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Lecithin:Retinol Acyltransferase Is Responsible for Amidation of Retinylamine, a Potent Inhibitor of the Retinoid Cycle

2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês

10.1074/jbc.m509351200

1083-351X

Marcin Golczak, Yoshikazu Imanishi, Vladimir Kuksa, Tadao Maeda, Ryo Kubota, Krzysztof Palczewski,

Photochromic and Fluorescence Chemistry

Lecithin:retinol acyltransferase (LRAT) catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to all-trans-retinol (vitamin A) and plays an essential role in the regeneration of visual chromophore as well as in the metabolism of vitamin A. Here we demonstrate that retinylamine (Ret-NH2), a potent and selective inhibitor of 11-cis-retinal biosynthesis (Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8162-8167), is a substrate for LRAT. LRAT catalyzes the transfer of the acyl group onto Ret-NH2 leading to the formation of N-retinylpalmitamide, N-retinylstearamide, and N-retinylmyristamide with a ratio of 15:6:2, respectively. The presence of N-retinylamides was detected in vivo in mice supplemented with Ret-NH2. N-Retinylamides are thus the main metabolites of Ret-NH2 in the liver and the eye and can be mobilized by hydrolysis/deamidation back to Ret-NH2. Using two-photon microscopy and the intrinsic fluorescence of N-retinylamides, we showed that newly formed amides colocalize with the retinyl ester storage particles (retinosomes) in the retinal pigment epithelium. These observations provide new information concerning the substrate specificity of LRAT and explain the prolonged effect of Ret-NH2 on the rate of 11-cis-retinal recovery in vivo. Lecithin:retinol acyltransferase (LRAT) catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to all-trans-retinol (vitamin A) and plays an essential role in the regeneration of visual chromophore as well as in the metabolism of vitamin A. Here we demonstrate that retinylamine (Ret-NH2), a potent and selective inhibitor of 11-cis-retinal biosynthesis (Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8162-8167), is a substrate for LRAT. LRAT catalyzes the transfer of the acyl group onto Ret-NH2 leading to the formation of N-retinylpalmitamide, N-retinylstearamide, and N-retinylmyristamide with a ratio of 15:6:2, respectively. The presence of N-retinylamides was detected in vivo in mice supplemented with Ret-NH2. N-Retinylamides are thus the main metabolites of Ret-NH2 in the liver and the eye and can be mobilized by hydrolysis/deamidation back to Ret-NH2. Using two-photon microscopy and the intrinsic fluorescence of N-retinylamides, we showed that newly formed amides colocalize with the retinyl ester storage particles (retinosomes) in the retinal pigment epithelium. These observations provide new information concerning the substrate specificity of LRAT and explain the prolonged effect of Ret-NH2 on the rate of 11-cis-retinal recovery in vivo. In vertebrates, the retinoid cycle is essential for regeneration of the chromophore 11-cis-retinal, which is an integral part of rhodopsin and cone visual pigments (1Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2003; 65: 851-879Crossref PubMed Scopus (196) Google Scholar). All-trans-retinol is generated by the photoisomerization of 11-cis-retinal bound to opsins in photoreceptor cells or supplemented from circulation. It is trapped in the retinal pigment epithelium (RPE) 3The abbreviations used are: RPEretinal pigment epitheliumRet-NH2retinylamineLCALeber congenital amaurosisLRATlecithin:retinol acyltransferaseRANN-retinylacetamideRPNN-retinylpalmitamideHPLChigh performance liquid chromatographyMSmass spectrometryHEKhuman embryonic kidneybis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolDHPC1,2-diheptanoyl-sn-glycero-3-phosphocholineDGATacyl CoA:diacylglycerol acyltransferaseWTwild typeABCRATP-binding cassette transporter, retina-specific.3The abbreviations used are: RPEretinal pigment epitheliumRet-NH2retinylamineLCALeber congenital amaurosisLRATlecithin:retinol acyltransferaseRANN-retinylacetamideRPNN-retinylpalmitamideHPLChigh performance liquid chromatographyMSmass spectrometryHEKhuman embryonic kidneybis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolDHPC1,2-diheptanoyl-sn-glycero-3-phosphocholineDGATacyl CoA:diacylglycerol acyltransferaseWTwild typeABCRATP-binding cassette transporter, retina-specific. in the form of insoluble fatty acid esters in subcellular structures known as retinosomes (2Imanishi Y. Batten M.L. Piston D.W. Baehr W. Palczewski K. J. Cell Biol. 2004; 164: 373-383Crossref PubMed Scopus (157) Google Scholar, 3Imanishi Y. Gerke V. Palczewski K. J. Cell Biol. 2004; 166: 447-453Crossref PubMed Scopus (85) Google Scholar). The enzyme responsible for the esterification of all-trans-retinol in the small intestine, liver, and eye, lecithin:retinol acyltransferase (LRAT) (4MacDonald P.N. Ong D.E. Biochem. Biophys. Res. Commun. 1988; 156: 157-163Crossref PubMed Scopus (91) Google Scholar, 5MacDonald P.N. Ong D.E. J. Biol. Chem. 1988; 263: 12478-12482Abstract Full Text PDF PubMed Google Scholar), was cloned (6Ruiz A. Winston A. Lim Y.H. Gilbert B.A. Rando R.R. Bok D. J. Biol. Chem. 1999; 274: 3834-3841Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and its function has been proven in vivo (2Imanishi Y. Batten M.L. Piston D.W. Baehr W. Palczewski K. J. Cell Biol. 2004; 164: 373-383Crossref PubMed Scopus (157) Google Scholar, 7Batten M.L. Imanishi Y. Maeda T. Tu D.C. Moise A.R. Bronson D. Possin D. Van Gelder R.N. Baehr W. Palczewski K. J. Biol. Chem. 2004; 279: 10422-10432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Later in the retinoid cycle the 11-cis configuration of the retinal is restored by enzymatic isomerization (8McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (315) Google Scholar). retinal pigment epithelium retinylamine Leber congenital amaurosis lecithin:retinol acyltransferase N-retinylacetamide N-retinylpalmitamide high performance liquid chromatography mass spectrometry human embryonic kidney 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 1,2-diheptanoyl-sn-glycero-3-phosphocholine acyl CoA:diacylglycerol acyltransferase wild type ATP-binding cassette transporter, retina-specific. retinal pigment epithelium retinylamine Leber congenital amaurosis lecithin:retinol acyltransferase N-retinylacetamide N-retinylpalmitamide high performance liquid chromatography mass spectrometry human embryonic kidney 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 1,2-diheptanoyl-sn-glycero-3-phosphocholine acyl CoA:diacylglycerol acyltransferase wild type ATP-binding cassette transporter, retina-specific. The key step in the transformation of all-trans-retinal to 11-cis-retinal is the isomerization reaction. Recently a candidate protein approach and expression cloning demonstrated that RPE65 exhibits Fe2+-dependent isomerization activity (9Redmond T.M. Poliakov E. Yu S. Tsai J.Y. Lu Z. Gentleman S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13658-13663Crossref PubMed Scopus (322) Google Scholar, 10Moiseyev G. Chen Y. Takahashi Y. Wu B.X. Ma J.X. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12413-12418Crossref PubMed Scopus (402) Google Scholar, 11Jin M. Li S. Moghrabi W.N. Sun H. Travis G.H. Cell. 2005; 122: 449-459Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). We have suggested that the regeneration of the chromophore might occur through a retinyl carbocation intermediate (12McBee J.K. Kuksa V. Alvarez R. de Lera A.R. Prezhdo O. Haeseleer F. Sokal I. Palczewski K. Biochemistry. 2000; 39: 11370-11380Crossref PubMed Scopus (87) Google Scholar) and demonstrated that isomerization is inhibited by positively charged retinoids (13Golczak M. Kuksa V. Maeda T. Moise A.R. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8162-8167Crossref PubMed Scopus (104) Google Scholar). This mechanism would be consistent with Fe2+-catalyzed alkyl cleavage of the retinyl esters. Retinylamine (Ret-NH2) potently and selectively inhibits the isomerization step of the retinoid cycle in vitro and in vivo, whereas modifications of the amino group lead to loss of inhibitory potency. Surprisingly Ret-NH2 has a long lasting effect, and when mice were treated with a single dose of the inhibitor, its inhibitory effect on the cycle was observed for several days (13Golczak M. Kuksa V. Maeda T. Moise A.R. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8162-8167Crossref PubMed Scopus (104) Google Scholar). Inhibition of the retinoid cycle may have implications in averting light damage to photoreceptors in some instances or preventing the accumulation of toxic condensation side products of bleached chromophore, all-trans-retinal, in the RPE. In Stargardt disease, a disease associated with mutations in the photoreceptor-specific ATP-binding cassette transporter (ABCR) (14Allikmets R. Singh N. Sun H. Shroyer N.F. Hutchinson A. Chidambaram A. Gerrard B. Baird L. Stauffer D. Peiffer A. Rattner A. Smallwood P. Li Y. Anderson K.L. Lewis R.A. Nathans J. Leppert M. Dean M. Lupski J.R. Nat. Genet. 1997; 15: 236-246Crossref PubMed Scopus (1093) Google Scholar) or elongation of the very long chain fatty acid-like 4 protein (ELOVL 4) (15Zhang K. Kniazeva M. Han M. Li W. Yu Z. Yang Z. Li Y. Metzker M.L. Allikmets R. Zack D.J. Kakuk L.E. Lagali P.S. Wong P.W. MacDonald I.M. Sieving P.A. Figueroa D.J. Austin C.P. Gould R.J. Ayyagari R. Petrukhin K. Nat. Genet. 2001; 27: 89-93Crossref PubMed Scopus (374) Google Scholar, 16Edwards A.O. Donoso L.A. Ritter III, R. Investig. Ophthalmol. Vis. Sci. 2001; 42: 2652-2663PubMed Google Scholar), the accumulation of all-trans-retinal is thought to be responsible for the formation of a component of a lipofuscin pigment called A2E. This fluorophore is accumulated during the course of the disease and, to a lesser extent, with age in normal individuals. It exerts a toxic effect on retinal cells, causing retinal degeneration and consequent loss of vision (17Finnemann S.C. Leung L.W. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3842-3847Crossref PubMed Scopus (230) Google Scholar, 18Sparrow J.R. Nakanishi K. Parish C.A. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1981-1989PubMed Google Scholar). In contrast with other less potent inhibitors such as 13-cis-retinoic acid or its metabolically active intermediates (19Radu R.A. Mata N.L. Nusinowitz S. Liu X. Sieving P.A. Travis G.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4742-4747Crossref PubMed Scopus (199) Google Scholar), Ret-NH2 does not activate the transcription of genes (13Golczak M. Kuksa V. Maeda T. Moise A.R. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8162-8167Crossref PubMed Scopus (104) Google Scholar), making it a safer candidate for therapeutic application. However, a total inhibition of 11-cis-retinoid production would resemble Leber congenital amaurosis (LCA), an autosomal recessive rod-cone dystrophy that presents itself at birth or the first few months of life. Rod and cone photoreceptor functions of the LCA patients are either absent or severely compromised at birth as evidenced by extinguished or barely detectable photopic and scotopic electroretinograms (20Perrault I. Rozet J.M. Gerber S. Ghazi I. Leowski C. Ducroq D. Souied E. Dufier J.L. Munnich A. Kaplan J. Mol. Genet. Metab. 1999; 68: 200-208Crossref PubMed Scopus (128) Google Scholar). LCA is highly heterogeneous; at least nine subtypes of LCA have been identified (see www.sph.uth.tmc.edu/RetNet/disease.htm) and linked to many unrelated genes. For enzymes of the retinoid cycle, numerous mutations in the RPE65 gene have been linked to LCA (21Perrault I. Hanein S. Gerber S. Barbet F. Ducroq D. Dollfus H. Hamel C. Dufier J.L. Munnich A. Kaplan J. Rozet J.M. Am. J. Hum. Genet. 2004; 75: 639-646Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 22Cremers F.P. van den Hurk J.A. den Hollander A.I. Hum. Mol. Genet. 2002; 11: 1169-1176Crossref PubMed Scopus (173) Google Scholar, 23Gu S.M. Thompson D.A. Srikumari C.R. Lorenz B. Finckh U. Nicoletti A. Murthy K.R. Rathmann M. Kumaramanickavel G. Denton M.J. Gal A. Nat. Genet. 1997; 17: 194-197Crossref PubMed Scopus (526) Google Scholar, 24Morimura H. Fishman G.A. Grover S.A. Fulton A.B. Berson E.L. Dryja T.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3088-3093Crossref PubMed Scopus (393) Google Scholar). Null mutations in the human LRAT and RDH12 genes lead to recessive early onset retinal dystrophy, a phenotype very similar to LCA (25Janecke A.R. Thompson D.A. Utermann G. Becker C. Hubner C.A. Schmid E. McHenry C.L. Nair A.R. Ruschendorf F. Heckenlively J. Wissinger B. Nurnberg P. Gal A. Nat. Genet. 2004; 36: 850-854Crossref PubMed Scopus (186) Google Scholar, 26Zernant J. Kulm M. Dharmaraj S. den Hollander A.I. Perrault I. Preising M.N. Lorenz B. Kaplan J. Cremers F.P. Maumenee I. Koenekoop R.K. Allikmets R. Investig. Ophthalmol. Vis. Sci. 2005; 46: 3052-3059Crossref PubMed Scopus (138) Google Scholar, 27Thompson D.A. Li Y. McHenry C.L. Carlson T.J. Ding X. Sieving P.A. Apfelstedt-Sylla E. Gal A. Nat. Genet. 2001; 28: 123-124Crossref PubMed Scopus (153) Google Scholar). Thus, a reduced but not blocked regeneration of rhodopsin could be beneficial for some forms of retinal dystrophies, particularly for a low dose and long lasting inhibitor. Here we provide evidence that Ret-NH2 is converted to pharmacologically inactive retinylamides in vitro and in vivo in the liver and RPE. We demonstrate that the enzyme responsible for the amidation is LRAT and that the amidation products are stored in retinosomes in the RPE. An equilibrium maintained between retinylamide and free Ret-NH2 results in the long lasting and highly potent inhibitory effect of Ret-NH2. Animals—All procedures using mice were approved by the Washington University Animal Care Committees and conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia and recommendations of the Association of Research for Vision and Ophthalmology. Animals were maintained in complete darkness, and all manipulations were done under dim red light using an Eastman Kodak Co. number 1 safelight filter (transmittance, >560 nm). Typically, 6-12-week-old mice were used in all experiments. Lrat-/- mice were obtained and genotyped as described previously (7Batten M.L. Imanishi Y. Maeda T. Tu D.C. Moise A.R. Bronson D. Possin D. Van Gelder R.N. Baehr W. Palczewski K. J. Biol. Chem. 2004; 279: 10422-10432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Animals were gavaged with 1 mg of Ret-NH2, N-retinylacetamide (RAN), and N-retinylpalmitamide (RPN) dissolved in 150 μl of vegetable oil 18 h prior to analysis. For gavage with radioactive retinoids, the total radioactivity per single dose did not exceed 10 μCi. Materials—Fresh bovine eyes were obtained from a local slaughter-house (Schenk Packing Co., Stanwood, WA). Preparation of bovine RPE microsomes was performed according to methods described by Stecher et al. (28Stecher H. Gelb M.H. Saari J.C. Palczewski K. J. Biol. Chem. 1999; 274: 8577-8585Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). 11,12-[3H]all-trans-retinol was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Retinoid Preparations—All retinoids were purified by normal phase HPLC (Beckman Ultrasphere-Si; 5 μm; 4.5 × 250 mm; detection at 325 nm; flow rate, 2 ml/min), and their concentrations were determined spectrophotometrically in EtOH. Absorption coefficients for Ret-NH2 and N-retinylamides were assumed to be equal to those of retinol and retinyl esters (29Hubbard R. Brown P.K. Bownds D. Methods Enzymol. 1971; 18: 615-653Crossref Scopus (191) Google Scholar, 30Robeson C.D. Cawley J.D. Weisler L. Stern M.H. Eddinger C.C. Chechak A.J. J. Am. Chem. Soc. 1955; 77: 4111-4119Crossref Scopus (89) Google Scholar). Chemical Synthesis—Ret-NH2 was prepared according to the method described by Golczak et al. (13Golczak M. Kuksa V. Maeda T. Moise A.R. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8162-8167Crossref PubMed Scopus (104) Google Scholar). N-Retinylacetamide and N-retinylpalmitamide were prepared by reacting Ret-NH2 with an excess of acetic anhydride and palmitoyl chloride, respectively, in anhydrous dichloromethane in the presence of N,N-dimethylaminopyridine at 0 °C for 1 h. After the reaction was completed (as judged by HPLC), water was added, and the product was extracted with hexane. The hexane layer was washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, filtered, and evaporated in a SpeedVac. Radioactive Ret-NH2 and N-retinylamides were synthesized starting from 11,12-[3H]all-trans-retinol, which was first oxidized with MnO2 (CH2Cl2, 20 °C, 4 h) to 11,12-[3H]all-trans-retinal. Ret-NH2 and retinylamides were then prepared using methods described previously (13Golczak M. Kuksa V. Maeda T. Moise A.R. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8162-8167Crossref PubMed Scopus (104) Google Scholar). N-Retinylheptanamide was prepared by dicyclohexylcarbodiimide-promoted coupling reaction. First dicyclohexylcarbodiimide was reacted with heptanoic acid in dichloromethane. Ret-NH2 in dichloromethane was added. The reaction mixture was incubated at room temperature for 3 h, extracted, and washed as described above. Mass spectrometry (MS) of synthesized retinoids was performed using a Kratos Analytical Instruments HV-3 direct probe mass spectrometer and electron-impact ionization. Inducible Expression of LRAT Protein in HEK Cells—Mouse LRAT cDNA was cloned as described elsewhere (7Batten M.L. Imanishi Y. Maeda T. Tu D.C. Moise A.R. Bronson D. Possin D. Van Gelder R.N. Baehr W. Palczewski K. J. Biol. Chem. 2004; 279: 10422-10432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). For expression, the LRAT coding region was amplified using the primers 5′-GCCACCATGAAGAACCCAATGCTGGAAGCT-3′ and 5′-ACATACACGTTGACCTGTGGACTG-3′. The PCR product was ligated into the pCR-Blunt II-TOPO vector (Invitrogen) and then subcloned into the EcoRI site of pcDNA4/TO. N-Acetylglucosaminyltransferase I-negative HEK293S cells (31Reeves P.J. Kim J.M. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13413-13418Crossref PubMed Scopus (172) Google Scholar), obtained from Dr. G. Khorana (Massachusetts Institute of Technology, Boston, MA), were transfected with the TetR expression plasmid pcDNA6-TR(blaR), and blasticidin-resistant colonies were selected and cloned. Cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum, and Zeocin and blasticidin antibiotics, and maintained at 37 °C, 5% CO2, and 100% humidity. TetR-expressing HEK 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 (7Batten M.L. Imanishi Y. Maeda T. Tu D.C. Moise A.R. Bronson D. Possin D. Van Gelder R.N. Baehr W. Palczewski K. J. Biol. Chem. 2004; 279: 10422-10432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). LRAT Activity Assay—The reaction was carried out in 10 mm bis-Tris propane buffer, pH 7.5, 1% bovine serum albumin. All-trans-retinol or Ret-NH2 was delivered in 0.8 μlof N,N-dimethylformamide to the final concentration of 20 μm. The reaction was initiated by adding 20 μl of bovine RPE microsomes or 50 μl of LRAT-expressing HEK cell lysate (∼150 mg of protein). The total volume of the reaction mixture was fixed at 200 μl. The reactions were incubated at 37 °C for the required times and then stopped by adding 300 μl of methanol followed by the same volume of hexane. Retinoids were extracted and analyzed on a Hewlett Packard 1100 series HPLC system equipped with a diode array detector. A normal phase column (Beckman Ultrasphere-Si, 5 μm, 4.5 × 250 mm) and a step gradient of ethyl acetate in hexane at a flow rate of 2 ml/min were used to elute N-retinylamides (10% ethyl acetate for 23 min and then 40% ethyl acetate up to 40 min). To detect Ret-NH2, retinoid separation was performed in 99.5% ethyl acetate with the addition of 0.5% of 7 n ammonia in methanol. Mouse Retinoid Extraction and Analysis—Two mouse eyes or 0.5 g of mouse liver were homogenized in a glass-to-glass homogenizer using 3 ml of 50% methanol in 20 mm bis-Tris buffer, pH 7.4. Retinoids were extracted with 4 ml of hexane. The organic phase was collected, dried down in a SpeedVac, and redissolved in 400 μl of hexane. In the case of the liver extract, 10 and 100 μl of retinoid solution were injected on an HPLC column for the detection of N-retinylamides and Ret-NH2, respectively. For samples from mouse eyes, 100 μl were analyzed for N-retinylamides and Ret-NH2. Separation conditions used for retinoid analysis were the same as described above. To determine the radioactivity distribution among retinoids found in the liver of animals gavaged with 11,12-[3H]all-trans-Ret-NH2 or 11,12-[3H]N-all-trans-retinylamide, products corresponding to the retinyl esters, retinol, retinylamides, and Ret-NH2 were collected during an HPLC run. Fractions containing retinoids were dried down in a SpeedVac and redissolved in 300 μl of N,N-dimethylformamide. The radioactivity of each fraction was examined by scintillation counting and normalized to total radioactivity of the sample injected on the column. Two-photon Vitamin A Imaging—Two-photon excitation microscopy was performed using a confocal/two-photon laser scanning microscope (LSM 510 MP-NLO; Carl Zeiss MicroImaging, Inc., Thornwood, NY) with LSM510 software version 3.0. Detailed methods were described in previous publications (2Imanishi Y. Batten M.L. Piston D.W. Baehr W. Palczewski K. J. Cell Biol. 2004; 164: 373-383Crossref PubMed Scopus (157) Google Scholar, 3Imanishi Y. Gerke V. Palczewski K. J. Cell Biol. 2004; 166: 447-453Crossref PubMed Scopus (85) Google Scholar). For localization of Ret-NH2 in the RPE cells, eyecup preparations were exposed to 0.25 mm Ret-NH2 caged with 100 mm (2-hydroxypropyl)-β-cyclodextrin for 15 min and washed briefly with Ames' medium (Sigma) for 3 min. Ames' medium was equilibrated with argon to purge O2 from the solution. Ret-NH2 Is Converted into N-Retinylamides upon Incubation with RPE Microsomes—HPLC analysis of the retinoids extracted from the standard isomerization assay (12McBee J.K. Kuksa V. Alvarez R. de Lera A.R. Prezhdo O. Haeseleer F. Sokal I. Palczewski K. Biochemistry. 2000; 39: 11370-11380Crossref PubMed Scopus (87) Google Scholar) showed no significant changes (within 5%) in the amount of Ret-NH2. When Ret-NH2 was incubated with RPE microsomes in the absence of all-trans-retinol, the level of Ret-NH2 dropped significantly with time concomitantly with the appearance of a new peak that eluted with the front of the HPLC column (Fig. 1A, top panel). Putative products of Ret-NH2 conversion were separated in 10% ethyl acetate/hexane, revealing the presence of three different compounds (Fig. 1A, bottom panel). Based on the elution time from a normal phase HPLC column and the shape and maximum of UV absorbance spectra, these products were retinoids less polar than Ret-NH2 (Fig. 1A). As measured by the disappearance of Ret-NH2, the observed reaction progress was much slower than the esterification of all-trans-retinol driven by LRAT (Fig. 1B). The unknown compounds 1, 2, and 3 were separated and purified by collecting the appropriate fractions from a normal phase HPLC column (Fig. 1A). The purity of biosynthetic products was examined by HPLC (Fig. 2A, a) prior to MS analysis. The biosynthetic compounds have an m/z of 551, 523, and 495 with respect to their order of elution from an HPLC column (Fig. 2B, a, b, and c). These observed masses correlate with the masses of N-retinylamides possessing C18, C16, and C14 acyl groups within their structure. To collect more evidence, the MS pattern of synthetic N-retinylpalmitamide was compared with the most abundant biosynthetic product (Fig. 2A, a, peak 2). The molecular ion peak and MS fragmentation patterns of both compounds are identical. Additionally comparison of elution time from the HPLC column and UV absorbance spectra revealed no differences between synthetic and biosynthetic products (Fig. 2A, b and c). Thus, we conclude that RPE microsomes converted Ret-NH2 into three main amides: RPN, N-retinylstearamide, and N-retinylmyristamide produced in a ratio of 15:6:2, respectively. N-Retinylamides Can Be Detected in Tissues of Mice Gavaged with Ret-NH2—The potential application of Ret-NH2 as an inhibitor of the retinoid cycle in vivo led us to investigate whether N-retinylamides can be detected in mouse tissue. Mice were gavaged with 1 mg of Ret-NH2 18 h prior to retinoid analysis. The presence of N-retinylamides in the liver and eye extracts could be easily detected by normal phase HPLC chromatography (Fig. 3). N-Retinylamides were recognized based on comparison of their UV absorbance spectrum and elution time. Interestingly, the amount of Ret-NH2 found in the examined tissues was smaller than the amount of N-retinylamides, 4 and 15 nmol, respectively, in the livers and 102 and 140 pmol found in the eyes (Fig. 3, insets). These results suggest that Ret-NH2 is efficiently converted into N-retinylamides in vivo. Ret-NH2 Is Amidated Due to LRAT Activity—To identify the enzyme that is responsible for amidation, we tested whether LRAT can utilize Ret-NH2 as a substrate. First we carried out qualitative analysis of retinyl esters and N-retinylamides formed in an excess of 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC). DHPC, present in millimolar concentration in the reaction mixture containing RPE microsomes and all-trans-retinol, serves as a donor of the C7 acyl group that is efficiently transferred onto all-trans-retinol. Consequently retinyl heptanoate was produced and was identified based on its mass spectrum and elution profile from normal phase HPLC in comparison with a synthetic standard (Fig. 4A). The same experiment performed in the presence of Ret-NH2 led to the formation of an amide containing a short acyl chain whose mass and elution time from the normal phase HPLC column perfectly matched the properties of N-retinylheptanamide (Fig. 4B). Considering transfer of the heptanoyl group from DHPC onto both all-trans-retinol and Ret-NH2, we conclude that LRAT could be responsible for both enzymatic activities. In addition, the production of N-retinylamides in LRAT-expressing HEK cells was investigated. Prior to the experiments, LRAT-expressing HEK cells were examined for expression and activity of the enzyme by immunoblotting and standard assays (Fig. 5B, a and b). Cells were harvested and homogenized, and the lysate was incubated in the presence of Ret-NH2. The products of the reaction were analyzed by normal phase HPLC. This analysis revealed striking differences between the retinoid composition extracted from untransfected and LRAT-expressing cells. In the presence of LRAT, products corresponding to N-retinylamides were observed (Fig. 5A, top panel) that correlate with the decrease of Ret-NH2 to below detection limits (Fig. 5A, bottom panel). In the control cells, the unreacted substrate was observed, and no amides were formed (Fig. 5A). Quantification of the retinoids found in the examined samples is shown in Fig. 5B, c. Additionally incubation of the cell lysate with Ret-NH2 led to an elevation of all-trans-retinol that was observed directly by HPLC in untransfected cells. In the case of LRAT-expressing cells, the amount of retinyl esters increased. Thus, as an alternative to amidation, Ret-NH2 can be also deaminated to all-trans-retinol. To further investigate the role of LRAT in Ret-NH2 acylation in vivo, we designed assays using Lrat-/- mice. Following gavage of Lrat-/- mice with synthetic Ret-NH2, we observed a complete lack of N-retinylamide formation in the examined livers (Fig. 6, top) concomitant with the detection of intact Ret-NH2 (Fig. 6, bottom). Together these observations demonstrate that LRAT utilizes Ret-NH2 as an acceptor of acyl groups. Additionally analysis of the chromatograms obtained from in vivo studies indicated that Ret-NH2 was transformed into vitamin A. This transformation was confirmed by the increased level of all-trans-retinol found in Lrat-/- mice gavaged with Ret-NH2 as well as the elevation of retinyl esters despite the absence of LRAT (Fig. 6, top). Without LRAT, retinol esterification most likely occurs due to the action of acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes (32O'Byrne S.M. Wongsiriroj N. Libien J.M. Vogel S. Goldberg I.J. Baehr W. Palczewski K. Blaner W.S. J. Biol. Chem. 2005; 280: 35647-35657Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 33Yen C.L. Monetti M. Burri B.J. Farese Jr., R.V. J. Lipid Res. 2005; 46: 1502-1511Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) or the recently described product of the GS2 gene (34Gao J. Simon M. J. Investig. Dermatol. 2005; 124: 1259-1266Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). N-Retinylamides Can Be Hydrolyzed Back to Ret-NH2 in Vivo—In contrast to Ret-NH2, N-retinylamides do not inhibit the formation of 11-cis-retinol in vitro (13Golczak M. Kuksa V. Maeda T. Moise A.R. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8162-8167Crossref PubMed Scopus (104) Google Scholar). We showed that Ret-NH2 has a long lasting affect on visual recovery (more than 48 h), which is surprising considering the efficient conversion of Ret-NH2 into N-retinylamides observed in vivo. It may be that once synthesized, N-retinylamides do not remain intact but are slowly hydrolyzed back to Ret-NH2. To address this hypothesis, WT mice were gavaged with 1 mg of RAN 18 h prior to HPLC analysis of retinoids extracted from liver. Based on the retention time from a normal phase HPLC column, RAN and C18, C16, and C14 N-retinylamides were identified (Fig. 7A). The presence