Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyof FATP4 is important for interacting with RPE65 and for inhibitingsynthesis of 11cROL.Commercial Relationships: Songhua Li, None; John F. Green,None; Jean T. Jacob, 4,865,601 (P), 5,282,851 (P); Minghao Jin,NoneSupport: EY021208Program Number: 3760 Poster Board Number: A0099Presentation Time: 2:45 PM - 4:30 PMCloning and Characterization of a Carboxylesterase from BovineRetinal Pigment EpitheliumGennadiy P. Moiseyev 1 , Bill X. Wu 2 , Yusuke Takahashi 5 , Ying Chen 1 ,Andrew T. Tsin 4 , Rosalie K. Crouch 3 , Jian-Xing Ma 1 . 1 Physiology,Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2 Biochemistry& Molecular Biol, Medical Univ of South Carolina, Charleston, SC;3 Ophthalmology, Medical Univ of South Carolina, Charleston, SC;4 Biology, University of Texas San Antonio, San Antonio, TX;5 Medicine-Endocrinology, Univ of Oklahoma Hlth Sci Ctr,Oklahoma City, OK.Purpose: Retinyl esters are stored in lipid droplets in retinal pigmentepithelium (RPE) and can serve as a substrate for RPE65isomerohydrolase for generation of 11-cis retinol, the precursorrhodopsin chromophore. At present, RPE65 is the only known retinylester hydrolase in the eye. Some carboxylesterases of the liver andother tissues have been shown to hydrolyze retinyl esters.Carboxylesterases may also participate in xenobiotic, drug and lipidmetabolism. However, little is known about carboxylesterasesexpression and function in the eye. The aim of this study was toexamine whether RPE carboxylesterase has retinyl ester hydrolyzingactivity or demonstrates other substrate preference.Methods: Carboxylesterase (CES1) has been cloned from bovineRPE cDNA library by PCR methods. A 293A cell line stablyexpressing CES1 with His-tag using pcDNA6 vector was generatedand used to express CES1. CES1 was purified from the culturemedium using N-NTA resin. CES1 activity was determinedspectrophotometrically in the reaction with various esters. Retinylester hydrolyzing activity was assayed by HPLC.Results: The cloned bovine CES1 cDNA sequence contains 1695 bp(accession number AY369075 in GenBank). The deduced protein hasa calculated molecular weight 61,780 Da and isoelectric point 6.79.CES1 mRNA was detected as a single transcript in the bovine RPE,kidney, liver and lung. No signal was detected in other bovine tissuesanalyzed, including the retina, pancreas, skeletal muscles, spleen,heart and brain. Immunohistochemistry showed the specificlocalization of CES1 in Bruch’s membrane. CES1 was purified tohomogeneity from culture medium of 293A cell line that stablyexpressed this protein. CES1 cleaves various esters producing alcoholand acid. Kinetic parameters kcat and Km were determined forparanitrophenyl acetate, vinyl acetate, vinyl butyrate, vinyl lauratewhich were found to be substrates for CES1. Lipid esters triolein andretinyl palmitate were not hydrolyzed by CES1.Conclusions: Bovine CES1 showed significant activity towardsesters with a small alcohol group. However, CES1 did not cleaveretinyl palmitate suggesting that it is not a retinyl ester hydrolase. Wehypothesize that CES1 may have a physiological role as a detoxifyingenzyme serving as a protector of RPE from various xenobiotic esters.Commercial Relationships: Gennadiy P. Moiseyev, CharlessonLLC (E); Bill X. Wu, None; Yusuke Takahashi, None; Ying Chen,None; Andrew T. Tsin, None; Rosalie K. Crouch, None; Jian-XingMa, NoneSupport: NIH grants EY018659, EY012231, EY019309 andP20GM104934Program Number: 3761 Poster Board Number: A0100Presentation Time: 2:45 PM - 4:30 PMThe rd12 allele of Rpe65 exerts a dominant-negative effect onvision because it may exhibit altered intracytoplasmic traffickingCharles B. Wright 1 , Micah A. Chrenek 1 , Stephanie L. Foster 1 ,Machelle T. Pardue 2 , Jeffrey H. Boatright 1 , John M. Nickerson 1 .1 Ophthalmology, Emory University, Atlanta, GA; 2 EmoryUniv/Ophthal, Atlanta VA Medical Center, Decatur, GA.Purpose: The rd12 allele of mouse Rpe65 has a nonsense mutation inexon 3 of the gene. The rd12 allele exerts a dominant-negative effecton vision that extends beyond the loss of visual function caused by alack of Rpe65 enzymatic activity. The purpose of this study is toattempt to define a possible mechanism-of-action for the rd12 allele.Methods: Optokinetic tracking (OKT) and electroretinography(ERG) were used to assess the visual acuity and retinal function ofC57BL/6J (+/+), Rpe65 KO (KO/KO), tvrm148 (tvrm148/tvrm148),rd12 (rd12/rd12), rd12/+, KO/+, tvrm148/+, KO/rd12, andKO/tvrm148 mice. Immunoblotting with an antibody specific to theN-terminus of Rpe65 was performed to detect protein, and qRT-PCRwas used to detect steady-state mRNA levels of genes of interest.RNAfold was used to predict the centroid structures of the Rpe65mRNAs from the +, tvrm148, and rd12 alleles. RNAimmunoprecipitation (RIP) was used to detect Rpe65 mRNA bindingto eIF4E.Results: Mice heterozygous for the rd12 allele lost 21% of theirvisual acuity by P210 while mice heterozygous for other mutationsdid not. Mice homozygous for the rd12 allele lost 100% visual acuityby P120, while mice homozygous for KO or tvrm148 alleles did notlose 100% visual acuity until P210. KO/rd12 mice had visualfunction loss that resembled rd12 homozygote mice. qRT-PCR for allRpe65 exon boundaries showed the rd12 allele has no splicing defectsin the mutant mRNA. No other coding mutations were found.Structural prediction of the mRNA predicted the rd12 allele mRNAdid not adopt a different secondary structure than wild type. qRT-PCR showed the same level of wild type and mutant mRNA in thecytoplasm, but RIPs showed the mutant mRNA was not bound byeIF4E, a translation initiation factor found in polysomes.Conclusions: Loss of visual function in mice homozygous andheterozygous for the rd12 allele shows the allele acts in a dominantnegativemanner to other Rpe65 mutant alleles, and that one copy ofthe rd12 allele is sufficient to drive this dominant-negative visualfunction loss. The rd12 allele does not produce a detectable protein,but does produce an mRNA that is appropriately transcribed, spliced,folded, and exported from the nucleus to the cytoplasm but does nottraffic to polysomes, suggesting the mutant mRNA may induce harmto the visual system because of altered intracytoplasmic trafficking.Commercial Relationships: Charles B. Wright, None; Micah A.Chrenek, None; Stephanie L. Foster, None; Machelle T. Pardue,None; Jeffrey H. Boatright, None; John M. Nickerson, NoneSupport: NIH GrantP30EY06360,R01EY016470,R24EY017045,R01EY014026,T32EY007092,RPBProgram Number: 3762 Poster Board Number: A0101Presentation Time: 2:45 PM - 4:30 PMMechanism for a Dominant-Acting D477G Mutation in RPE65Leading to Vision ImpairmentOlga Nikolaeva 1 , Gennadiy P. Moiseyev 1 , Yusuke Takahashi 2 , Jian-Xing Ma 1 . 1 Department of Physiology, OUHSC, Oklahoma City, OK;2 Endocrinology, OUHSC, Oklahoma City, OK.Purpose: RPE65 is a key enzyme in the visual cycle. Multiplerecessive point mutations in the RPE65 gene have been shown to leadto impaired vision in patients with retinitis pigmentosa (RP) and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.