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. 2011 Feb 2;133(4):849-57.
doi: 10.1021/ja107195u.

Enzymatic Degradation of A2E, a Retinal Pigment Epithelial Lipofuscin Bisretinoid

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Enzymatic Degradation of A2E, a Retinal Pigment Epithelial Lipofuscin Bisretinoid

Yalin Wu et al. J Am Chem Soc. .
Free PMC article

Abstract

Some forms of blinding macular disease are associated with excessive accumulation of bisretinoid lipofuscin in retinal pigment epithelial (RPE) cells of the eye. This material is refractory to lysosomal enzyme degradation. In addition to gene and drug-based therapies, treatments that reverse the accumulation of bisretinoid would be beneficial. Thus, we have examined the feasibility of degrading the bisretinoids by delivery of exogenous enzyme. As proof of principle we report that horseradish peroxidase (HRP) can cleave the RPE bisretinoid A2E. In both cell-free and cell-based assays, A2E levels were decreased in the presence of HRP. HRP-associated cleavage products were detected by ultraperformance liquid chromatography (UPLC) coupled to electrospray ionization mass spectrometry, and the structures of the aldehyde-bearing cleavage products were elucidated by 18O-labeling and 1H NMR spectroscopy and by recording UV−vis absorbance spectra. These findings indicate that RPE bisretinoids such as A2E can be degraded by appropriate enzyme activities.

Figures

Figure 1
Figure 1
HPLC quantitation of A2E pigment after incubation in the presence and absence of horseradish peroxidase (HRP)/H2O2. (a–d) HPLC chromatograms generated with samples of A2E, A2E incubated with H2O2, A2E incubated with HRP, and A2E incubated with H2O2 (0.2%) and HRP for 24 h; reversed-phase HPLC. (e) Quantitation of A2E and A2E isomers. Chromatographic peak areas were measured, A2E and isomers were summed and are presented as percent of pigment in the absence of HRP and H2O2; mean (SEM of three experiments; +, presence of compound/reagent. HPLC quantitation of A2E in ARPE-19 cells to which HRP was delivered using Bioporter reagent; +, presence of compound/reagent; mean ± SEM of 4–7 experiments. (f) HPLC quantitation of A2E in ARPE-19 cells to which HRP was delivered using Bioporter reagent; +, presence of compound/reagent. Values (pmol/106 cells) are expressed as percent of A2E in absence of Bioporter and HRP; mean ± SEM of 4–7 experiments. (g) A2E: structure, mass-to-charge ratio (m/z), UV-vis absorbance (nm), and electronic transition assignments (↔).
Figure 2
Figure 2
Effects of pH on HRP-mediated degradation of the bisretinoid A2E. A2E (20 µM) was incubated with HRP (200 units/mL) and H2O2 (0.6%) at the indicated pH for 5 h or was incubated in the absence of HRP/H2O2. Residual A2E and isomers were quantified by reversed-phase HPLC. Mean ± SEM of three experiments.
Figure 3
Figure 3
Intracellular horseradish peroxidase (HRP) delivered to ARPE-19 cells accumulated in lysosomes. (a–c) Intracellular horseradish peroxidase introduced to the cells via Bioporter delivery is detected immunocytochemically using primary antibody to HRP, an avidin-biotin-alkaline phosphatase complex, and color development with Vector Red. The reaction product is visible with both fluorescence (a) and bright field (b) optics. HRP is present (+) or absent (−). (d–k) Colocalization of HRP with a lysosomal marker in ARPE-19 cells. Imaging by laser scanning confocal microscopy (x-y scans). Lysotracker (d and h), HRP present (+) or absent (−) (e and i), DAPI-stained nuclei (f and j), and overlay (g and k).
Figure 4
Figure 4
Cell viability with HRP-mediated degradation of intracellular A2E. (a) Cell viability 3 and 14 days after introduction of HRP was probed by MTT assay. Bar height is indicative of MTT absorbance (at 570 nm) and reflects cell viability; mean ± SEM of four experiments. (b) A two-color fluorescence assay was employed to quantify percent nonviable cells; mean ± SEM of two experiments. Cells accumulated A2E in culture, and HRP was delivered to the cells using Bioporter reagent; +, presence of compound/reagent.
Figure 5
Figure 5
UPLC–ESI-MS analysis of samples of A2E–bromine (A2E–Br) following oxidation and cleavage by horseradish peroxidase (HRP). (a–c) Representative reversed-phase UPLC profiles (Acquity BEH C18 column, monitoring at 430 nm) obtained after 3 and 18 h incubations of A2E–Br, HRP, and H2O2 (0.2%), and A2E–Br alone. Insets in panels a and b (top): UV-vis absorbance spectra of A2E–Br, monooxo-A2E–Br, and bisoxo-A2E–Br. Inset in panel b (right): chromatogram expanded between retention time 2–4 min for detection of HRP-associated cleavage products (F1-F4); the chromatogram is magnified in Figure 6a. (d–h) MS spectra of peaks a–e present in panels a–c. The molecular ions at m/z 730/732, 746/748, and 762/764 correspond to A2E–Br, monooxo-A2E–Br, and bisoxo-A2E–Br, respectively. Note bromine isotope (79Br and 81Br) peaks differing by 2 m/z units in panels d–h. Inset in panel d: A2E–Br structure, UV-vis absorbance (nm), and electronic transition assignments (↔).
Figure 6
Figure 6
UPLC–MS analysis of HRP-induced cleavage products of A2E–Br. (a) UPLC chromatogram (monitoring at 430 nm) of A2E–Br incubated with HRP and H2O2 (0.2%) for 3 h; expanded between retention times 2–4 min (full chromatogram in Figure 5b). Top insets: UV-vis absorbance spectra of cleavage products F1-F4. (b–g) Extracted ion monitoring chromatograms of A2E–Br incubated in water (b–d) and with HRP and H2O2 (e–g) for 3 h. Data were acquired in ESI mode with selection for mass-to-charge ratios (m/z) 610, 570, and 544 and recorded as a function of retention time in a reversed-phase UPLC column. In panels e–g four prominent ion peaks corresponding to UPLC peaks F1-F4 (panel a) were observed. (h–k) Assignment of m/z to chromatographic peaks (F1-F4) by coupled electrospray ionization analyses. The bromine tag was indicative of cleavage products that included the pyridinium headgroup of A2E–Br. Insets: proposed structures of cleavage products and electronic transition assignments (↔) of intact arms of A2E–Br.
Figure 7
Figure 7
Elucidation of aldehyde-containing cleavage products. (a–d) UPLC–MS analysis of 18O-labeled cleavage products generated from A2E–Br incubated with HRP in 18O-labeled water (H218O) and hydrogen peroxide (H218O2). (a and b) Selected ion chromatograms with detection set for mass-to-charge ratios (m/z) of 614 and 574. Two peaks (F2a and F3a) were observed that corresponded to 18O-labeled peaks F2 and F3 according to the identical retention time. (c and d) ESI-MS spectra of the chromatographic peaks, F2a and F3a. Insets: structures of 18O-labeled cleavage products. In the use of H218O and H218O2, the mass of F2 (m/z 610/612) and F3 (m/z 570/572) is shifted by 2 m/z units to 612/614 (F2a) and 572/574 (F3a), respectively, due to 16O-18O exchange. (e) 1H NMR spectra recorded in the region of 10–5 ppm (400 MHz; MeOD): upper, A2E; lower, A2E incubated with HRP and H2O2 for 5 h. The signal at ~9.4 ppm (arrow) is characteristic of the aldehyde proton.
Figure 8
Figure 8
Proposed mechanisms for HRP-catalyzed oxidation and degradation of A2E; L, ligand. The HRP catalytic cycle involves two active species, compound I (Cpd I) and compound II (Cpd II), that possess high-valence iron ions storing two and one oxidation equivalents, respectively (ref 31). Two-electron oxidation of HRP by H2O2 (step 1) generates the first active species Cpd I; the latter mediates oxygen-transfer reactions to yield C–H hydroxylation (step 2) and/or C=C epoxidation (step 3). Cpd I then returns to ground state (step 4). At epoxidation sites, opening and hydration of the epoxide ring would give rise to a diol (step 5) that would undergo a periodate-like enzymatic carbon-carbon fission reaction to form two carbon-centered alcoholic radicals (step 6), one that includes the positively charged pyridinium ring (⊕) with the residual component being without charge (neutral) (step 6). Cpd I (step 1) initiates one-electron oxidation on one of these radicals to generate an aldehyde or methylketone-bearing products (step 7) and Cpd II (step 8). The latter forms by transfer of a hydrogen atom from the hydroxyl group of the radical to the iron-oxo complex (Cpd I). Subsequently one-electron oxidation of another radical by Cpd II yields an aldehyde or methylketone-bearing product (step 7), and the enzyme (Cpd II) returns to ground state (ref 19) (step 9). Note that enzyme cleavage at the 9–10 double bond would generate an aldehyde-bearing fragment and a ketone-bearing fragment; cleavage at 7–8, 7′–8′, or 11′–12′ double bonds would lead to two aldehyde-bearing fragments.

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