Release of 11-cis-Retinal From CRALBP

Abstract

Purpose:: To develop assays for the release of 11-cis-retinal from cellular retinaldehyde-binding protein (CRALBP) and to explore conditions leading to release of the retinoid.

Methods:: 9-cis-retinal (used in place of 11-cis-retinal) was added to human recombinant CRALBP (rCRALBP) and excess retinoid removed by Ni-chromatography. Assays developed exploited the protection of the carbonyl of 9-cis-retinal from reaction with water-soluble carbonyl reagents when the retinoid is bound to native CRALBP. One assay followed the change in absorption when bound 9-cis-retinal (410 nm max abs) was released from CRALBP and reacted with NH₂OH to form 9-cis-retinal oxime (355 nm max abs). A second assay employed HPLC to quantify the amount of retinal oxime formed upon incubation of CRALBP·9-cis-retinal with various RPE extracts and agents in the presence of 2 mM NH₂OH.

Results:: After 3 hours of incubation of CRALBP·9-cis-retinal with NH₂OH at room temperature and pH 7, 90% of the chromophore was recovered as 9-cis-retinal by HPLC analysis, demonstrating the stability of native CRALBP. Addition of 50% ethanol, which denatured the protein and released the retinoid, resulted in the detection of >95% 9-cis-retinal oxime. Urea (4 M) resulted in a perturbation of the ligand-binding domain as evidenced by the slow release of 9-cis-retinal (t_1/2 12 min). Incubation of CRALBP·9-cis-retinal with subcellular fractions from bovine RPE homogenates did not release the retinoid. Lowering the pH from 7 to 5 resulted in a pronounced blue shift in the absorbance (pKa ~ 6), indicating that the ligand-binding domain had been perturbed.

Conclusions:: CRALBP is a water-soluble protein found in relatively high abundance in RPE and Muller cells. Based on in vitro enzymology, studies of CRALBP knockout mice, and human mutations, it is likely that apo-CRALBP accepts 11-cis-retinol from the isomerohydrolase reaction in the visual cycle and facilitates its oxidation to 11-cis-retinal. If this mechanism is correct, 11-cis-retinal must then be released from CRALBP. In this study we demonstrate that 11-cis-retinal is released from CRALBP at the approximate pH of an early endosome.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium