Interactions of all-trans-retinol and long-chain fatty acids with interphotoreceptor retinoid-binding protein

Abstract

Interphotoreceptor retinoid-binding protein (IRBP), a predominant protein in the interphotoreceptor matrix of the retina, has been implicated in transfer of retinoids between retinal pigment epithelium and photoreceptor cells. The interactions of IRBP with all-trans-retinol have been studied by three fluorescence-based methods and by measurements of binding of 3[H]-labeled all-trans-retinol to this protein. It was found that IRBP contains two sites with similar but not identical affinities for all-trans-retinol. The dissociation constant of the all-trans-retinol-IRBP complex at the first site was 0.1 microM, which is about 10-fold lower than previously reported values. The second site had about 2.5-fold lower affinity for all-trans-retinol as compared to the first site. Long-chain fatty acids were found in this study to displace all-trans-retinol from the stronger retinol-binding site on IRBP. Displacement of all-trans-retinol was used to study the interactions of fatty acids with this protein. It was found that docosahexaenoic acid (DHA C22:6n-3), an essential fatty acid which plays an important role in vision, had the highest apparent affinity for the site probed on IRBP of all the fatty acids studied.

Docosahexaenoic Acid Modulates the Interactions of the Interphotoreceptor Retinoid-binding Protein with 11-cis-Retinal

“…The FA:IRBP molar ratio of the protein was found to be 5.5. This value is about 2-fold lower than the value we previously reported based on a microfluorometric assay (14). However, we recently noticed that the microfluorometric assay is susceptible to several artefacts and may yield values that vary by up to 2-3-fold.…”

“…The effects of 11-cis-retinal and of the FAs on the interactions of all-trans-retinol with IRBP at this site were additive. These observations and the similarity of the values of K d for 11-cisretinal obtained in the absence and in the presence of DHA (Table III (14,15). These observations are likely to reflect that a tryptophan or tyrosine residue(s) are located near both of the sites and that ligand binding leads to quenching of the fluorescence of these residues.…”

“…Binding of retinol in one of the sites results in a significant enhancement of the fluorescence of the ligand, a behavior indicative of a hydrophobic and/or restrictive nature of the binding pocket. In contrast, binding in a second IRBP retinoid-binding site does not affect the fluorescence of retinol, suggesting that the site is located at the surface of the protein and may interact with the ligand via its polar head group (14). Examination of the interactions of IRBP with several forms of retinoids revealed that the protein displays the highest affinity for the all-trans form of retinol and the 11-cis isomer of retinal (15).…”

“…IRBP contains two binding sites for retinoids (3,13,14), and fluorescence studies of binding of retinoids to the protein indicated that the two sites are quite different. Binding of retinol in one of the sites results in a significant enhancement of the fluorescence of the ligand, a behavior indicative of a hydrophobic and/or restrictive nature of the binding pocket.…”

“…Of all the FAs tested, docosahexaenoic acid (c22:6 n-3; DHA) was the most effective in displacing retinoids from IRBP (14). This observation is of particular interest, since DHA is essential for vision and composes a large fraction of the acyl chains of phospholipids in the membranes of ROS (16 -18).…”