Extra-retinal localization of retinol binding protein 3 (RBP3) suggests novel functions beyond the visual cycle

Abstract

Purpose : Most studies of retinol binding protein 3 (RBP3) have focused on its role in the vitamin A cycle or transport of lipids in the retina. RPB3 is the major protein component of the interphotoreceptor matrix (IPM) and plays a role in maintaining the health and integrity of the retina. However, studies have detected RPB3 in other compartments of the eye, and this study seeks to further explore its presence in the humors of the eye as a step in determining extra-retinal function.

Methods : Intact human globes were used with postmortem times from 3-12 hrs. Aqueous was obtained by inserting a 30-gauge syringe through the cornea into the anterior chamber. Vitreous was obtained via a syringe after removing the anterior structures by a circumferential incision at the level of the ciliary body. 7 vitreous and 7 aqueous samples were collected from 14 different individuals without history of ocular disease. To challenge human sample data, aqueous and soluble IPM fractions were collected from wildtype (WT) and RBP3-/- adult albino Sprague Dawley rats reared under cyclic dim light. Aqueous was collected by the same methods as human eyes. To collect IPM, retinas were detached under chilled PBS and gently agitated. The extract was centrifuged to remove cell fragments. Western blot analysis used rabbit monoclonals directed against human and rat RPB3s.

Results : RBP3 (124 kDa) was easily detected in human aqueous and vitreous. Minor immunoreactive bands were detected, particularly in vitreous, at 100, 75 and 60 kDa. Intensity of bands did not correlate with postmortem interval. In the aqueous, RBP3 was 65% less concentrated vs. vitreous and typically lacked lower bands. Western blot analysis showed an immunospecific full-length 140 kDa band in the WT IPM which was not seen in RBP3-/- animals. Minor bands were not detected. In the aqueous, RBP3 was detected by Western blot and appeared to be the most prominent protein overall, although its level corresponded to ~2% of that in the IPM.

Conclusions : RBP3 is not restricted to the IPM but is a prominent component of the aqueous and vitreous. Our data indicates that there is a gradient in RBP3’s concentration, with levels decreasing from the IPM to vitreous, with the lowest in aqueous. These distribution patterns across species suggest extra-retinal roles and merit further investigation of RBP3’s expression and function throughout the eye.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.