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SK Bhattacharya, Z Wu, M Miyagi, KA West, Z Jin, M Nawrot, JC Saari, JW Crabb; Interactions of CRALBP With Other Visual Cycle Proteins . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4567.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To demonstrate an interaction between cellular retinaldehyde-binding protein (CRALBP) and 11-cis-retinol-dehydrogenase (RDH5) and to identify other visual cycle protein interactions. The visual cycle is the enzymatic pathway by which all-trans-retinal from photoreceptor bleaching is isomerized to 11-cis-retinal in the retinal pigment epithelium (RPE) for visual pigment regeneration. Methods:Recombinant CRALBP and RDH5 were produced and purified to apparent homogeneity. Kinetic parameters for RDH5 catalyzed reactions with retinoid substrates were determined in the presence and absence of CRALBP. Protein-protein interactions were sought in bovine RPE microsomes by reciprocal immunoprecipitations and other affinity isolation methods. Proteins were identified by Western analyses, MALDI TOF MS and LC MS/MS. Results:Wildtype CRALBP enhances the affinity of RDH5 for 9-cis- and 11-cis-retinoids by 2-3 fold over free retinoid. Mutant M225K rCRALBP, which lacks ligand binding capability, has no effect on RDH5 enzyme kinetics. CRALBP, RDH5, RPE65 and RGR opsin co-precipitate from RPE microsomes with anti-CRALBP antibodies. Similar results were obtained with anti-RDH5 and anti-RPE65 antibodies. Conclusion:A stable, functional interaction has been demonstrated between CRALBP and RDH5. Visual cycle proteins CRALBP, RDH5, RPE65 and RGR opsin appear to interact in a protein complex. Further proteomic and affinity isolation approaches are directed toward identifying other possible RPE components of a visual cycle protein complex. CR: N. Supported in part by NIH grants EY06603, EY01730, EY02317, a Research Center Grant from The Foundation Fighting Blindness, and funds from the Cleveland Clinic Foundation and Research to Prevent Blindness, Inc.
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