Beyond the IPM – understanding the extraretinal roles of interphotoreceptor retinoid-binding protein (IRBP / RBP3)

Abstract

Purpose : Most studies of RBP3 have focused on its function within the interphotoreceptor matrix (IPM). RBP3 is thought to be entrapped in the subretinal space due to the RPE zonula occludens and adherens junctions of the external limiting membrane. Although the [RBP3] in the vitreous is important to the etiology of diabetic retinopathy (DR) nothing is known regarding the function of RBP3 in the vitreous/aqueous and what factors control its expression in those compartments.

Methods : Human cadaveric globes had postmortem intervals of 3-12 hrs. Aqueous was obtained by inserting a 30-gauge syringe through the cornea. Vitreous was obtained via a 16-gauge syringe inserted at the limbus. 25 samples were obtained from donors without history of ocular disease. Samples were subjected to SDS 4-20% PAGE. The RBP3 band was quantified by “stain-free” imaging (BioRad), Western Blot analysis (WB), and ELISA. The latter two used a monoclonal antibody. Purified bovine RBP3 was used as the internal standard for WB. A human RBP3 peptide was used for the ELISA calibration.

Results : The RBP3 band (124 kDa) was slightly lower than that of bovine RBP3 (140 kDa). Degradation fragments were not characteristic of most samples and when present did not correlate with postmortem intervals. [RBP3] in the vitreous and aqueous was 3.4 nM +/- 0.13 and 1.9 nM +/- 0.44 (N=4 SD) respectively. The concentration by WB was ~5 x higher, and by ELISA ~20 fold lower. The overestimate by WB may be due to the antibody being more sensitive to the human protein to which it is directed. The lower sensitivity by ELISA indicates that epitope is buried and therefore not accessible within the native protein.

Conclusions : Vitreal [RBP3] is ~2 fold higher compared to the aqueous. The most reliable estimation at present of its absolute concentration in these two compartments is by nonimmunological methods. These values are several orders of magnitude lower than its concentration in the IPM. This is consistent with a model that RBP3 diffuses into the vitreous and then is further diluted as it passes into the anterior chamber. This indicates that the subretinal compartment can communicate with the vitreous chamber. Alterations in the integrity of the junctional complexes bordering the subretinal space could affect the vitreal and aqueous RBP3 concentrations in disease states such as DR.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.