Inhibition of the visual cycle by A2E through direct interaction with RPE65 and implications in Stargardt disease

Abstract

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.

Fig. 1.

Inhibition of the isomerohydrolase activity by A2E in mouse eyes. BALB/c mice received a subretinal injection of 1 μL of 30 mM of A2E dissolved in DMSO or 1 μL of DMSO alone as control. Twenty-four hours after the injection, eyes were enucleated, and the eyecups homogenized in the grinder and incubated with 0.2 μM of all-trans [³H]-retinol for 2 h at 37 °C. The retinoids generated were analyzed by HPLC. (A) HPLC elution profile for the mice injected by 1 μL DMSO; (B) HPLC elution profile for the mice injected by 1 μL A2E dissolved in DMSO. (C) Amounts of 11-cis retinol generated were quantified based on the standard and averaged (mean ± SD, n = 4). Peaks were identified by coelution with corresponding retinoid standards. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, all-trans retinol.

Fig. 2.

Inhibition of the isomerohydrolase activity by A2E in cell culture. The 293A-LRAT cells infected by Ad-RPE65 at MOI 100 were preincubated for 6 h with the indicated concentrations of A2E followed by the addition of 2 μM of all-trans retinol and another 2 h incubation. At approximately 24 h after infection, the cells were harvested, and retinoids extracted with methanol and hexane and saponified. Production of 11-cis retinol was monitored by a normal phase HPLC. (A) HPLC elution profile. Peak 1, retinyl esters; 2, 11-cis retinol; 3, 13-cis retinol; 4, all-trans retinol. (B) Western blot analysis with an antibody specific for RPE65. Equal amounts of total cell lysate were loaded in each lane. (C) Dependence of the amount of generated 11-cis retinol on A2E concentration in culture medium (mean ± SD, n = 4).

Fig. 3.

Inhibition of the isomerohydrolase activity by A2E in vitro. The same amount (62 μg) of total proteins from 293A-LRAT cells infected by Ad-RPE65 at MOI 100 was incubated with 0.2 μM of all-trans [³H]-retinol in the presence or absence of A2E for 2 h at 37 °C. The retinoids generated were analyzed by HPLC. (A) HPLC elution profile without A2E; (B) with 13.5 μM A2E. Peak 1, retinyl esters; 2, 11-cis retinol; 3, all-trans retinol. (C) A2E concentration-dependent inhibition of 11-cis retinol generation (mean ± SD, n = 4).

Fig. 4.

Competitive inhibition of RPE65 isomerohydrolase by A2E in a liposome-based isomerohydrolase assay. All-trans retinyl ester incorporated in liposomes was used as a substrate for RPE65 expressed in 293A cells infected by Ad-RPE65 at MOI 100. (A) HPLC elution profile without A2E; (B) with 6.8 μM A2E. Peak 1, retinyl esters; 2, 11-cis retinol; 3, all-trans retinol. (C) Lineweaver–Burk plot of 11-cis retinol generation by RPE65. Liposomes with increasing concentrations (S) of all-trans retinyl palmitate were incubated with equal amounts (25 μg) of purified chicken recombinant RPE65 in the absence (♦) or presence (▪) of A2E (6.7 μM).

Fig. 5.

Fluorescence measurement of binding of A2E to RPE65. (A) Binding of A2E to purified chicken RPE65. Fluorescence emission spectra of 1 μM A2E in PBS, 0.1% CHAPS or after the addition of 1 μM RPE65 or 1 μM RNase A (control). Excitation peaked at 400 nm. (B) Titration of chicken RPE65 with A2E as measured by the increase in fluorescence intensity of A2E. Excitation was recorded at wavelength 400 nm, emission at wavelength 576 nm. The titration system consisted of 2 ml of 0.1 μM of RPE65 (●) or 0.1 μM pancreatic ribonuclease A (□) in 0.1% CHAPS, PBS buffer, pH 7.4. (C) Titration of chicken RPE65 with A2E as measured by the quenching of protein fluorescence. Excitation was recorded at wavelength 278 nm, emission at wavelength 340 nm. The titration system consisted of 2 mL of 0.1 μM of RPE65 in 0.1% CHAPS in PBS, pH 7.4 (mean ± SD, n = 4).