Drusen Proteome Analysis: an Approach to the Etiology of Age-Related Macular Degeneration

Drusen proteome analysis: An approach to the etiology of age-related macular degeneration John W. Crabb*†‡§, Masaru Miyagi*†, Xiaorong Gu*†‡, Karen Shadrach*†, Karen A. West*†, Hirokazu Sakaguchi*†, Motohiro Kamei*†¶, Azeem Hasan*†, Lin Yan*†ʈ, Mary E. Rayborn*†, Robert G. Salomon‡, and Joe G. Hollyfield*†§ *Cole Eye Institute and †Lerner Research Institute, Cleveland Clinic Foundation, and ‡Department of Chemistry, Case Western Reserve University, Cleveland, OH 44195 Communicated by Thaddeus P. Dryja, Harvard Medical School, Boston, MA, September 11, 2002 (received for review July 19, 2002) Drusen are extracellular deposits that accumulate below the retinal The causal events responsible for AMD are not known. With pigment epithelium on Bruch’s membrane and are risk factors for age, debris-like material, referred to as drusen, accumulates developing age-related macular degeneration (AMD). The progres- below the RPE on Bruch’s membrane. Drusen can be observed sion of AMD might be slowed or halted if the formation of drusen during a funduscopic eye examination, and these deposits are could be modulated. To work toward a molecular understanding of referred to as either soft drusen or hard drusen, clinical terms drusen formation, we have developed a method for isolating defining their relative size, abundance, and shape (reviewed in microgram quantities of drusen and Bruch’s membrane for pro- refs. 8 and 9). Normal eyes may have maculas free of drusen, yet teome analysis. Liquid chromatography tandem MS analyses of drusen may be abundant in the retinal periphery (10). The drusen preparations from 18 normal donors and five AMD donors presence of numerous and͞or confluent, soft drusen in the identified 129 proteins. Immunocytochemical studies have thus macula is considered a major risk factor for developing AMD. far localized Ϸ16% of these proteins in drusen. Tissue metal- This correlation is so well established that many clinicians refer loproteinase inhibitor 3, clusterin, vitronectin, and serum albumin to individuals with soft drusen in the macula, in the absence of were the most common proteins observed in normal donor drusen any loss of macular vision, as an early stage of AMD (8, 11). whereas crystallin was detected more frequently in AMD donor Although drusen are widely accepted as contributors to the drusen. Up to 65% of the proteins identified were found in drusen etiology of AMD, their composition and the mechanism of their from both AMD and normal donors. However, oxidative protein formation is not understood. Histochemical and immunocyto- modifications were also observed, including apparent crosslinked chemical studies have shown that drusen contain a variety of species of tissue metalloproteinase inhibitor 3 and vitronectin, and lipids, polysaccharides, and glycosaminoglycans (8, 9) and have carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole ad- identified Ϸ20 drusen proteins (12–14). Better definition of the ducts are uniquely generated from the oxidation of docosa- molecular composition of drusen should provide important clues hexaenoate-containing lipids. By Western analysis they were regarding drusen biogenesis as well as possible leads for drug found to be more abundant in AMD than in normal Bruch’s targets and therapeutic agents to prevent AMD. We report here membrane and were found associated with drusen proteins. direct proteomic analysis of drusen isolated from normal and Carboxymethyl lysine, another oxidative modification, was also AMD donor eyes and present evidence of oxidative protein detected in drusen. These data strongly support the hypothesis modifications that may be linked to drusen formation and that oxidative injury contributes to the pathogenesis of AMD and underlie the pathogenesis of AMD. suggest that oxidative protein modifications may have a critical role in drusen formation. Methods Human Tissue Procurement. Eyes from 29 normal human donors ge-related macular degeneration (AMD) is the most com- and 16 AMD donors, all between 56 and 98 years of age, were Amon cause of legal blindness in the elderly population of used in this study, including 23 eyes (18 normal, five AMD) used developed countries, constituting a growing health problem for drusen isolation and protein identification and 22 eyes (11 (reviewed in ref. 1). Between 6 million and 10 million Americans normal, 11 AMD) used for Western analyses. Normal eyes were are blind from AMD, and hundreds of thousands of new cases obtained through the Cleveland Eye Bank or the National are diagnosed in the U.S. each year. Macular degeneration is Disease Research Interchange (Philadelphia). AMD donor eyes characterized by the breakdown of the macula, the small portion were from clinically diagnosed patients registered through the of the central retina (Ϸ2 mm in diameter) responsible for Eye Donor Program of the Foundation Fighting Blindness high-acuity vision. Genetic studies have shown that identical (Owings Mills, MD). Eyes were enucleated between 1.5 and 7 h twins exhibit 100% concordance for AMD (2) and that gene after death and frozen in liquid nitrogen. Tissues obtained from mutations cause early-onset forms of macular degeneration sources outside Cleveland were transferred to the laboratory on Ϫ (3–7). Late-onset macular degeneration (i.e., AMD) is usually dry ice, and then stored at 80°C until used. defined as either ‘‘dry’’ or ‘‘wet’’ and is a slow, progressive disease with genetic influences as well as environmental risk Protein Identification. Lipids were extracted from isolated drusen ͞ factors such as cigarette smoking and perhaps diet and lifetime preparations with chloroform methanol (15). After lipid extrac- ␮ light exposure (1). The wet, exudative, neovascular form of tion, the aqueous fraction was dried and dissolved in 5 lof400 AMD affects only Ϸ10% of those with the disease and is characterized by abnormal blood vessels growing from the Abbreviations: AGE, advanced glycation end product; AMD, age-related macular degen- choriocapillaris through the retinal pigment epithelium (RPE), eration; CEP, carboxyethyl pyrrole; LC MS͞MS, liquid chromatography tandem MS; RPE, typically resulting in hemorrhage, exudation, scarring, and͞or retinal pigment epithelium; TIMP3, tissue metalloproteinase inhibitor 3. serous retinal detachment (8). Approximately 90% of patients See commentary on page 14619. with AMD have the non-neovascular dry form characterized by §To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. atrophy of the RPE and loss of macular photoreceptors (8). At ¶Present address: Department of Ophthalmology, Osaka University Medical School, Suita, present there is no cure for any form of AMD, although some Osaka 565-0871, Japan. success in attenuating choroidal neovascularization has been ʈPresent address: Department of Cell Biology and Molecular Medicine, University of Med- obtained with photodynamic therapy (8). icine and Dentistry of New Jersey–New Jersey Medical School, Newark, NJ 07103. 14682–14687 ͉ PNAS ͉ November 12, 2002 ͉ vol. 99 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.222551899 Downloaded by guest on September 30, 2021 mM ammonium bicarbonate containing 8 M urea. Cysteine was antibodies to calgranulin A and B were obtained from Santa reduced by adding 1 ␮l of 100 mM DTT, and after 30 min at Cruz Biotechnology and used at 2 ␮g͞ml; sheep polyclonal room temperature under argon, 1 ␮l of 200 mM iodoacetamide antibodies to psoriasin were from Fitzgerald Industries Interna- was added and the alkylation continued 30 min under the same tional, Concord, MA, and used at 10 ␮g͞ml. Monoclonal conditions. The preparation was then diluted 3-fold with water, antibody to carboxymethyllysine was purchased from Wako incubated with trypsin (100 ng) overnight at 37°C, and centri- Chemical, Richmond, VA, and used at 5 ␮g͞ml. Monoclonal fuged to remove insoluble material by using established methods anti-carboxyethyl pyrrole (CEP) antibodies to CEP adducts were (16). Soluble tryptic peptides were analyzed by liquid chroma- prepared in the Hybridoma Core Facility, Lerner Research tography tandem MS (LC MS͞MS) with a CapLC system Institute, Cleveland Clinic Foundation and used at 5 ␮g͞ml (21). (Micromass, Beverly, MA) and a quadrupole time-of-flight mass spectrometer (QTOF2, Micromass) as described (17). Peptides Western Analysis. For 1D or 2D Western analysis, Bruch’s mem- were separated on a 75 ␮m ϫ 5 cm Biobasic C18 column (New brane͞RPE choroid was dissected from frozen AMD and nor- Objective, Cambridge, MA) by using aqueous formic acid͞ mal eyes as described (12). Tissues were homogenized in 7 M acetonitrile solvents, a flow rate of 250 nl͞min, and a gradient urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammo- of 5–40% acetonitrile over 120 min followed by 80% acetonitrile nio]-1-propanesulfonate, 0.5% Triton X-100, 2% ampholytes for 5 min. Protein identifications from MS͞MS data used (pH 3–10), and 1% DTT, and subjected to either 1D or 2D PROTEINLYNX GLOBAL SERVER and MASSLYNX 3.5 software electrophoresis. Electrophoresis and Western blotting methods (Micromass) and the Swiss-Protein and National Center for were as described (17, 18). Rabbit polyclonal anti-CEP antibod- Biotechnology Information protein sequence databases. Identi- ies that recognize CEP adducts (21) were used as a purified IgG fication of proteins by peptide mass mapping using in-gel tryptic fraction (at 1 ␮g͞ml) in Western analyses of dissected Bruch’s digestion and a Voyager DE Pro matrix-assisted laser desorption membrane͞RPE

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