Lecithin-retinol Acyltransferase Is Essential for Accumulation of All-trans-Retinyl Esters in the Eye and in the Liver
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m312410200
ISSN1083-351X
AutoresMatthew L. Batten, Yoshikazu Imanishi, Tadao Maeda, Daniel C. Tu, Alexander R. Moise, Darin Bronson, Daniel E. Possin, Russell N. Van Gelder, Wolfgang Baehr, Krzysztof Palczewski,
Tópico(s)Photoreceptor and optogenetics research
ResumoLecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6–8-week-old Lrat-/- mice revealed that the rod outer segments are ∼35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation. Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6–8-week-old Lrat-/- mice revealed that the rod outer segments are ∼35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation. Lecithin-retinol acyltransferase (LRAT) 1The abbreviations used are: LRAT, lecithin-retinol acyltransferase; ANOVA, analysis of variance; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ERG, electroretinogram; HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid; PLR, pupillary light response(s); ROS, rod outer segment(s); RPE, retinal pigmented epithelium; Rpe65, gene for 65-kDa retinal pigment epithelium cell protein; WT, wild-type.1The abbreviations used are: LRAT, lecithin-retinol acyltransferase; ANOVA, analysis of variance; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ERG, electroretinogram; HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid; PLR, pupillary light response(s); ROS, rod outer segment(s); RPE, retinal pigmented epithelium; Rpe65, gene for 65-kDa retinal pigment epithelium cell protein; WT, wild-type. converts all-trans-retinol (vitamin A) to all-trans-retinyl esters in several tissues, including the liver, lung, pancreas, intestine, testis, and the retinal pigmented epithelium (RPE) (1Saari J.C. 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All-trans-retinyl esters are intermediate compounds in a metabolic pathway (“visual cycle” or “retinoid cycle”) that recycles 11-cis-retinal, the chromophore of rhodopsin and cone pigments (for review, see Refs. 8McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (314) Google Scholar, 9Bok D. Investig. Ophthalmol. Vis. Sci. 1985; 26: 1659-1694PubMed Google Scholar, 10Saari J.C. Investig. Ophthalmol. Vis. Sci. 2000; 41: 337-348PubMed Google Scholar). In this cycle, all-trans-retinal dissociates from rhodopsin and cone pigments after photobleaching. In the photoreceptors, all-trans-retinal is reduced to all-trans-retinol and subsequently exported to the adjacent RPE. In the RPE, all-trans-retinol is esterified by LRAT and stored. All-trans-retinyl esters have been suggested to be the substrate for a putative isomerohydrolase in the RPE (11Rando R.R. Biochemistry. 1991; 30: 595-602Crossref PubMed Scopus (76) Google Scholar) and for a retinyl ester hydrolase that produces all-trans-retinol, a substrate for the putative isomerase (for review, see Ref. 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). Ultimately, 11-cis-retinol is produced, oxidized to 11-cis-retinal, and exported to the photoreceptors. In the rod and cone photoreceptor outer segments, 11-cis-retinal recombines with opsins to form rhodopsin and cone pigments (for review, see Ref. 8McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (314) Google Scholar). Human LRAT cDNA was cloned from a retinal-RPE cDNA library (7Ruiz 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 (211) Google Scholar) and rodent Lrat cDNA from liver and other tissues (13Zolfaghari R. Wang Y. Chen Q. Sancher A. Ross A.C. Biochem. J. 2002; 368: 621-631Crossref PubMed Google Scholar, 14Zolfaghari R. Ross A.C. J. Nutr. 2002; 132: 1160-1164Crossref PubMed Scopus (43) Google Scholar, 15Zolfaghari R. Ross A.C. J. Lipid Res. 2000; 41: 2024-2034Abstract Full Text Full Text PDF PubMed Google Scholar). Lrat mRNA was shown to be a 5.0-kb species expressed in the RPE, and the multiple transcripts based on differential polyadenylation were detected in several other tissues known for the highest LRAT activity (13Zolfaghari R. Wang Y. Chen Q. Sancher A. Ross A.C. Biochem. J. 2002; 368: 621-631Crossref PubMed Google Scholar). The human LRAT polypeptide consisted of 230 amino acids and is predicted to be an integral membrane protein with two transmembrane domains. Based on the primary sequence, LRAT is a member of a large N1pC/P60 superfamily involved in murein degradation, amide hydrolysis, and acyl transfer (16Anantharaman V. Aravind L. Genome Biol. 2003; http://genomebiology.com/2003/2/3/reviews/0011Google Scholar). Eukaryotic members of this family include the class II tumor suppressor genes and the nematode developmental regulator Egl-26 (17Jahng W.J. Xue L. Rando R.R. Biochemistry. 2003; 42: 12805-12812Crossref PubMed Scopus (37) Google Scholar). Site-specific mutagenesis of recombinant human LRAT revealed that of four Cys residues, Cys161 is a nucleophile creating a thioacylenzyme intermediate, which is then transferred to all-trans-retinol (18Mondal M.S. Ruiz A. Bok D. Rando R.R. Biochemistry. 2000; 39: 5215-5220Crossref PubMed Scopus (42) Google Scholar, 19Mondal M.S. Ruiz A. Hu J. Bok D. Rando R.R. FEBS Lett. 2001; 489: 14-18Crossref PubMed Scopus (15) Google Scholar). Further mutagenesis experiments identified two His residues essential for LRAT catalytic activity (19Mondal M.S. Ruiz A. Hu J. Bok D. Rando R.R. FEBS Lett. 2001; 489: 14-18Crossref PubMed Scopus (15) Google Scholar). The human LRAT gene consists of three exons and is located as a single copy on chromosome 4q31.2 (20Ruiz A. Kuehn M.H. Andorf J.L. Stone E. Hageman G.S. Bok D. Investig. Ophthalmol. Vis. Sci. 2001; 42: 31-37PubMed Google Scholar). Because of its key role in the retinoid cycle, defects in the LRAT gene are predicted to have severe consequences on vision and retinoid metabolism. Screening of 267 retinal dystrophy patients identified two disease-associated mutations inherited recessively (21Thompson 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 (152) Google Scholar). The missense mutation S175R and a frameshift mutation 396delAA inactivated LRAT. Patients carrying the S175R LRAT null gene developed normally, suggesting that the lack of LRAT in the liver and other tissues had no deleterious effects. However, their visual functions are severely attenuated, causing early onset severe retinal dystrophy. To characterize further the consequences of deleting the LRAT gene, we generated Lrat+/- and Lrat-/- mice by the standard gene replacement technique. The homozygous knock-out Lrat-/- animals exhibit slow degeneration of the retina. The RPE of Lrat-/- mice is devoid of all-trans-retinol or all-trans-retinyl esters, photoreceptors have no functional rhodopsin, and electroretinogram (ERG) responses are highly attenuated. Lrat-/- mice are an important experimental model for human retinal dystrophies and for vitamin A deprivation. Animals—All animal experiments employed procedures approved by the University of Washington, University of Utah, and 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 employing a Kodak no. 1 safelight filter (transmittance >560 nm). Rpe65-/- mice, obtained from Dr. M. Redmond (NEI, National Institutes of Health), were genotyped as described previously (22Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (773) Google Scholar). Typically, 6–12-week-old mice were used in all experiments. Construction of Targeting Vector and Generation of Lrat-/- Mice—A ∼14-kb genomic clone was isolated from a 129SvEvλ library. The probe used for screening was amplified with PCR primers LRAT2 (5′-GACGTGTTGGAGGTGTCACGGAC-3′) and LRAT4 (5′-CTTGTGTGTGCAGACCAAGATGAC-3′), fragment size 660 bp. The targeting vector was constructed by using a 1,350-bp DNA fragment as a short arm, which was PCR product amplified using the LRATSA1 and LRATSA2 primers. LRATSA1 is located 3.6 kb upstream of exon 1 (containing ATG), with the sequence 5′-GTACCCAGGAGTAGGACCAAC-3′. LRATSA2 is located 2.2 kb upstream of exon 1, with the sequence 5′-TGTGAGTGAGAGGCCACATCC-3′. The short arm was inserted into the 5′-end of the Neo gene cassette using an MluI site. The long arm was a 10-kb genomic fragment that starts from SmaI site (inside exon 1) to the end of the 14-kb λ clone. In this strategy, the part of the coding sequence of exon 1 (including ATG) and the 2.2-kb upstream sequence was replaced by a Neo gene cassette. 10 μg of the targeting vector was linearized by NotI and then transfected by electroporation of 129 SvEviTL1 embryonic stem cells. After selection in neomycin-containing medium G418, surviving colonies were expanded. PCR analysis was performed to identify clones that had undergone homologous recombination. PCR was done using primer pairs LRATSA6 and Neo1. Primer LRATSA6 is located outside of short arm, 80 bp upstream of LRATSA1, with the sequence 5′-AAAGAATCAATAGGACAAAGAACTGG-3′. Primer Neo1 is located in the 5′-promoter region of the Neo gene cassette and has the sequence 5′-TGCGAGGCCAGAGGCCACTTGTGTAGC-3′. The positive clones were identified based on the 1.4-kb PCR fragment. The correctly targeted embryonic stem cell lines were microinjected into C57BL/6J blastocysts. The chimeric mice were generated, and they gave germ line transmission of the disrupted Lrat gene. Genotyping Lrat-/- and Lrat+/- Mice—To identify the wild-type allele, primer pairs LRATSA1/LRATWT1 (1.4-kb fragment) or LRAT1S/LRATWT1 (300-bp fragment) were used. LRATWT1 is located 44 bp downstream of LRATSA2, with the sequence 5′-AAGTGCTGGGCATGGTGACTTGTG-3′. The knock-out gene was identified with LRAT1S (5′-TCCAGTTCCAGACTCTTTCCACCCAC-3′) and Neo-1 (370-bp fragment). The PCR conditions were 94 °C for 30 s; 60 °C for 30 s; 72 °C for 120 s; total of 30 cycles. The mice were outbred into the C57BL/6J strain. Mouse Anti-Lrat Monoclonal Antibody Production—We isolated mouse RPE RNA using the MicroAqueous RNA Isolation Kit (Ambion). Lrat cDNA was amplified using Hotstart Turbo Pfu Polymerase (Stratagene) using the primers 5′-GCTCACCTCGTACAGAACAGTTGC-3′ and 5′-ACATACACGTTGACCTGTGGACTG-3′. A fragment of Lrat corresponding to the residues Gln89–Glu179 in the polypeptide sequence of mouse Lrat was amplified using the primers 5′-CATATGCAGAAGGTGGTCTCCAACAAGCGT-3′ and 5′-GGATCCTCACTCAGCCTGTGGACTGATCCGAGA-3′ and cloned downstream of a His6 tag between the NdeI and BamHI sites of the inducible bacterial expression vector pET15b (Invitrogen). The plasmid was transformed into BL-21RP cells (Stratagene), and expression was induced with isopropyl-1-thio-β-d-galactopyranoside. The His6-tagged fragment of the mouse Lrat protein (10 kDa) was purified by nickel-nitrilotriacetic acid affinity chromatography using the manufacturer’s protocol (Qiagen). The purified protein was examined by gel electrophoresis. After in-gel trypsin digestion, the eluted tryptic peptides were examined by microsequencing by liquid chromatography-mass spectrometry to verify the identity of the recombinant Lrat fragment. The purified protein was used to immunize mice as described before (23Haeseleer F. Jang G.F. Imanishi Y. Driessen C.A. Matsumura M. Nelson P.S. Palczewski K. J. Biol. Chem. 2002; 277: 45537-45546Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), and the monoclonal antibody was produced by established methods (24Adamus G. Zam Z.S. Emerson S.S. Hargrave P.A. In Vitro Cell. Dev. Biol. 1989; 25: 1141-1146Crossref PubMed Scopus (3) Google Scholar). The antibody was tested for its specificity by immunocytochemical testing of the Lrat+/+ and Lrat-/- mouse retinas. Rhodopsin Measurements—All procedures were performed under dim red light as described previously (25Van Hooser J.P. Aleman T.S. He Y.G. Cideciyan A.V. Kuksa V. Pittler S.J. Stone E.M. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8623-8628Crossref PubMed Scopus (223) Google Scholar, 26Jang G.F. Van Hooser J.P. Kuksa V. McBee J.K. He Y.G. Janssen J.J. Driessen C.A. Palczewski K. J. Biol. Chem. 2001; 276: 32456-32465Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 27Palczewski K. Van Hooser J.P. Garwin G.G. Chen J. Liou G.I. Saari J.C. Biochemistry. 1999; 38: 12012-12019Crossref PubMed Scopus (136) Google Scholar). Typically, two mouse eyes were used per rhodopsin measurement. Mouse eyes were enucleated and rinsed with double-stilled H2O. The lenses were removed, and the eyes were cut into three or four pieces and frozen immediately on a dry ice/EtOH bath. Rhodopsin was extracted with 0.9 ml of 20 mm BisTris propane, pH 7.5, containing 10 mm dodecyl-β-maltoside and 5 mm freshly neutralized NH2OH·HCl. The sample was homogenized with a Dounce tissue homogenizer and shaken for 5 min at room temperature (Eppendorf mixer 5432). The sample was then centrifuged at 14,000 rpm for 5 min at room temperature (Eppendorf centrifuge 5415C). The supernatant was collected, and the pellet was extracted one more time. The combined supernatants were centrifuged at 50,000 rpm for 10 min (Beckman Optima TLX centrifuge/TLA100.3 fixed angle rotor), and absorption spectra were recorded before and after a 12-min bleach (60-watt incandescent bulb). The concentration of rhodopsin was determined by the decrease in absorption at 500 nm using the molar extinction coefficient ∈ = 42,000 m-1 cm-1. Retinoids—All experimental procedures related to extraction, derivatization, and separation of retinoids from dissected mouse eyes were carried out as described previously (25Van Hooser J.P. Aleman T.S. He Y.G. Cideciyan A.V. Kuksa V. Pittler S.J. Stone E.M. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8623-8628Crossref PubMed Scopus (223) Google Scholar, 28Van Hooser J.P. Liang Y. Maeda T. Kuksa V. Jang G.F. He Y.G. Rieke F. Fong H.K. Detwiler P.B. Palczewski K. J. Biol. Chem. 2002; 277: 19173-19182Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 29Kuksa V. Bartl F. Maeda T. Jang G.F. Ritter E. Heck M. Van Hooser J.P. Liang Y. Filipek S. Gelb M.H. Hofmann K.P. Palczewski K. J. Biol. Chem. 2002; 277: 42315-42324Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30McBee J.K. Van Hooser J.P. Jang G.F. Palczewski K. J. Biol. Chem. 2001; 276: 48483-48493Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). All reactions involving retinoids were carried out under dim red light. Retinoids were separated by normal phase HPLC (Beckman, Ultrasphere-Si, 4.6 × 250 mm) with 10% ethyl acetate and 90% hexane at a flow rate of 1.4 ml/min with detection at 325 nm using an HP1100 HPLC with a diode array detector and HP Chemstation A.03.03 software. ERGs—Mice were anesthetized by intraperitoneal injection using 20 μl/g body weight of 6 mg/ml ketamine and 0.44 mg/ml xylazine diluted with 10 mm sodium phosphate, pH 7.2, containing 100 mm NaCl. The pupils were dilated with 1% tropicamide. A contact lens electrode was placed on the eye, and ground electrodes were placed in the ear and tail. ERGs were recorded with the universal testing and electrophysiologic system UTAS E-3000 (LKC Technologies, Inc.). The light intensity was calibrated and computer-controlled. The mice were placed in a Ganzfeld chamber, and scotopic and photopic responses to flash stimuli were obtained from both eyes simultaneously. Flash stimuli had a range of intensities (-3.7–2.8 log cd·s·m-2), and white light flash duration was adjusted according to intensity (from 20 μs to 1 ms). Three to five recordings were made with >10-s intervals, and for higher intensity intervals, intervals were 10 min or as indicated. There were no significant differences between the first and fourth flashes. Light-adapted responses were examined after bleaching at 1.4 log cd·m-2 for 15 min. Typically, four to eight animals were used for recording of each point in all conditions. Leading edges of the ERG responses were fitted with a model of rod phototransduction activation as described previously (25Van Hooser J.P. Aleman T.S. He Y.G. Cideciyan A.V. Kuksa V. Pittler S.J. Stone E.M. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8623-8628Crossref PubMed Scopus (223) Google Scholar). The results were examined using the one-way ANOVA test. Pupillometry—Mice were dark-adapted for at least 1 h prior to recordings. Pupillary light responses were recorded under infrared conditions using a CCD video camera; data analysis was performed by video pupillometry. Light stimuli were provided by a halogen source. Wavelength and intensity were manipulated with neutral density and narrow bandwidth (10 nm) interference filters (Oriel). Irradiance measurements (watts/m2) were made using a calibrated radiometer (Advanced Photonics International). Light Microscopy—For light microscopy, eyecups were fixed in 2% glutaraldehyde, 2% paraformaldehyde for 18 h, infiltrated with 20% sucrose in 0.1 m sodium phosphate, pH 7.4, and then embedded in 33% OCT compound (Miles) diluted with 20% sucrose in 0.1 m sodium phosphate, pH 7.4. Thin sections were cut at 5 μm and subjected to light microscopy. Immunocytochemistry—All procedures have been described previously (23Haeseleer F. Jang G.F. Imanishi Y. Driessen C.A. Matsumura M. Nelson P.S. Palczewski K. J. Biol. Chem. 2002; 277: 45537-45546Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Sections were analyzed under an episcopic fluorescent microscope (Nikon). Digital images were captured with a digital camera (Diagnostic Instruments; Sterling Heights, MI). Transmission Electron Microscopy (EM)—For transmission EM, mouse eyecups were fixed primarily by immersion in 2.5% glutaraldehyde, 1.6% paraformaldehyde in 0.08 m PIPES, pH 7.4, containing 2% sucrose initially at room temperature for ∼1 h then at 4 °C for the remainder of 24 h. The eyecups were then washed with 0.13 m sodium phosphate, pH 7.35, and secondarily fixed with 1% OsO4 in 0.1 m sodium phosphate, pH 7.35, for 1 h at room temperature. The eyecups were dehydrated through a methanol series and transitioned to the epoxy embedding medium with propylene oxide. The eyecups were embedded for sectioning in Eponate 812. Ultrathin sections (60–70 nm) were stained with aqueous saturated uranium acetate and Reynold’s formula lead citrate prior to survey and micrography with a Philips CM10 EM. Mouse Lrat Gene and Targeting Construct—The mouse Lrat gene appears to be present as a single copy in the mouse (and human) genomes. The entire mouse Lrat gene sequence can be found at the NCBI web site as a complement (18,757,074.18,766,035) at locus NT_039234 (containing 26,830,222 bp). The published mouse Lrat cDNA sequence (15Zolfaghari R. Ross A.C. J. Lipid Res. 2000; 41: 2024-2034Abstract Full Text Full Text PDF PubMed Google Scholar) and an expressed sequence tag (accession BY705162) have a 250–267-nucleotide 5′-untranslated region that perfectly matches the upstream gene sequence. Thus, in contrast to human LRAT, the mouse Lrat gene most likely has no upstream untranslated exon and consists only of two coding exons. The intervening sequence in the mouse Lrat gene is ∼6,040 bp in length (human ∼4,080 bp). LRAT expressed sequence tags have been identified in multiple tissues, including the colon, testis, liver, spleen, and mammary gland (e.g. see www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm&CID=33921). In the Lrat targeting vector, part of the coding sequence of exon 1 (including ATG) and a 2.2-kb upstream sequence was replaced by a Neo gene cassette, disabling translation of a functional product (Fig. 1A). To generate chimeric mice, correctly targeted embryonic stem cell lines were microinjected into C57BL/6J blastocysts. Heterozygous knock-out mice with germ line transmission were generated by standard outbreeding procedures (Ingenious Targeting, Stony Brooks, NY). The wild-type allele was identified using the primer pair of LRAT1S and LRATWT1 (diagnostic fragment ∼300 bp) and the knockout gene using LRAT1S and Neo1 (370 bp) (Fig. 1B). The expression of Lrat is abolished in the eye of Lrat-/- mice as determined by immunocytochemistry (Fig. 1C). Immunoblotting revealed two major molecular forms of Lrat (28 kDa and 25 kDa) in the eye (Fig. 1D) or in the liver (data not shown) of Lrat+/+ mice which could arise by alternative splicing/posttranslational modifications. Both forms are absent in Lrat-/- mice. The reason for the difference between the forms was not investigated further. The Lrat-/- mice develop normally, have similar weight, females are fertile at a normal level, whereas males appear to be frequently infertile. This observation is consistent with the role of retinoids in the reproductive system of males, as observed employing vitamin A-deprived mice (32Niederreither K. Subbarayan V. Dolle P. Chambon P. Nat. Genet. 1999; 21: 444-448Crossref PubMed Scopus (875) Google Scholar). Histology and Ultrastructure of the Retina from Lrat+/- and Lrat-/- Mice—At 6–8 weeks of age, the only major change in histology of the retina observed at the light microscopy levels is an ∼35% reduction in the length of ROS in the retina when Lrat-/- mice are compared with Lrat+/+ mice (Fig. 2, A and B, n = 3). The outer nuclear layer and inner retina appeared to be comparable between these mice (Fig. 2C). Quantitative calculations of the thickness of different retinal layers showed no significant variation between these two genetic backgrounds within the experimental error (Fig. 2D). Retinas of Lrat+/- mice were indistinguishable from those of Lrat+/+ mice (data not shown). EM analysis of the retina and the RPE layer revealed no gross changes between Lrat-/- mice with the exception of shorter ROS as noted by light microscopy (Fig. 3). ROS are shorter and thinner, and they are less tightly packed. Higher resolution images of the RPE did not unveil any differences between Lrat-/- mice and WT mice (data not shown). Higher resolution at the synaptic terminal of the photoreceptors showed less developed synaptic ribbons (Fig. 3, C and D). In 4.5-month-old Lrat-/- mice, ROS are ∼one-half length compared with normal, and there is a minor reduction in the number of photoreceptor nuclei ( 95%) of all-trans-retinyl esters were present in RPE (data not shown). Retinoid Analysis from Liver, Blood, and Lung of Lrat-/- Mice—The results of the analysis of retinoids from the liver and blood of Lrat-/- mice mirrored the lack of all-trans-retinyl esters in the RPE of Lrat-/- mice, whereas the all-trans-retinyl ester levels of Lrat+/- mice were comparable with those from Lrat+/+ mice (Fig. 5 and Table I). Similar amounts of all-trans-retinol in the blood were found in Lrat+/+ and Lrat+/- mice although slightly reduced in Lrat-/- mice (Table I). A similar dramatic decrease of all-trans-retinyl esters was observed in the lung of Lrat-/- mice (data not shown). This reduction in the ester content is consistent with the high activity of LRAT in the lung (14Zolfaghari R. Ross A.C. J. Nutr. 2002; 132: 1160-1164Crossref PubMed Scopus (43) Google Scholar).Table IAll-trans-retinyl esters and all-trans-retinol in the liver and blood in mice from different genetic backgrounds The results are presented ± S.D., and n was between 3 and 5.RetinoidLrat+/+Lrat+/-Lrat-/-LiveraExpressed per whole liver (623-780 mg)BloodbExpressed per 100 μl of bloodLiveraExpressed per whole liver (623-780 mg)BloodbExpressed per 100 μl of bloodLiveraExpressed per whole liver (623-780 mg)BloodbExpressed per 100 μl of bloodμmolpmolμmolpmolμmolpmolAll-trans-retinyl esters291.1 ± 30.621.10 ± 6.10353.1 ± 20.77.80 ± 3.61TraceTraceAll-trans-retinol0.60 ± 0.1317.4 ± 5.090.98 ± 0.2225.4 ± 12.50.45 ± 0.208.02 ± 2.2a Expressed per whole liver (623-780 mg)b Expressed per 100 μl of blood Open table in a new tab ERG Analysis—To evaluate the light response, Lrat-/- mice were studied using ERG
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