Abstract
Purpose :
Interphotoreceptor retinoid binding protein (IRBP, RBP3) has been implicated to play an important role in diverse conditions including recessive RP, diabetic retinopathy, AMD, myopia, uveitis, and Kawasaki’s disease. Although a component of the aqueous, vitreous and the most abundant soluble protein of the interphotoreceptor matrix, little is known regarding its function beyond solubilizing/targeting retinoids in the vitamin A cycle. Here we explore the idea that IRBP goes beyond solubilizing retinoids but functions as a multifunctional free radical scavenger.
Methods :
3 approaches were used to monitor: 1) IRBP’s ability to protect retinoids from photodecomposition. Here, the ability of purified bovine IRBP to protect all-trans retinol and retinal were evaluated using an integrating cavity as both a light toxicity chamber and the measuring cuvette (Gonzalez-Fernandez et al., Mol. Vis. 2016; EER, 2023). Actinic light from an LED firing at 100 pulses/sec enters through a single port; Additional ports bring in the scanning beam at 100 scans/sec via an OLIS RSM 1000 UV/Vis rapid-scanning spectrophotometer, and allow light exit to the detector PMT. The actinic pulses and scanning beams were interleafed. This allowed the retinoid photodecomposition to be followed in real time. 2) IRBP’s ability quench free radical formation in the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ABTS assay. 3) Cellular vulnerability and pattern of IHC expression of 4-HNE, acrolein and nitrotyrosine in IRBP(-/-) mice.
Results :
The pump-probe system allowed retinoid photodecomposition to be followed in real time. We found that bIRBP significantly reduces the rate of retinoid photodecomposition. In the ABTS assay bIRBP showed significant free radical scavenging activity > synthetic vitamin E, trolox. Absence of IRBP affects the ONL with sparing of the INL and ganglion cells. Reduction in ONL thickness was biphasic with an initial reduction in between p5 – p20 in both WT and mutant strains but more so in the IRBP (-/-) mice. A further marked degeneration occurred in the IRBP(-/-) retina over the next 2.5 months. Increased HNE deposition in the inner/outer segments suggests that absence of IRBP increases oxidative stress through a different mechanism(s) than in light toxicity.
Conclusions :
Taken together our data suggests that IRBP has significant in vitro free radical scavenging that is relevant in vivo.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.