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Mary Alice Garlipp, Federico Gonzalez-Fernandez; Association of Interphotoreceptor Retinoid-Binding Protein (IRBP) with Pericellular Outer Segment and Müller cell Domains. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1999.
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© ARVO (1962-2015); The Authors (2016-present)
How IRBP accurately targets the delivery of 11-cis and all-trans retinol, and 11-cis retinaldehyde between the RPE, rods, cones, and Muller cells in the rod/cone visual cycles remains largely a mystery. Here we address whether IRBP can associate with specific anatomic compartments along the cells bordering the interphotoreceptor matrix.
Adult Xenopus lavis were selected for these experiments because of their large photoreceptors, and that their retina can be detached in light and maintained in organ culture. Retinas were detached directly under 4% paraformaldehyde ("unwashed"), or Ringer’s saline followed by 3 saline washes and fixation ("washed"). The distribution of IRBP was characterized by indirect confocal immunofluorescence in wholemount and cross-section using rabbit anti-Xenopus IRBP serum, and double labeling with anti-opsin (mAb COS-1), and fluorescently labeled lectins (PNA and WGA). The distribution of IRBP in the rod and cone associated matrix was also studied by immunogold electron microscopy in undetached retinas. Washed retinas were also incubated with purified Xenopus IRBP-647, ovalbumin-Alexa 647, and Alexa-647 dye alone. Full-length Xenopus IRBP was expressed in a soluble form as a thioredoxin fusion protein. The thioredoxin was removed and the Xenopus IRBP purified by a combination of ion exchange, and Ni2+ affinity chromatography.
In unwashed retinas, IRBP was distributed throughout the IPM. Washing removed the diffuse protein leaving behind a residual IRBP associated with specific anatomic domains. This wash resistant IRBP typically rimmed the outer segments of rods and in particular to that of cones. The above may correspond to a diffuse and peri-cellular IRBP noted by immunogold EM. Recombinant Xenopus IRBP-647 preferentially targeted the pericellular domain surrounding cone outer segments, and to a lesser extent the rod outer segments. Binding of Xenopus IRBP-647 was also observed in the region of the Muller cell villi. Immunogold showed that IRBP is normally sequestered in this region. The above interaction was abolished by incubating with a 50-fold excess of unlabeled Xenopus IRBP. A similar binding was observed with bovine IRBP. No binding was detected with ovalbumin Alexa-647, or Alexa-647 dye alone.
Our data indicates that IRBP is not simply loosely present in retina as previously thought. Rather, it interacts with specific domains associated with the pericellular region of the outer segments particularly that of the cones, and the Müller cell villi. These findings may provide a mechanism through which IRBP targets specific retinoids to the correct cells during the visual cycle.
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