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Originally published In Press as doi:10.1074/mcp.D200001-MCP200 on January 16, 2003.
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Molecular & Cellular Proteomics 2:37-49, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Protein Database, Human Retinal Pigment Epithelium*

Karen A. West, Lin Yan, Karen Shadrach, Jian Sun, Azeem Hasan, Masaru Miyagi, John S. Crabb, Joe G. Hollyfield, Alan D. Marmorstein and John W. Crabb{ddagger}

From the Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome.

The RPE1 is a simple cuboidal epithelium that separates the photoreceptor cells of the retina from their principal blood supply in the choroid (1). In all vertebrates, the RPE forms an integral part of the blood-retinal barrier and is responsible for vectorial transport of nutrients to rod and cone photoreceptors and removal of waste products to the blood. In addition, the RPE phagocytizes shed photoreceptor outer segments, absorbs scattered light, and functions in the retinoid visual cycle and regeneration of bleached visual pigment (2, 3). To facilitate studies of retina and RPE in health and disease (4), we have initiated the development of a human RPE protein database.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
In Vivo RPE Samples—
Forty-two normal human eyes were used in this study and were obtained from the Cleveland Eye Bank within 3–12 h postmortem. Following bisection of the globe behind the limbus, the anterior segment and vitreous were discarded, and the retina was removed. RPE cells were gently brushed from the eye cup using an artist’s 7-mm angular paint brush (Langnickel L7160) and Ca2+- and Mg2+-free phosphate-buffered saline containing 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (two to three times with 500 µl). The RPE cells were then washed two to three times by centrifugation in phosphate-buffered saline. For select preparations, red blood cells were removed by centrifugation in a Percoll gradient (1.0–1.1 g/ml, Amersham Biosciences); the pigmented RPE cells float near the top of the gradient and were collected with a Pasteur pipette and then washed two times in phosphate-buffered saline. Whole cell lysates were prepared by homogenizing RPE cell pellets in isoelectric focusing (IEF) solvent B (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100, 2% carrier ampholyte, 1% dithiothreitol), the solution was clarified by centrifugation, and protein was quantified by a modified Bradford assay (5). About 95 µg of soluble whole cell RPE protein was recovered per eye (n = 11 eyes).

Subcellular Fractionation—
Subcellular RPE fractions were prepared according to Saari et al. (6). Briefly, freshly isolated RPE cells were suspended in 1–4 ml of 25 mM Tris acetate, pH 7, 0.25 M sucrose, 1 mM dithiothreitol, homogenized with 25–125 manual passes of a glass homogenizer, and clarified in a microcentrifuge for 10 min at 1,000 x g. The clarified RPE lysate was centrifuged at 27,000 x g at 5 °C for 20 min, yielding the P2 membrane fraction. The supernatant was centrifuged again at 150,000 x g for 1 h at 5 °C, yielding the microsomal and cytosolic cell fractions. The microsomal and P2 pellets were resuspended in IEF solvent B. The cytosolic fraction was exchanged into IEF solvent B using Centricon concentrators (Amicon, 10-kDa molecular mass cut-off). Average recovery per eye was about 18 µg of cytosolic protein, 16 µg of P2 membrane protein, and 9 µg of microsomal protein (n = 31 eyes) based on the modified Bradford assay (5).

Electrophoresis—
One- and two-dimensional electrophoresis was performed as described previously using the Bio-Rad Mini-Protein II, Bio-Rad Protein IIxi, Amersham Biosciences IPGphor, and Amersham Biosciences IsoDalt systems (7–9). Isoelectric focusing was performed with non-linear pH 3–10 or linear pH 4–7 immobilized pH gradients (18-cm IPG strips, Amersham Biosciences) in 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100, 2% carrier ampholytes, 1% dithiothreitol. Second dimension electrophoresis utilized 23.5- x 18- x 0.1-cm gels (12% acrylamide). Colloidal Coomassie Blue- (Pierce Code Blue) or silver-stained gel patterns were recorded with Quantity One and PDQuest gel analysis software (Bio-Rad). Protein from multiple eyes was utilized for most electrophoretic separations, and amounts varied from ~30 to ~500 µg/gel.

Protein Identification by Mass Spectrometry—
Identification of proteins by peptide mass mapping and/or liquid chromatography electrospray tandem mass spectrometry (LC MS/MS) were as described elsewhere (7–9). Briefly, gel spots and bands were excised, stain was washed away, proteins were digested in-gel with trypsin, and peptides were extracted for mass spectrometric analysis. For MALDI-TOF MS, peptides were adsorbed onto C18 ZipTips (Millipore, Bedford, MA), eluted with 75% acetonitrile, 0.02% trifluoroacetic acid, and analyzed using a Voyager DE Pro MALDI-TOF mass spectrometer (PE Biosystems, Framingham, MA). Measured peptide masses were used to query the Swiss Protein, TrEMBL, and National Center for Biotechnology Information (NCBI) sequence databases for matches using MS-Fit and Profound search programs and a mass tolerance of 50 ppm. Positive identification by peptide mass mapping required four to five peptide matches under the search conditions used (10).

For analysis by LC MS/MS, tryptic digests were injected by autosampler onto a 0.3- x 1-mm trapping column (PepMap C18, LC Packings) using a CapLC system (Micromass, Beverly, MA). Peptides were eluted at 250 nl/min and chromatographed on Biobasic C18 columns (50 µm x 5 cm or 75 µm x 5 cm, New Objective, Cambridge, MA) directly into a quadrupole time-of-flight mass spectrometer (QTOF2, Micromass). Protein identifications from MS/MS data utilized Micromass software ProteinLynxTM Global Server, MassLynxTM, version 3.5, and the Swiss Protein and NCBI protein sequence databases. MS/MS spectra were examined manually to verify determined sequences.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Two hundred seventy-eight proteins were identified in proteomic analyses of the human RPE and are listed in Table I with database accession numbers, cell subfraction of origin, and summaries of electrophoretic and mass spectrometric data supporting the identifications. A total of 17 2D gels were performed with RPE cell fractions, including eight for method development and nine in which spots were excised and analyzed (five for whole cell, three for cytosolic, three for microsomal, and one for P2 cell fractions). Representative 2D gels for each cell fraction are shown in Figs. 1–4. In addition, bands were excised and analyzed from two 1D SDS-PAGE separations of the P2, microsomal, and cytosolic RPE cell fractions (Fig. 5). One hundred sixty proteins were identified following 2D PAGE, 180 proteins were identified following 1D PAGE, and 62 proteins were identified from both 1D and 2D gels. The 1D and 2D PAGE methods were complimentary, each contributing unique protein identifications to this preliminary proteome analysis. Other profiling of human RPE gene expression has been obtained through serial analysis of gene expression (SAGE), which yielded the identity of 445 genes (227 with assigned functions) and 333 unknown sequence tags (11). Expressed sequence tag analysis of combined human RPE and choroid tissue has also been reported (12); however, the identify of RPE components was obscured by the choroid vasculature. Less than half (44%) of the proteins recently identified from in vitro cultures of rat RPE-J cells (7) were also observed in the present analysis of human in vivo RPE.


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  		TABLE I Proteins identified in human retinal pigment

 


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  		FIG. 1. Human RPE whole cell lysate. Approximately 150 µg of protein were applied to 2D gel analysis (75,202 V-h IEF), and the gel was stained with colloidal Coomassie Blue. Numbered spots were excised for protein identification (Table I).

 


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  		FIG. 2. Human RPE cytosol. Approximately 150 µg of RPE cytosolic protein were applied to 2D gel analysis (87,550 V-h IEF), and the gel was stained with silver. Numbered spots were excised for protein identification (Table I).

 


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  		FIG. 3. Human RPE P2 membrane subcellular fraction. Approximately 80 µg of RPE P2 membrane protein were applied to 2D gel analysis (87,550 V-h IEF), and the gel was stained with silver. Numbered spots were excised for protein identification (Table I).

 


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  		FIG. 4. Human RPE microsome subcellular fraction. Approximately 190 µg of RPE microsomal protein were applied to 2D gel analysis (88,400 V-h IEF), and the gel was stained with colloidal Coomassie Blue. Numbered spots were excised for protein identification (Table I).

 


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  		FIG. 5. SDS-PAGE of human RPE subcellular fractions. One-dimensional SDS-PAGE was performed with the RPE P2 membrane (~30 µg) and the microsomal fractions (~30 µg) on a minigel (10 x 8 cm) and with the RPE cytosolic fraction (~40 µg) on a 20- x 20-cm gel. Gels were stained with colloidal Coomassie Blue, and consecutive ~1-mm bands were excised from the top to the bottom of the lane for protein identification (Table I). A total of 65 bands was cut and analyzed from each of the P2 and microsomal sample lanes, and 80 bands were cut from the cytosolic sample lane.

 
In addition to housekeeping proteins common to many cell types, a number of proteins were identified that are known to be associated with specialized functions of the RPE. These included proteins associated with retinoid metabolism and the visual cycle proteins such as cellular retinaldehyde-binding protein, cellular retinol-binding protein, retinal pigment epithelium 65, 11-cis-retinol dehydrogenase 5, retinal G protein-coupled receptor, and interphotoreceptor retinoid-binding protein. Interphotoreceptor retinoid-binding protein is synthesized in the photoreceptor cells; however, it appears to be part of a visual cycle protein complex in the RPE (13). The RPE is the most active phagocytic tissue in humans, and each RPE cell daily phagocytizes the shed outer segments tips from ~50 photoreceptor cells. Identified proteins involved in macromolecular degradation included cathepsins B, D, and Z; lysozyme; and several proteasome components. The photooxidative environment in the retina and active phagocytic processing provide abundant reactive oxygen species to the RPE. Identified antioxidant proteins included thioredoxin-dependent peroxide reductase 1 and 2, catalase, peroxiredoxin 6, superoxide dismutase, glutathione S-transferase, and thioredoxin peroxidase. Ten of the 278 identified proteins are currently designated hypothetical or of unknown function, and ~6% were identified based on homology with other species. Despite significant efforts to purify RPE cells free of extracellular debris and other cell types, low level blood contamination (e.g. hemoglobin) was detected in three gels. Photoreceptor-specific proteins such as phosducin, recoverin, and rhodopsin were also identified and may be in the RPE due to phagocytosis. The RPE proteins identified here provide an initial reference library for targeted studies of this important visual tissue.


   FOOTNOTES
 
Received, June 28, 2002, and in revised form, January 10, 2003.

Published, MCP Papers in Press, January 16, 2003, DOI 10.1074/mcp.D200001-MCP200

1 The abbreviations used are: RPE, retinal pigment epithelium; 1D, one-dimensional; 2D, two-dimensional; IEF, isoelectric focusing; IPG, immobilized pH gradient; LC MS/MS, liquid chromatography tandem electrospray mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back

* This work was supported in part by National Institutes of Health Grants EY06603, EY02362, EY13160, EY014239, and EY014240; a Research Center grant from The Foundation Fighting Blindness; and funds from the Cleveland Clinic Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Cole Eye Inst. (i31), Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: crabbj{at}ccf.org


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Marmor, M. F., and Wolfensberger, T. J. (1998) The Retinal Pigment Epithelium, Function and Disease, Oxford University Press, New York

  2. Bok, D. (1993) The retinal pigment epithelium: a versatile partner in vision. J. Cell Sci. Suppl. 17, 189 –195[Medline]

  3. Marmorstein, A. D. (2001) The polarity of the retinal pigment epithelium. Traffic 2, 867 –872[CrossRef][Medline]

  4. Crabb, J. W., Miyagi, M., Gu, X., Shadrach, K., West, K. A., Sakaguchi, H., Kamei, M., Hasan, A., Yan, L., Rayborn, M. E., Salomon, R. G., and Hollyfield, J. G. (2002) Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc. Natl. Acad. Sci. U. S. A. 99, 14682 –14687[Abstract/Free Full Text]

  5. Ramagli, L. S. (1999) in Methods in Molecular Biology, 2D-Proteome Analysis Protocols (Link, A. J., ed) Vol. 112, pp. 99–103, Humana Press, Totawa, NJ

  6. Saari, J. C., and Bredberg, D. L. (1988) CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium. J. Biol. Chem. 263, 8040 –8090

  7. West, K. A., Yan, L., Miyagi, M., Crabb, J. S., Marmorstein, A. D., Marmorstein, L., and Crabb, J. W. (2001) Proteome survey of proliferating and differentiating rat retinal pigment epithelial J-cells. Exp. Eye. Res. 73, 479 –491[CrossRef][Medline]

  8. Aulak, K. S., Miyagi, M., Yan, L., West, K. A., Massillon, D., Crabb, J. W., and Stuehr, D. J. (2001) Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc. Natl. Acad. Sci. U. S. A. 98, 12056 –12061[Abstract/Free Full Text]

  9. Miyagi, M., Sakaguchi, H., Darrow, R. M., Yan, L., West, K. A., Aulak, K. S., Stuehr, D. J., Hollyfield, J. G., Organisciak, D. T., and Crabb J. W. (2002) Evidence that light modulates protein nitration in rat retina. Mol. Cell. Proteomics 1, 293 –303[Abstract/Free Full Text]

  10. Jensen, O. N., Wilm, M., Schevchenko, A., and Mann, M. (1999) in Methods in Molecular Biology, 2D-Proteome Analysis Protocols (Link, A. J., ed) Vol. 112, pp. 513–530, Humana Press, Totawa, NJ

  11. Sharon, D., Blackshaw, S., Cepko, C. L., and Dryja, T. P. (2002) Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE). Proc. Natl. Acad. Sci. U. S. A. 99, 315 –320[Abstract/Free Full Text]

  12. Wistow, G., Bernstein, S. L., Wyatt, M. K., Farris, R. N., Behal, A., Touchman, J. W., Bouffard, G., Smith, D., and Peterson, K. (2002) Expressed sequence tag analysis of human RPE/choroid for the NEIBank project: over 6000 non-redundant transcripts, novel genes and splice variants. Mol. Vis. 8, 205 –220[Medline]

  13. Bhattacharya, S. K., Wu, Z., Jin, Z., Yan, L., Miyagi, M., West, K., Nawrot, M., Saari, J. C., and Crabb, J. W. (2002) Proteomic approach to identification of a mammalian visual cycle protein complex. FASEB J. 16, A14 (Abstr. 16.2)


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