Interphotoreceptor retinoid-binding protein (IRBP) binding in the interphotoreceptor matrix (IPM) and its potential role in the cone visual cycle
Garlipp, Mary Alice
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The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. Retinoids and fatty acids are exchanged across the IPM by interphotoreceptor retinoid-binding protein (IRBP). IRBP is thought not to bind the retina but freely distribute itself within the subretinal space, because it is readily extracted by saline wash. Therefore, it is prudent to ask: are there specialized IPM domains which could facilitate a role for the exchange of these molecules across the IPM via IRBP? In this study, the prevailing hypothesis that IRBP does not bind the retina or IPM is challenged by determining the association of IRBP in chicken ( Gallus domesticus ), turkey ( Meleagris gallopavo ), pig ( Sus scrofa ); and Xenopus laevis : (1) the distribution of native IRBP in retina following saline wash; (2) tissue localization of expressed Xenopus IRBP-Alexa 647 binding in Xenopus retina; (3) effect of light (LD)- and dark (DD)-adaptation on IRBP distribution/binding by fluorescence microscopy and Western blot. Retinas were detached and fixed directly, or washed in saline prior to fixation. Wholemounts and cryo-sections were compared by immunofluorescence from retinas detached under Ringer's followed by saline washes, or detached directly under 4% paraformaldehyde without saline wash. The distribution of IRBP and cone opsin were localized by confocal fluorescence microscopy, using monoclonal anti-IRBP or anti- Xenopus IRBP serum, and monoclonal COS-1, respectively. The distribution of IPM glycans was determined by using the lectins peanut agglutinin (PNA, cone matrix), and wheat germ agglutinin (WGA, rod/cone matrix), and also defined by fluorescence. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). For IRBP tissue binding studies, Xenopus IRBP was expressed in E. coli as a soluble thioredoxin (trx) fusion protein. Xenopus IRBP was freed of trx, purified by nickel affinity and anion exchange chromatography, and conjugated to Alexa 647. Disassociated photoreceptors with adherent matrix were also prepared from chicken and turkey retinas. Under hypotonic buffer, insoluble matrix was delaminated from saline washed retinas from chicken and turkey retinas. Retina flat mounts (chicken, turkey, and pig) showed IRBP diffusely distributed in an interconnecting, lattice-like pattern throughout the entire matrix. Saline wash replaced this pattern with fluorescent annuli surrounding individual cone outer segments. In isolated cones and matrix sheets, IRBP also co-localized with the PNA-binding matrix glycans. Ultrastructurally, immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments and Müller microvilli. In Xenopus, immunofluorescence showed saline wash removed most of the diffuse IRBP, but not that coating the cone outer segments and Müller microvilli. IRBP-Alexa 647 bound to the cone outer segment and Müller microvilli, and to a much lesser degree to the rods. IRBP labeling was markedly reduced when coincubated with unlabeled IRBP; ovalbumin-Alexa 647 and dye alone showed no binding. DD retinas showed diminished IRBP-Alexa 647 binding compared with that of LD retinas. Quantitative Western blot of LD and DD retinas washes showed no apparent difference in the amount of IRBP extracted. Cones are critically dependent on IRBP for retinoid delivery in the visual cycle. The cone-dominant vertebrates and Xenopus animal model system, offer an opportunity to uncover the molecular basis of IRBP's role in this process. Our results reveal a wash-resistant association of IRBP with a matrix domain immediately surrounding cone outer segments. The cone matrix sheath may be responsible for IRBP mediated cone targeting of 11- cis retinoids. Our data suggest the pericellular matrix of the cone outer segments and Müller cells may provide specialized domains to facilitate molecular interactions of the IPM, and could be key to IRBP's role in the cone visual cycle.