 24 h; b, LiAlH₄, Et₂O, 0 °C, 30 min; c, Ph₃P·HBr, MeOH, room temperature, 24 h; d, H₂ (bar), MeOH, Pd/C, room temperature, 24 h; e, tert-BuOK, 18-crown-6, CH₂Cl₂, room temperature to −78 °C to room temperature, 12 h.

SCHEME 3. Reaction catalyzed by RetSat converting all-trans-retinol into all-trans-13,14-dihydroretinol