Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyT J Hollingsworth, Alecia K. Gross. Vision Sciences, University ofAlabama at Birmingham, Birmingham, AL.Purpose: The Ter349Glu rhodopsin mutant causes a severe retinalphenotype characterized by early-onset and rapid degeneration. Priorin vitro assays showed that Ter349Glu rhodopsin is able to fold andfunction similarly to wild type (WT) while mislocalizing in polarizedcells. To determine the origin of the severe phenotype, we examinedthe retina of a knock-in mouse bearing the mutation for receptorlocalization, rod cell morphogenesis and degeneration, andinflammation.Methods: Retinas from Ter349Glu heterozygote and homozygotealbino mice were examined for receptor localization, photoreceptordegeneration, and inflammation using fluorescentimmunohistochemistry, and the in vivo retinal condition usingfunduscopy and optical coherence tomography (OCT).Results: Rods of the Ter349Glu homozygote mice degenerate rapidlyand lack a clear delineation between inner and outer segments. Inheterozygote animals, the degeneration seen is slowed withapproximately two nuclei lost in the outer nuclear layer. In theserods, mislocalization of both WT and the mutant receptor wasobserved. Labeling for both phospho-STAT3 and F4/80 antigen, eacha marker of retinal inflammation, show activation of both theJAK/STAT pathway and macrophages, respectively. Funduscopyreveals hallmarks of retinitis pigmentosa including attenuated retinalvessels and patches of light reflection indicating a loss of neuralretina. Examination by OCT reveals detachments between thephotoreceptors and the pigmented epithelium.Conclusions: The rapid degeneration and RP phenotypes observed inhomozygote animals are consistent with what is seen in humanpatients. The lack of obvious rod outer segments is indicative of aloss of proper morphogenesis, possibly due to the occlusion of the C-terminal sorting motif. Mislocalization of the photoreceptor proteincould be due to aggregate formation and/or endoplasmic reticulumretention of both the mutant and WT proteins. The presence ofactivated macrophages in the retinas of homozygote knock-in animalsindicates an inflammatory response possibly associated with aninfiltration of blood-borne macrophages from the choroid. Theactivation of the JAK/STAT pathway is indicative of retinalinflammation and may be responsible for lowering the expressionlevels of rhodopsin through cytokine signaling, possibly in aninterleukin-6 dependent manner.Commercial Relationships: T J Hollingsworth, None; Alecia K.Gross, NoneSupport: NIH/NEI R01 EY019311Program Number: 697 Poster Board Number: D0254Presentation Time: 10:30 AM - 12:15 PMUNC119B, an UNC119/RG4 paralog expressed in retina andRPECecilia D. Gerstner 1 , Houbin Zhang 1 , Jeanne M. Frederick 1 ,Wolfgang Baehr 1, 2 . 1 Ophthalmology, University of Utah, Salt LakeCity, UT; 2 biology, University of Utah, Salt Lake City, UT.Purpose: UNC119B is related closely to UNC119/HRG4, a homologof C. elegans Unc-119, and more distantly to the prenyl-bindingprotein, PrBP/δ. Each of these (UNC119, UNC119B and PrBP/δ)forms an immunoglobulin-like β-sandwich fold for lipid binding. Ourpurpose was to identify expression and function of the UNC119Bisoform in retina and RPE.Methods: GST-pulldowns, preparation of monospecific antibodiesraised in rabbit and chicken, western blotting, immunohistochemistry,generation of an Unc119b knockout mouse.Results: By gene and EST blasting, UNC119B was determined to beexpressed in both vertebrates and invertebrates. Polyclonal antibodieswere generated using N-terminal peptide and recombinant full-lengthUNC119B as antigens. By multiple mouse tissue immunoblot, weshow that UNC119B is expressed strongly in testis, ovary, lung, brainand thymus, but weakly in whole eye, heart and muscle. UNC119B isexpressed prominently in the RPE and weakly in retina.Immunohistochemistry using frozen mouse and monkey (Macacamulatta) retina/RPE sections show partial colocalization with RDH5in RPE cell peripheral margins. Both GST-UNC119B and GST-UNC119/RG4 pull down Tα from retina lysates, suggesting closelyrelatedfunctions as acyl binding proteins. Both UNC119 isoformsreveal perfect conservation of all amino acids known to interact withthe acyl side chain and N-terminal residues of Tα, and predict nearlyidentical structures. An Unc119b-/- mouse was generated using aMMRCC (UC Davis) genetrap. The trap, located in exon3, blocksUNC119B protein expression. The Unc119b-/- mouse did not reveala retina degeneration phenotype at one-month postnatally.Conclusions: UNC119B is detected prominently in the RPE andweakly in the retina. UNC119B may function as an acyl bindingprotein with specificity for a novel G protein α-subunit expressed inthe RPE.Commercial Relationships: Cecilia D. Gerstner, None; HoubinZhang, None; Jeanne M. Frederick, None; Wolfgang Baehr, NoneSupport: EY08123; EYEYo14800A2;EY019298Program Number: 698 Poster Board Number: D0255Presentation Time: 10:30 AM - 12:15 PMIn Depth Analysis of Retinal Proteins using a CombinatoryHPLC based Mass Spectrometric PlatformSebastian Funke, Dominik Wolters, Lukas Weiler, Francis K. Mwiiri,Katharina Bell, Corina Wilding, Norbert Pfeiffer, Franz H. Grus.Experimental Ophthalmology, University Medical Center, Mainz,Germany.Purpose: The retina proteome with special focus on retinal ganglioncell proteins is of important interest regarding clinical research onneurodegenerative diseases like glaucoma. To intensivelycharacterize retinal proteins we developed protein extraction andfractionation techniques coupled to LC MALDI and ESI methods toincrease the sensitivity towards retinal sample species. Detailedreference maps should be generated by use of the developedworkflow providing new insights to the understanding of the retinaproteome.Methods: Retinal sample species were used as study material.Different extraction techniques were developed encircling non-ionicdetergent extraction, stepwise differential extraction and methanolchloroformextraction for sample fractionation. Extract analysisincludes gel-based RP-RP-2D-capillary-LC MALDI TOF/TOF andRP-capillary LC ESI-LTQ Orbitrap mass spectrometry. The mediummolecular weight range was directly investigated by top downmicrobore-LC MALDI TOF MS. Results were used to generatereference protein maps of so far identified retinal proteins.Results: By use of non-ionic detergent extraction more than 1000proteins per lysate could be identified with high reproducibility bythe LC ESI approach in a single sample lysate.The gel-based MALDIapproaches detected more than 300 proteins. More than 856 mediummolecular weight components per sample were detected by top downMALDI analysis. Furthermore ESI and MALDI methods incombination could distinctly increase the output emphasizing thecomplementary power of MALDI and ESI techniques. By use ofstepwise differential extraction and chloroform-methanol extraction adistinct and reproducible fractionation of proteins could be achieved.Moreover post-translational modification sites of retinal proteinshave been determined by mass spectrometric analysis.Conclusions: The use of the developed platform allowed us to get a©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.