Localization of Serum Albumin, Serum Retinol–Binding Protein, and Interphotoreceptor Retinoid–Binding Protein in the Bovine Retina

Abstract

Abstract: : Purpose: Recent work (Tsina et al., 2004) has shown that the transport and/or clearance of retinol from isolated rod photoreceptors requires an extracellular factor(s). Interphotoreceptor retinoid–binding protein (IRBP) is a component of the interphotreceptor matrix (IPM) and known to bind visual cycle retinoids. Serum albumin, a protein capable of binding retinoids, has also been reported to be a component of the IPM (Adler et al., 1988; Edwards et al., 2000). It is of interest to know the components present in the IPM that are capable of binding visual cycle retinoids and that also facilitate rhodopsin regeneration. The purpose of this study was to determine the localization of serum albumin, serum retinol binding protein (sRBP), and IRBP in the bovine retina using immunofluorescence analysis. Methods: Fresh bovine eyes, obtained from a local abattoir, were immediately processed for immunolabeling. Cryosections were incubated with primary antibodies to bovine serum albumin, sRBP, and IRBP. Sections were then incubated with DAPI, Alexa Fluor® 488 goat anti–mouse, and Alexa Fluor® 568 goat anti–rabbit secondary antibodies.Sections were analyzed using a laser scanning confocal microscope equipped with Nomarski optics. Western blot analysis of bovine retinal tissues and purified protein standards was performed using the primary antibodies to BSA, sRBP, and IRBP to show specificity to their respective antigens. Results: Immunoblotting analysis showed that monoclonal anti–BSA was highly specific for bovine serum albumin detecting only a single band at ∼67 kD. Anti–human sRBP and anti–bovine IRBP were also highly specific recognizing a single band at ∼25 and ∼133 kD, respectively. Immunofluorescence analysis showed labeling for IRBP throughout the IPM. IRBP labeling was especially associated with the outer segments of photoreceptors and also with the apical surface of the RPE. Immunofluorescence labeling for serum albumin was associated only with the lumen of retinal and choroidal blood vessels. Staining for both serum albumin and sRBP in the IPM was negative. Conclusions: Immunofluorescence analysis of fresh bovine eyes using antibodies to BSA and sRBP clearly shows that serum albumin and sRBP are not components of bovine IPM. IRBP, on the other hand, is localized to the IPM where it is available for the binding and transport of visual cycle retinoids. From these data we conclude that serum albumin and sRBP are not factors that could participate in the binding and/or transport of visual cycle retinoids in the IPM of bovine retina.

Keywords: retinoids/retinoid binding proteins