Drusen proteome analysis: an approach to the etiology of age-related macular degeneration

Abstract

Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximately 16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.

Fig. 1.

Isolation of drusen. (A) Bruch's membrane/choroid dissected from an 82-year-old donor eye. (B) Macular region of Bruch's membrane containing drusen (white particulate material), diameter = 3 mm. (C) Drusen on surface of Bruch's membrane. (D) Drusen on Bruch's membrane. (E) Histological section of drusen on Bruch's membrane (embedded in plastic, stained with toluidine blue). Asterisks represent the location of the RPE before removal and the arrows indicate Bruch's membrane. (F) Isolated drusen. (G) Isolated drusen in pipette. (H) Isolated drusen in test tube. (Bars = A, 5 mm; C, 100 μm; D, 50 μm; E and F, 25 μm.)

Fig. 2.

SDS/PAGE analysis of drusen. Drusen isolated from a 93-year-old male, normal donor was dissolved in Laemmli sample buffer and ≈10 μg was fractionated by SDS/PAGE (4% stacking, 7.5% separating polyacrylamide gel). The gel was stained with Coomassie blue, bands were excised, and proteins were identified by LC MS/MS as described in Methods.

Fig. 3.

Western analysis of AMD and normal Bruch's membrane/RPE/choroid tissues. Human Bruch's membrane/RPE/choroid from the macular region of AMD and normal donor eyes was subjected to SDS/PAGE (≈20 μg protein/lane), electroblotted to poly(vinylidene difluoride), and probed with the rabbit polyclonal anti-CEP antibody to CEP adducts from docosahexaenoic acid. Human serum albumin modified with CEP (HSA-CEP, 20 ng) was used as a positive control. The age and sex of the donor eyes are listed at the top of each lane. More immunoreactivity can be seen in the AMD samples than in the normals.