Purification of Interphotoreceptor Retinoid-Binding Protein (IRBP, RBP3) from globes of separate donors

Abstract

Purpose : IRBP is a retina specific 124 kDa lipoglycoprotein with an unusual 4-module structure. Although abundant in the interphotoreceptor matrix (IPM) and present in the vitreous and aqueous, its structure and function is not understood. Renewed interest in IRBP comes from its emerging role in diabetic retinopathy and AMD. However, research has relied only on IRBP purified from bovine retina and various recombinant IRBPs. Aspects of its function in man may not be represented in IRBP from bovine due to differences in its aa sequence and/or post-translational modifications not in recombinant proteins. Our long-term goal is to determine if IRBP can be purified from human globes and what are the postmortem parameters and disease states that might affect its purification.

Methods : Our approach was to scale down our standard prep for 200 bovine retinas down to one human globe. Eye bank protocols were established for fresh and frozen globes were established. The retina was gently detached from the RPE and transferred to a dish with chilled PBS with broad coverage protease inhibitors. The retina was gently agitated under PBS and 3 washes pooled (total vol = 12 ml). The wash was then centrifuged at 20,000 xg for 10 min. Aliquots of the supernatant were saved, and the remainder incubated with 2.5 ml of Con-A Sepharose for 2 hrs at 4C with gently shaking in a 10 ml spin column. The column was then washed 4 x in PBS followed by incubation overnight with methyl alpha-D-glucopyranoside. The elution was subjected to anion exchange under a linear salt gradient (20 ml bed volume). Protein profiles were compared by SDS 4-29% gradient PAGE.

Results : The matrix extraction was an important step affording an initial enrichment. Long post-mortem intervals increased “leakage” of serum and cellular proteins. Con-A provided a significant enrichment of the IRBP albeit only ~80%. Anion exchange was required to achieve >90% purification. Pooled matrix of 8 globes yielded 0.06 mg/eye (0.48 nmoles/eye).

Conclusions : We developed a strategy to purify IRBP from cadaveric globes from individual Donors. This will facilitate studies of the structure and function of IRBP in health and disease.

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