Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynegative regulator of alternative pathway complement activation),native properdin (P2 dimer, P3 trimer, and P4 tetramer; a positiveregulator of the same pathway), and complement C3b binding toCEP-BSA.Methods: SPR experiments were performed using a Biacore 3000.CEP-BSA with pyrrole to protein (P/P) ratio 7 (as measured by theEhrlich assay) and BSA were immobilized on CM5 chips by aminecoupling. The human CFH, C3b, P2, P3, or P4, or recombinant CFH(rCFH) fragments diluted in HBS-P buffer were injected over thechips, and the sensorgrams analyzed. rCFH fragments includedcomplement control protein (CCP) repeat domains 1-3, 5-7, 6-8, 7-9,17-19 (with 6XHis tag), as well as CCPs 18-20 and 19-20.Results: CFH, native properdin forms, and C3b bound to CEP-BSAwhile they didn’t bind to the reference surface where BSA wasimmobilized. Kinetic rate constants were determined usingBiaevaluation software (V4.0.1) by the global-fit method. The datashowed affinity values (KD) in the range 10 -6 M (C3b) to 10 -7 M(P3). CFH appears to have the highest association-rate constant (ka),while the native properdins have the slowest dissociation kinetics(kd). The rCFH fragments CCPs 5-7, 6-8, 18-20 and 19-20 bound toCEP-BSA P/P 7, with affinity values in the range of 1.4 X 10 -6 M to4.4 X 10 -6 M. The other recombinants did not bind.Conclusions: CFH likely binds to CEP moieties through its “heparinbinding”domains, CCPs 18-20 and 6-8. Properdin, a positivelychargedmultimer, avidly bound to the CEP moieties. Therefore, thenegative charges of CEP are probably responsible for attractionthrough electrostatic interactions. As the CEP-BSA was bound byboth positive and negative regulators of the complement pathway, abalance may be necessary for allowing complement activation(promoted by properdin), while regulating the extent of activation (byCFH) to a level that facilitates opsonization and removal of lipidoxidation products, but not tissue damage.Commercial Relationships: Lisa Kuttner-Kondo, None; VivianaP. Ferreira, None; Claudio Cortes, None; Satya P. Yadav, None;Joe G. Hollyfield, NoneSupport: Research to Prevent Blindness Unrestricted Grant & NIHR01EY014240-10 (JGH); NIH RR016789-01A1 (SPY);AHA12GRNT10240003 (VPF)Program Number: 4585 Poster Board Number: A0104Presentation Time: 11:00 AM - 12:45 PMThe aging retina: Macula-less rat and macula-bearing monkeyretinae exhibit common age-related changes in their retinalproteinsMichael R. Boehm 1, 2 , Sonja Mertsch 1 , Harutyun Melkonyan 1 , SolonThanos 1 . 1 Institute for Experimental Ophthalmology, School ofMedicine, WWU Muenster, Muenster, Germany; 2 CurrentAffiliation: Department of Ophthalmology, University of MuensterMedical Center, Muenster, Germany.Purpose: The visual consequences of age-related alterations in theneural retina are well documented, but little is known about theirmolecular bases. We performed a comparative proteomic analysis ofthe retinae in marmosets and rats, in order to identify proteins forwhich the expression profiles in retinas are altered with age.Methods: Protein profiles were compared in the newborn, juvenile,middle-aged, and aged retinae using two-dimensional gelelectrophoresis. Seven proteins exhibited changes in contentthroughout the life of rats, and 21 proteins that exhibited age-relatedcontent changes in marmosets were identified by matrix-assisteddesorption ionization-time-of-flight mass spectrometry. Western-blot(WB), quantitative reverse-transcriptase PCR (qRT-PCR), andimmunohistochemistry (IHC) analyses of selected proteins and theirmRNA were employed to determine whether changes identified byproteomics were verifiable at the cellular and molecular levels.Results: We found four proteins common to both species [Parkinsondisease (autosomal recessive, early onset) 7/DJ-1, stathmin,peroxiredoxin, and β-synuclein] whose contents were regulated withage. WB, IHC, and qRT-PCR analyses confirmed these findings. Theproteins were localized in certain layers and subsets of cells withinthe retinae of both species. Alterations in the expressions of theseproteins between the localization of the macular and retinal peripherywere analyzed with IHC. Detection of found proteins in the adulthuman retina were confirmed with IHC.Conclusions: This study is the first to provide evidence that theaging retina is physiologically characterized by specific changes in itsproteome. These changes occur in key functional pathways during theprocessing of visual signals and may be involved in the developmentof age-related pathologies, such as age-related macular degeneration.Commercial Relationships: Michael R. Boehm, None; SonjaMertsch, None; Harutyun Melkonyan, None; Solon Thanos, NoneSupport: Supported by Interdisciplinary Centre for Clinical Researchof Muenster (IZKF) grants to MRRB and ST (Tha3/002/09)Program Number: 4586 Poster Board Number: A0105Presentation Time: 11:00 AM - 12:45 PMGene expression profile of Retinal Pigment Epithelium derivedARPE-19 cells under serum starvationSanghamitra Mishra 1 , Katherine M. Peterson 1 , Alan E. Berger 2 ,Graeme J. Wistow 1 . 1 Section for Molecular Structure and FunctionalGenomics, National Eye Institute, Bethesda, MD; 2 JHBMC LoweFamily Genomics Core, Johns Hopkins University - School ofMedicine, Baltimore, MD.Purpose: Age Related Macular Degeneration (AMD) is associatedwith formation of subretinal deposits (drusen) and damage to Bruch’smembrane (BM) basal to the retinal pigment epithelium (RPE). Thiscould lead to serum deprivation of overlying RPE. Here we studiedgene expression in the human RPE derived cell line, ARPE-19,during a seven day time course of serum starvation.Methods: Gene expression levels in serum starved ARPE-19 cellswere measured for 0, 1, 3, 5, and 7 days using cDNA microarrays.Standard statistical tools and pathway analysis software were used toidentify regulated genes and altered pathways. Differentiallyexpressed genes and pathways, identified from microarray analysis,and the effect of supplements were confirmed by qPCR, western blotanalysis and confocal microscopy.Results: Differentially expressed genes at days 1, 3, 5, and 7 wereidentified. The most notable changes in gene expression were upregulationof cholesterol and lipid gene pathways and downregulationof methallothionein genes. This was confirmed by qPCRand western blotting. Supplementing LDL reversed the change incholesterol pathway genes. Uptake of LDL in the serum starved cellswas also tested. Cells at days 3, 5 and 7 of serum starvation showedincreasing uptake of LDL-Dy549, whereas no uptake was seen atDay 0. The labeled LDL uptake was abolished in the presence ofunlabeled LDL. Downregulation of metallothionein genes along withupregulation of zinc binding genes is consistent with a reduction inthe intracellular levels of bio-available zinc. Zinc supplementationreversed expression changes of the metallothionein and zinctransportergenes.Conclusions: Serum starved ARPE-19 cells showed significantchanges in expression patterns of cholesterol and zinc-relatedpathway genes. Cholesterol synthesis enzymes were upregulated andcells showed increased uptake of exogenous cholesterol. Thissuggests that serum deprivation initiates a cellular program requiringcholesterol in RPE cells. The cells also have an increasedrequirement of zinc. Zinc-binding proteins frequently function as©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.