Biochemical Characterization of Retinoid Isomerases in the RPE and Retina

Abstract

Purpose: Enzyme mediated conversion of all-trans-retinoids to 11-cis-retinoids plays a key role in the biochemical process that regenerates visual pigment for photoreceptor cells. RPE65, a known protein in RPE cell, has been confirmed as the isomerase in retinal pigment epithelium cells. Recently we identified dihydroceramide desaturase 1 (DES1) as the retinoid isomerase for regenerating cone opsins in daylight. However the biochemical mechanism of the enzyme still remains elusive. Here we characterize the biochemical properties of DES1 using a variety of biochemical and biophysical methods.

Methods: GST fused DES1 and His-CRALBP protein were purified by chromatography. Pull-down assays were carried out with the purified proteins. The isomerase activities of RPE65 and DES1 in corresponding animal tissues and cell expression in the presence of a variety of specific inhibitors have been examined.

Results: The physiological and reciprocal pull-down assays confirm the direct interaction between DES1 and CRALBP. DES1 isomerase activity is dependent on the cytochrome b5 electron transfer chain. Inhibition of DES1 isomerase activity by spin traps and probes demonstrate a possible radical intermediate pathway. DES1 is a Fe(III) dependent retinol isomerase.

Conclusions: Similarity towards different inhibitors for chicken retina and HEK-DES1 suggests DES1 is the isomerase-2 responsible for the alternative visual cycle in retina. The reaction mechanism of DES1 appears to involve a free-radical intermediate.

Keywords: 705 retinoids/retinoid binding proteins • 658 protein purification and characterization