Abstract
Purpose:
We reported Fundus albipunctatus in patients caused by mutations in RDH5R238W and A294P and RPE65I115T (Retina 2010 and Ophthalmology 2011). We hypothesize disturbed interactions of the retinol cycle enzymes RDH5, RLBP1, and RPE65 as a possible reason for the reduced rate of conversion to 11-cis-retinal. Here we report on investigations of RDH5, RLBP1, and RPE65 under heterologous expression of wild type and mutant RDH5 and RPE65.
Methods:
RDH5, RLBP1, and RPE65 were cloned into pCDNA3.1 vectors and mutations were introduced into RDH5 and RPE65 by mutation specific primers and PCR. The constructs were expressed in HeLa cells, and probed with anti-human RDH5 (Santa Cruz Biotechnologie), anti-human RLBP1, and RPE65 (Novus Biologicals) and Alexa Fluor coupled secondary antibodies (Invitrogen). Protein was extracted from human RPE specimen using the Proteo Extract Native Membrane Protein Kit (Merck, Darmstadt) and after heterologous expression of constructs in HEK293 cells. The protein samples were resolved on Clear Native (CN)-PAGE gels (Serva). RDH5 and RLBP1 were probed ON Western-blots with primary antibodies and HRP-labled secondary antibodies (Sigma-Aldrich).
Results:
Wild type RDH5 and RLBP1 were comparably distributed throughout the cytoplasma. RDH5R238W and RDH5A294P did not considerably change the intracellular localization but showed additional local accumulation of immunoreactivity close to the plasma membrane. RPE65 was distributed throughout the cytoplasma but focused close to the nucleus. RPE65I115T did not change this distribution. CN-PAGE revealed immunopositive bands comparable with dimeric RDH5 in protein extracts from human RPE specimen and additional bands in the higher molecular range. RLBP1 immunoreactivity was detected predominantly at sizes according to monomeric protein and at smaller amounts in the higher molecular range. Heterologous expression revealed immunoreactivity at sizes according to dimeric and higher molecular weight complexes with RDH5 from wild type and mutant constructs overlapping with RLBP1 immunopositive signals.
Conclusions:
We could show that RDH5 forms dimeric and higher molecular weight complexes which were localized throughout the cytoplasma in HeLa cells. The interactions and localizations were not completely abolished by the mutant proteins. Mutant RPE65 localized comparable to wild type RPE65 in HeLa cells. The higher molecular weight complexes detected in CN-PAGE require further work-up.
Keywords: 696 retinal degenerations: hereditary •
701 retinal pigment epithelium •
705 retinoids/retinoid binding proteins