Confocal immunolocalization of bovine serum albumin, serumretinol-binding protein, and interphotoreceptor retinoid-binding proteinin bovine retina

[摘要] Purpose: Recently it has been shown that the transport as well asclearance of retinol from isolated rod photoreceptors requires anextracellular factor. Interphotoreceptor retinoid-binding protein (IRBP)is a component of the interphotoreceptor matrix (IPM) and is known tobind visual cycle retinoids. Serum albumin and serum retinol-bindingprotein (sRBP), proteins capable of binding retinoids, have also beenreported to be components of the IPM. It is of interest to know thecomponents present in the IPM that are capable of binding visual cycleretinoids and that also facilitate rhodopsin regeneration. The purposeof this study was to determine the localization of serum albumin, sRBP,and IRBP in bovine retina using immunofluorescence analysis.Methods: Fresh bovine eyes, obtained from a local abattoir, werefixed immediately after enucleation. Tissue sections (100 μm) wereincubated with primary antibodies to bovine serum albumin (BSA), sRBP,and IRBP. Sections were washed then incubated 4 h with4'-6-Diamidino-2-phenylindole (DAPI), Alexa Fluor® 488 goatantimouse, and Alexa Fluor® 568 goat antirabbit secondaryantibodies. Sections were analyzed using a laser scanning confocalmicroscope equipped with Nomarski optics. Western immunoblot analysis ofbovine retinal tissues and protein standards was performed using theprimary antibodies to BSA, sRBP, and IRBP to show specificity to theirrespective antigens.Results: Immunoblot analysis showed that monoclonal anti-BSA washighly specific for BSA detecting only a single band at about 67 kDa.Antihuman sRBP and antibovine IRBP were also highly specific,recognizing a single band at about 25 and about 133 kDa, respectively.No immunopositive bands were observed in bovine neural retinal whenprobed with the anti-sRBP antibody; however, a single immunoreactiveband at about 67 and about 133 kDa was detected in bovine neural retinaby the anti-BSA and IRBP antibodies, respectively. Immunofluorescenceanalysis showed labeling for IRBP throughout the IPM. IRBP labeling wasespecially associated with the outer segments of photoreceptors and alsowith the apical surface of the retinal pigment epithelium.Immunofluorescence labeling for serum albumin was associated only withthe lumen of retinal and choroidal blood vessels. Staining for bothserum albumin and sRBP in the IPM was negative.Conclusions: Immunofluorescence analysis of fresh bovine eyes usingantibodies to BSA and sRBP clearly shows that serum albumin and sRBP arenot components of bovine IPM. IRBP, on the other hand, is localized tothe IPM where it is available for the binding and transport of visualcycle retinoids. From these data we conclude that serum albumin and sRBPare not factors that could participate in the binding as well astransport of visual cycle retinoids in the IPM of bovine retina.

[学科分类] 生物化学/生物物理