Inhibition of the visual cycle by A2E through direct interaction with RPE65 and implications in Stargardt disease.
Stargardt disease (STGD) is the major form of inherited juvenile macular degeneration. Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in retinal pigment epithelium (RPE) cells in STGD and contributes to the disease pathogenesis. However, the precise role of A2E in vision loss is unclear. Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key enzyme in the visual cycle. Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase activity in mice. Likewise, A2E also inhibited isomerohydrolase activity in cells coexpressing RPE65, lecithin retinol acyltransferase (LRAT), and cellular retinaldehyde-binding protein. In vitro isomerohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in a concentration-dependent manner, but did not inhibit LRAT activity. The inhibition type for isomerohydrolase was found to be reversible and competitive with K(i) = 13.6 μM. To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were performed. As shown by fluorimetric titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence. Consistently, the fluorescence of RPE65 decreased upon incubation with A2E. Both of these experiments suggest a direct, specific binding of A2E to RPE65. The binding constant for A2E and purified RPE65 was calculated to be 250 nM. These results demonstrate that A2E inhibits the regeneration of 11-cis retinal, the chromophore of visual pigments, which represents a unique mechanism by which A2E may impair vision in STGD.
Moiseyev
Gennadiy
G
Department of Medicine Endocrinology, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. gennadiy-moiseyev@ouhsc.edu
Nikolaeva
Olga
O
Chen
Ying
Y
Farjo
Krysten
K
Takahashi
Yusuke
Y
Ma
Jian-xing
JX
D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000037:antagonists & inhibitors*; D005453:Fluorescence; D008268:Macular Degeneration / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011726:Pyridinium Compounds / Q000378:metabolism* / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism* / Q000494:pharmacology; D014785:Vision, Ocular / Q000502:physiology*; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D000217:Acyltransferases; D002352:Carrier Proteins; D005136:Eye Proteins; D011726:Pyridinium Compounds; D012176:Retinoids; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C112040:A2-E (N-retinylidene-N-retinylethanolamine); C056942:lecithin-retinol acyltransferase