Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologycell number by whole mount staining with calbindin antibody andcone cell numbers by a combination of opsin and peanut agglutininstaining.Results: PRMT8 staining was confined to cone photoreceptors,horizontal cell nuclei and processes, subset of amacrine cells andinner plexiform layer. Co-labeling study with cell type specificmarkers and absence of PRMT8 signal in knockout animalsconfirmed our PRMT8 localization in retina. PRMT1, a closehomologue of PRMT8 was found mainly in all inner nuclei andganglion cells indicating a distribution pattern that is different thanPRMT8. Interestingly, our preliminary study showed an increase(40%) in horizontal cell density in central part of retina in animallacking PRMT8 compared to littermate controls. Increase inhorizontal cell density was supported by increased levels of calbindinby western blotting.Conclusions: Our results demonstrate that PRMT8 and PRMT1localization differs in the retina suggesting that each enzyme has aunique functional role independent of other. Additionally our resultsshowing differences in the horizontal cell number in animals lackingPRMT8 suggest that intercellular spacing and density of horizontalcells may be controlled by epigenetic mechanisms or posttranslational protein modification by arginine methylation.Commercial Relationships: Ratnesh Singh, None; Yasutake Mori,None; Shingo Miyata, None; Masaya Tohyama, None;Visvanathan Ramamurthy, NoneProgram Number: 2451 Poster Board Number: D0056Presentation Time: 2:45 PM - 4:30 PMCharacterization of recombinant intra-melanosomal domain ofhuman tyrosinaseMonika B. Dolinska 1 , Peter S. Backlund 2 , Brian P. Brooks 1 , Yuri V.Sergeev 1 . 1 OGVFB, National Eye Institute, Bethesda, MD;2 NICHD/NIH, Bethesda, MD.Purpose: Mutations in tyrosinase gene cause an autosomal recessivedisorder, oculocutaneous albinism Type 1 (OCA1). OCA1 isclassified into a disorder with complete absence of tyrosinase activity(OCA1A) and with residual tyrosinase activity (OCA1B). Tyrosinaseis an enzyme which catalyzes the rate-limiting conversions oftyrosine to L-DOPA and dopachrome in melanin production in theskin and eye. Here we characterized intra-melanosomal domain ofhuman tyrosinase, hTyrCtr (residues 19 - 469 of the native protein).Methods: Recombinant hTyrCtr was produced in larvae (C-PERL,MD) and purified from the soluble fraction by immobilized metalaffinity and size-exclusion chromatography. The effect of variousinhibitors and activators, kinetics parameters and optimumtemperature and pH were examined by using L-DOPA and L-tyrosineenzymatic reactions. N-glycosylation sites and a composition ofsugar components in hTyrCtr were determined using MS/MS, Maldi-Tof MS, and Q-Tof MS/MS mass spectroscopy combined withtrypsin, asp-proteinase (peptide sequencing) and PNGase-Fproteinase digests (sugar composition).Results: Recombinant hTyrCtr show maximum enzymatic activity atoptimal temperature of 37°C and neutral pH. The hTyrCtr is N-glycosylated as shown by the PNGase de-glycosylation. Five N-glycosylation sites are either modified (N86, N337) or partiallymodified (N111, N230, N290) by carbohydrates. Expected asparaginsN161 and N371 are not glycosylated. Molecular modeling of humantyrosinase suggests that all observed N-glycosylation sites are locatedat the surface of intra-melanosomal domain with sugar chainsaccessible for inter-molecular interactions within the melanosome.Conclusions: Recombinant hTyrCtr is a functional N-glycosylatedenzyme. In contrast to current observation that genetic mutationsaffect just a few N-glycosylation sites our work suggests that a widerspectrum of sequence positions associated with N- glycosylationsequons, 84-91, 109-116, 228-235, 288-295, and 335-342, and mightbe associated with genetic defects. The recombinant hTyrCtr could beused to search for small molecule activators of tyrosinase mutantvariants as a potential treatment of OCA1 type of albinism.Commercial Relationships: Monika B. Dolinska, None; Peter S.Backlund, None; Brian P. Brooks, None; Yuri V. Sergeev, NoneProgram Number: 2452 Poster Board Number: D0057Presentation Time: 2:45 PM - 4:30 PMInteractions between Dopamine Receptor D4 and VisualArrestinsJanise D. Deming 1, 2 , Kayleen Lim 1, 2 , Bruce M. Brown 1, 2 , JosephPak 1, 2 , Kathleen Van Craenenbroeck 3 , Cheryl M. Craft 1, 2 . 1 Mary D.Allen Lab for Vision Research, Doheny Eye Institute, Los Angeles,CA; 2 Ophthalmology & Cell & Neurobiology, Keck School ofMedicine of Univ. Southern Calif., Los Angeles, CA; 3 Physiology,University of Gent, Gent, Belgium.Purpose: Dopamine (DA) and its G-protein coupled receptors(GPCR) contribute to normal photopic vision, including lightadaptation and contrast sensitivity, but the specific cellular pathwaysby which they contribute are unclear. Similar defective visualphenotypes are observed when either the DA receptor D4 (DRD4) orcone arrestin (ARR4) is not expressed. The purpose of this study wasto determine the contribution of ARR4 in the desensitization ofDRD4 in vitro and in photopic vision.Methods: Immunohistochemistry (IHC) was performed usingspecific antibodies on frozen sections of mouse retinas or fixedtransfected cells. C57Bl/6J mice were used, along with Drd4 -/- andthe four ARR knockout mice (J. Chen, USC; R. Lefkowitz, Duke) asnegative controls. Co-immunoprecipitation (co-IP) followed byimmunoblot analysis was used for protein-protein interactions. DRD4desensitization was measured using in vitro internalizationexperiments with tagged plasmid constructs of human DRD4 andfour ARRs in transfected HEK 293T cells incubated with or without10 µM DA.Results: IHC of control compared to Drd4 -/- retinas revealed the coexpressionof DRD4 and ARR4 in cone pedicles, with higher DRD4expression in dark vs. light adapted mice. Both βARR1 and βARR2were also expressed in photoreceptors. DRD4 interaction withβARR2 is DA independent, but our co-IP data demonstrated a DAdependent interaction between DRD4 and ARR4 but not ARR1. Invitro studies showed DRD4 internalization only when a βARR and avisual ARR were co-transfected with DRD4. Internalization washighest with βARR2 and ARR4, but other combinations revealedlimited internalization; however, none was detectable with twoβARRs or two visual ARRs.Conclusions: DRD4 and ARR4 are required for normal photopicvision and are co-expressed with ARR1 and βARRs in conephotoreceptors, particularly in the pre-synaptic terminals. Our studiesdemonstrate that for complete desensitization of the DRD4, indicatedby internalization after DA stimulation, at least one visual ARR mustbe co-expressed with a βARR. To our knowledge, no GPCR haspreviously been shown to require the expression of two differentARRs for desensitization. Our in vitro experiments indicate that notonly is the interaction of DRD4, a visual arrestin, and a βARRpossible in the cone photoreceptors, but their interaction may play anessential role in maintaining normal high acuity vision.Commercial Relationships: Janise D. Deming, None; KayleenLim, None; Bruce M. Brown, None; Joseph Pak, None; KathleenVan Craenenbroeck, None; Cheryl M. Craft, NoneSupport: NIH Grant EY015851(CMC), P30EY03040(DEI),Research to Prevent Blindness (DEI), Mary D. Allen Endowment,©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.