Characterization of RPE65 and RDH12, Two Enzymes Associated with Retinal Dystrophy and Retinoid Processing.
ABSTRACT Phototransduction in vertebrate vision is mediated by visual pigments composed of opsin apoproteins covalently attached to a light-sensitive chromophore, 11-cis retinal. Absorption of light isomerizes 11-cis retinal to all-trans retinal, initiating a signal transduction cascade. A complex set of enzyme reactions occurring in the retinal pigment epithelium and the retina is responsible for the synthesis and regeneration of the chromophore and is termed the visual cycle. Several forms of inherited retinal degeneration and dysfunction manifest as a result of mutations in the genes associated with visual cycle function, such as RPE65 and RDH12. RPE65 is essential for the synthesis of 11-cis retinal and was recently confirmed to function as the visual cycle isomerase. Cone photoreceptors have been proposed to possess an exclusive chromophore regenerative pathway. Using a monoclonal antibody approach, we have now mapped antigenic determinants of the protein surface, shown that RPE65 is not expressed in cone cells, and confirmed that RPE65 is associated with the visual cycle enzyme RDH5. Rdh12 has an in vitro activity and localization profile that made it an excellent candidate to serve as the all-trans retinal reductase of the visual cycle. Immunochemical analysis localized RDH12 protein to the photoreceptor inner segments and outer nuclear layer in both humans and mice, suggesting an equivalent physiological role for RDH12 in both species. However, analysis of the phenotype of Rdh12-deficient mice revealed no differences in histology, retinoid processing or electroretinogram response compared to wild-type. Rdh12-deficient mice did show a decreased ability to reduce all-trans retinal and 11-cis retinal as measured by in vitro activity assays of retinal homogenates. These findings suggest that RDH12 function in mice does not directly contribute to visual cycle function. Instead, a critical function of RDH12 is likely the reduction of retinaldehydes that exceed the reductive capacity of the photoreceptor outer segment and gain access to the inner segments in conditions of high illumination. The study of these genes is important not only to gain a better understanding of visual cycle mechanism, but also to elucidate mechanisms of pathogenesis and to develop targeted forms of therapeutic intervention.
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ABSTRACT: RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K(D) = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K(D) = 14 nM, 11-cis-retinol binds with a K(D) = 3.8 nM, and all-trans-retinol (vitamin A) binds with a K(D) = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by binding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.Biochemistry 11/2003; 42(40):11824-30. · 3.38 Impact Factor
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ABSTRACT: RPE65 is essential for the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin. Here, we show that the membrane-associated form (mRPE65) is triply palmitoylated and is a chaperone for all-trans-retinyl esters, allowing their entry into the visual cycle for processing into 11-cis-retinal. The soluble form of RPE65 (sRPE65) is not palmitoylated and is a chaperone for vitamin A, rather than all-trans-retinyl esters. Thus, the palmitoylation of RPE65 controls its ligand binding selectivity. The two chaperones are interconverted by lecithin retinol acyl transferase (LRAT) acting as a molecular switch. Here mRPE65 is a palmitoyl donor, revealing a new acyl carrier protein role for palmitoylated proteins. When chromophore synthesis is not required, mRPE65 is converted into sRPE65 by LRAT, and further chromophore synthesis is blocked. The studies reveal new roles for palmitoylated proteins as molecular switches and LRAT as a palmitoyl transferase whose role is to catalyze the mRPE65 to sRPE65 conversion.Cell 07/2004; 117(6):761-71. · 31.96 Impact Factor
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ABSTRACT: Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.Journal of Biological Chemistry 07/2001; 276(24):21098-104. · 4.65 Impact Factor
CHARACTERIZATION OF RPE65 AND RDH12, TWO ENZYMES
ASSOCIATED WITH RETINAL DYSTROPHY AND RETINOID PROCESSING
Jared D. Chrispell
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
in The University of Michigan
Professor Debra A. Thompson, Chair
Professor Jairam Menon
Professor Anand Swaroop
Associate Professor Raymond C. Trievel
Associate Professor Anne Vojtek
© Jared D. Chrispell
All rights reserved
To my mother, father and sister
for all of your love and support over the years
I would like to thank first and foremost my mentor, Debra Thompson for all of
her guidance and assistance throughout my tenure in the lab. Her tireless efforts have
without a doubt helped me to become a better scientist and only served to increase my
love for research. I would also be remiss if I did not thank the influence and
encouragement of my high school chemistry teacher Mr. Doug Getman, my
undergraduate teaching advisor Dr. Clyde Herreid, and my undergraduate research
advisors Drs. Christine Campbell and Richard Gronostajski.
I would also like to thank my committee members Drs. Anand Swaroop, Jairam
Menon, Ray Trievel and Ann Vojtek for their invaluable input and criticisms over the
course of my research. This research would not have been possible without the aid of
those who laid the groundwork and contributed as co-authors. Dr. Andreas Janecke group
at the University of Innsbruck, Austria performed the initial patient screening and Dr.
Peter Nurnberg’s group at the Max Delbrück Center in Berlin, Germany performed the
linkage analysis. Drs. Ingo Kurth, Christian Hübner and Andreas Gal of the Institut für
Humangenetik in Hamburg, Germany created of the Rdh12-knockout mouse and anti-
mouse Rdh12 antibodies and Klaus Ruther of Augenklinik Campus Virchow-Klinikum
Charité in Berlin, Germany performed the ERG analysis. Dr. Alan Mears generated the
Nrl-knockout mouse and performed the subsequent microarray analysis, the results of
which were analyzed by Ritu Khanna of the Swaroop lab. Drs. Maureen Kane and
Jospeh Napoli of the University of California, Berkeley performed the retinal retinoic
acid analysis and Dr. Janet Sparrow performed the lipofuscin analysis for our studies.
Matt Brooks of the Swaroop lab performed the microarray analysis on the Rdh12-
knockout mouse and whose knowledge was invaluable assistance in several other aspects
of our research. I would also like to thank Drs. Hemant Khanna and Carlos Murga for
their constructive criticism of my presentations and work in general. I must also thank
members of the Thompson lab, past and present. Thanks go to Nahid Hemati for her help
in teaching me many immunohistochemical techniques as well as helping to generate a
number of RDH constructs. Dr. Christina McHenry’s expertise was invaluable in helping
me to perform organic extractions and showing me the use of the HPLC machine.
Shameka Shelby was of great assistance when performing early protein purification
assays. I must also thank Kecia Feathers for her exceeding helpfulness in almost every
aspect of my research, and for being such a great lab buddy over the last few years.
I must also thank my many friends, bother here and afar, for their years of
constant support and camaraderie, all of which helped to make the low points of the
research experience bearable and the high points memorable. And finally I must thank
my family, whose unending support and encouragement has allowed me excel at
whatever it is I attempt, and are the best role models I could ever hope to have.