RPE45 Is the Caspase-Mediated Fragment of RPE65 and an Oxidative Stress Biomarker

Abstract

Purpose:: Previous studies of oxidant-induced retinal pigment epithelium (RPE) cell death indicate that reactions between oxidants and caspase-initiated cascades lead to apoptosis. However, the specific biochemical mediator that links oxidative stress to molecular changes in the visual cycle remains unclear. The present study provides evidence that RPE65, a peripheral membrane protein of the RPE, is cleaved in the presence of an oxidizing agent as well as active recombinant caspases. This cleavage results in the generation of RPE45, the caspase-mediated fragment of RPE65.

Methods:: Crude extracts of RPE membrane proteins from B6.129S7-Per2tm1Brd/J (B6) mice were prepared using a mild solution of 1%Triton X-100 in PBS. Bovine and zebrafish samples were used for comparison as well. Proteins were incubated with an oxidative stress agent (hydrogen peroxide) or active recombinant caspases. After incubation, proteins were separated on one- or two-dimensional SDS-PAGE followed by Western blot and mass spectrometry analyses. Bovine and zebrafish samples were used for comparison as well.

Results:: The protein samples which were treated with hydrogen peroxide and caspases generated RPE45, a fragment of RPE65 as well as other smaller domains of RPE65. The increase in the RPE45 fragment was dependent on the amount of oxidant and caspases. The sequence of RPE45 fragment was confirmed by electrospray tandem mass spectrometry. Interestingly, retinoid binding assay showed that RPE45 has the retinoid binding capacity.

Conclusions:: This study indicates that the oxidative stress and apoptosis in visual cycle can induce a cleavage of RPE65. This causes a decrease in the amount of all-trans-retinoid to 11-cis- retinoid which down-regulates the process in the visual cycle. Our results implicate that caspase-mediated RPE45 is a biomarker for oxidative stress and apoptosis in the RPE.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • apoptosis/cell death