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. 2015 Nov 6;290(45):27215-27227.
doi: 10.1074/jbc.M115.680363. Epub 2015 Sep 22.

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Jilin Zhou  1 , Keiko Ueda  1 , Jin Zhao  1 , Janet R Sparrow  2
Affiliations

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Jilin Zhou et al. J Biol Chem. .

Abstract

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch's membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch's membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4(-/-) and Rdh8(-/-)/Abca4(-/-) mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4(-/-) but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4(-/-) mice revealed that carbonyl adduct deposition in Bruch's membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4(-/-) mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch's membrane that can confer risk of age-related macular degeneration.

Keywords: aging; basement membrane; bisretinoid; dicarbonyl; eye; lipofuscin; photodegradation; retina; retinal degeneration; retinal pigment epithelium.

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Figures

FIGURE 1.
FIGURE 1.
Detection of carbonyl-modified protein. A, carbonylation of Rpe65 in Abca4−/− mice. Rpe65 was immunoprecipitated from posterior eyecups of mice. Samples were subjected to SDS-PAGE, derivatized with DNPH, probed with antibody to DNP (panel i), and re-probed with antibody to Rpe65 (panel ii). M indicates molecular weight markers. Abca4−/−, 7 months; Abca4+/+, 12 months; Rpe65rd12, 7 months. Data are representative of three independent immunoblots; eight eyes are from four mice. B, immunoblot detection of carbonyl-modified protein in ARPE-19 cells that accumulated A2E and were irradiated at 430 nm. 1st lane, RPE; 2nd lane, A2E-containing RPE; 3rd lane, A2E-containing RPE irradiated with 430 nm light. Carbonyl content in total protein was analyzed by SDS-PAGE using a gradient gel and immunoblotting with anti-DNP antibody (panel i). M indicates molecular weight marker; +, carbonyl-BSA (positive control). The additional bands in the 3rd lane and in the BSA lane (+) represent higher molecular weight derivatives related to adduct formation. Loading was controlled by re-probing with anti-β-actin antibody (42 kDa) (panel ii). Densitometric analysis (panel iii) of immunoreactive bands generated in two experiments. Carbonyl protein/actin ratios were normalized to untreated control and presented as mean ± S.E. *, p < 0.05 as compared with the other values; one-way ANOVA and Tukey's multiple comparison test. C, protein-carbonyl burden was measured by ELISA. ARPE-19 cells that had accumulated A2E were not irradiated (A2E) or irradiated (A2E + 430 nm) and then incubated for 2 or 6 h before the carbonyl was assayed. Mean ± S.E., three experiments. *, p < 0.05 as compared with A2E-containing cells that were not irradiated; one-way ANOVA and Newman-Keuls multiple comparisons test.
FIGURE 2.
FIGURE 2.
Detection of MG adducts. A, MG-derived hydroimidazolone protein adducts (MG-H1) in posterior eyecups of Abca4+/+ wild type (WT), Rdh8−/−/Abca4−/−, and Abca4−/− mice (age 6 months) measured by competitive indirect ELISA. Mice were exposed (+) or not exposed −) to blue light (430 nm) as a test of specificity. Detection using anti-MG antibody with MG-H1 quantification by interpolation from an MG-BSA standard curve (MG-BSA equivalents). Mean ± S.E., three experiments; four mice/eight eyes per group. *, p < 0.05 as compared with unexposed groups and wild-type 430 nm-exposed mice; one-way ANOVA and Tukey's multiple comparison test. B, UPLC-MS detection of DNPH-derivatized photocleavage products of A2E released upon irradiation (430 nm) of an A2E sample. Absorbance monitoring at 355 nm detects DNPH (trace 1), methylglyoxal-DNPH (trace 2), and glyoxal-DNPH (trace 3). MS-selected ion monitoring chromatograms at m/z 197 (DNPH) (trace 1), m/z 251 (methylglyoxal-DNPH) (trace 2), m/z 237 (glyoxal-DNPH) (trace 3), and an isomer of glyoxal-DNPH (trace 4). Data are representative of two experiments. C, measurement of MG-H1 protein adducts in lysates prepared from ARPE-19 cells that had accumulated A2E and were irradiated at 430 nm (RPE A2E 430 nm). Controls were RPE only and A2E-containing unirradiated RPE (RPE A2E). Mean ± S.E., three experiments. *, p < 0.05 as compared with other values; one-way ANOVA and Tukey's multiple comparison test.
FIGURE 3.
FIGURE 3.
Immunohistochemical labeling of methylglyoxal adducts and carbonyl adducts (DNP) in murine outer retina. A, labeling with antibodies that recognize MG-derived hydroimidazolone protein adducts (MG-H1) (MG) and antibodies that recognize carbonyl groups derivatized with DNPH. Mutant mice and ages are indicated. Control, unreacted section. Two-headed arrow, choroid; arrowhead, RPE nuclei; single-headed arrows, Bruch's membrane. Data are representative of three experiments. The dark line on the basal side of RPE is indicative of Bruch's membrane staining. The interface between choroid and sclera is also visible. B, immunodot blot detection of MG adducts. Nitrocellulose membrane was visualized under white light (Nikon DSLR camera) immediately after samples were applied to the membrane (row 1) and by exposure to x-ray film following immune reaction (row 2). Blotted samples were A2E incubated with BSA (a and d) and A2E and BSA irradiated at 430 nm (b and e); commercial MG-BSA was positive control (c and f). Blotted samples (one experiment) were reacted with rabbit polyclonal anti-MG adduct (a–c) and mouse monoclonal anti-MG adducts (d–f) followed by HRP-conjugated secondary antibody and a luminol-based chemiluminescent substrate.
FIGURE 4.
FIGURE 4.
Collagen (human type IV) serving as substrate for A2E-containing ARPE-19 cells is modified by MG after 430 nm irradiation. Detection by Western blotting with rabbit polyclonal antibody to MG adducts. Cells grown on collagen substrate (lane 1), ARPE-19 cells on collagen substrate accumulated A2E (lane 2), A2E-containing RPE cells grown on collagen and irradiated at 430 nm for 20 min (lane 3), and collagen exposed to MG (positive control) (lane 4). Data are representative of three experiments.
FIGURE 5.
FIGURE 5.
Protein cross-linking by exposure to photodegradation products of all-trans-retinal dimer and A2E. These photo-products are known to include MG and GO. A, RNase A (∼14 kDa) as model protein was incubated (5 days; 37 °C) with A2E photodegraded by light irradiation at 430 nm (A2E 430 nm). Analysis was by SDS-PAGE. GO, MG-positive controls; C, RNase only control; AG, aminoguanidine. M, molecular mass markers. Data are representative of three experiments. B, RNase activity assay performed in absence (C, control) or presence of A2E that had been irradiated at 430 nm (A2E 430 nm) or MG, positive control. Mean ± S.E., four replicates; *, p < 0.05 as compared with control; one-way ANOVA, Newman-Keuls multiple comparison test. C, RNase A was incubated (5 days; 37 °C) with A2E or all-trans-retinal dimer (atR-di) (left) or with A2E photodegraded by light irradiation at 430 nm (A2E 430 nm) or with all-trans-retinal dimer photodegraded (atR-di 430 nm). C, RNase-only control; MG, positive control. Data are representative of three experiments. D, arginine-free (somatostatin) and lysine-free (renin) peptides. Conditions as in A. Dimers and smearing are indicative of higher molecular weight oligomers. M, molecular mass markers; C control. Data are representative of three experiments.
FIGURE 6.
FIGURE 6.
Reduction in MMP-2 and MMP-9 activity induced by products of A2E photodegradation. A, MMP-2 digestion of fluorescein-conjugated collagen IV is reduced after collagen is modified by dicarbonyls released by A2E photodegradation. Collagen and A2E mixture were irradiated at 430 nm and incubated with human recombinant MMP-2 (rMMP-2) at indicated concentration. MG, methylglyoxal (positive control). Fluorescence intensity (excitation/emission, 495/515 nm) is indicative of MMP-2 activity. Mean ± S.E., four replicates. *, significantly different from rMMP-2 digestion in absence of A2E/430 nm irradiation or MG; one-way ANOVA and Tukey's multiple comparison test. The value for MG treatment and the value for A2E/430 nm/0.1 μg of rMMP-2 treatment are not significantly different from untreated fluorescein-conjugated collagen. B, human collagen IV exposed to photodegradation products of A2E is modified by MG adducts. Immuno-dot blot detection. Nitrocellulose membrane was visualized under white light (Nikon DSLR camera) immediately after samples were applied to the membrane (row 1) and by exposure to x-ray film following immune reaction (row 2). All blotted samples (one experiment) were reacted with a rabbit polyclonal antibody that recognizes MG-modified protein, followed by HRP-conjugated secondary antibody, and a luminol-based chemiluminescent substrate. Lane a, A2E irradiated at 430 nm and then blotted in the absence of collagen; lane b, collagen in the absence of A2E; lane c, collagen incubated with non-irradiated A2E; lane d, collagen incubated at 37 °C with irradiated A2E; lane e, collagen incubated with methylglyoxal as positive control. C, zymography assay. Modification of collagen IV under conditions of A2E photodegradation reduces MMP-2 and MMP-9 cleavage of collagen IV. hMMP-2 and hMMP-9 activity was measured by densitometric analysis as described under “Experimental Procedures,” and normalized (GLMMP band ÷ GLMMP-free) gray level intensities in the MMP lysis bands are presented. Cross-hatched bar, GLMMP-free. Mean ± S.E. of three experiments; * p < 0.05, one-way ANOVA and Newman-Keuls multiple comparison test. D, MMP-2- and MMP-9-mediated cleavage was assayed by measuring the hydroxyproline content remaining in the zymography bands after MMP digestion. A decrease in hydroxyproline release from MMP bands is indicative of increased cross-linking of collagen and thus reduced MMP activity. Mean ± S.E. of two experiments.
FIGURE 7.
FIGURE 7.
RAGE, glyoxylase-1, and glyoxylase-2 expression. RT-qPCR was performed with RNA purified from posterior eyecups of mice. A, RAGE expression was examined in Abca4+/+ wild-type, Abca4−/−, Rdh8−/−/Abca4−/− mice, and Rpe65rd12 mice at the ages indicated. The expression of each gene was normalized to 18S, and fold change in expression is presented relative to WT 4 months; three experiments. B, RAGE expression in ARPE-19 cells that had accumulated A2E and were irradiated at 430 nm (A2E 430 nm). Controls were untreated cells only and A2E-containing unirradiated RPE (A2E). Mean ± S.E., three experiments. *, p < 0.05 as compared with other values; one-way ANOVA and Dunnett's multiple comparison test. C, glyoxylase-1 expression was examined in Rpe65rd12 (8 months), Abca4+/+ (10 months), Abca4−/− 430 nm exposed (7 months), and Abca4−/− unexposed (11 months) mice and expressed relative to Rpe65rd12. *, p < 0.05, as compared with Rpe65rd12; **, p < 0.05 as compared with Abca4+/+; one-way ANOVA and Tukey's multiple comparison test; two experiments. D, glyoxylase-2 expression was examined as in C. Mean ± S.E.; three experiments.
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