Differential Protein Profiling of Primary versus Immortalized Human

Differential Protein Profiling of Primary versus Immortalized Human RPE Cells Identifies Expression Patterns Associated with Cytoskeletal Remodeling and Cell Survival Claudia S. Alge,† Stefanie M. Hauck,‡ Siegfried G. Priglinger,† Anselm Kampik,† and Marius Ueffing*,‡ Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany, and GSF Research Center for Environment and Health, Neuherberg, Germany Received November 25, 2005

Functional research of retinal pigment epithelium (RPE) most often relies on utilization of RPE-derived cell lines in vitro. However, no studies about similarities and differences of the respective cell lines exist so far. Thus, we here analyze the proteome of the most popular RPE cell lines: ARPE-19 and hTERT and compare their constitutive and de novo synthesized protein expression profiles to human early passage retinal pigment epithelial cells (epRPE) by 2-D electrophoresis and MALDI-TOF peptide mass fingerprinting. In all three cell lines the baseline protein expression pattern corresponded well to the de novo synthesized cellular proteome. However, comparison of the protein profile of epRPE cells with that of hTERT-RPE cells revealed a higher abundance of proteins related to cell migration, adhesion, and extracellular matrix formation, paralleled by a down-regulation of proteins attributed to cell polarization, and showed an altered expression of detoxification enzymes in hTERT-RPE. ARPE-19 cells, however, exhibited a higher abundance of components of the microtubule cytoskeleton and differences in expression of proteins related to proliferation and cell death. epRPE cells, hTERT-RPE, and ARPE-19 therefore may respond differently with respect to certain functional properties, a finding that should prove valuable for future in vitro studies. Keywords: retinal pigment epithelium • ARPE-19 • hTERT-RPE • cell culture • proteomics

Introduction The retinal pigment epithelium (RPE) is a monolayer of highly differentiated, polarized epithelial cells interposed between the outermost layer of the retina and the choroid in the eye. Defects in RPE-specific functions and dedifferentiation have been implicated in the development of a number of ocular pathologies. These range from inherited and degenerative retinal disease such as Best’s disease and age-related macular degeneration to proliferative vitreoretinopathies. The RPE serves a major component of the blood retinal barrier, synthesizes numerous enzymes to digest debris from photoreceptors, and is involved in visual pigment recycling.1 RPE cells also not only are an important source of trophic factors and growth factors involved in tissue maintenance, homeostasis and inflammation, at the same time they are the central cell type acting in and regulating these processes.2 Much of our current understanding about RPE function has arisen from in vitro studies using different RPE cell lines. Although well characterized with respect to single RPE cell markers, our comprehensive understanding of cellular and * To whom correspondence should be addressed. Tel: ++49-89-31873567. Fax: ++49-89-3187-4426. E-Mail: [email protected]. † Department of Ophthalmology, Ludwig-Maximilians-University. ‡ GSF Research Center for Environment and Health.

862

Journal of Proteome Research 2006, 5, 862-878

Published on Web 02/21/2006

molecular constraints of the different RPE cell lines is still very limited. A more detailed understanding of presumable intercell line variations at the global protein expression level under defined in vitro conditions may thus be a prerequisite for an appropriate application of the different in vitro RPE cell models. Once brought into culture, the differentiated RPE cells loose their specialized properties after multiple passages. They develop into a variety of cellular morphologies, loose RPE-specific markers and, in some instances, synthesize intermediate filaments that are more characteristic of cells of mesenchymal origin.3-5 For this reason, primary or early passage cells (epRPE) have been commonly utilized for RPE research. Primary cultures of RPE cells from various species have been shown to retain normal physiological functions, including polarization,6 the ability to transport retinoids,7 or to phagocytose rod outer segments.8,9 However, despite their inherent advantages, primary cultures of human RPE cells have a finite lifespan, the limited availability of human donor eyes makes them difficult to obtain, and such cultures usually require purification before a uniform population of cells is established.10-12 To overcome the limitations imposed by primary human RPE cell cultures, several immortalized human RPE cell lines have been developed such as hTERT-RPE,13 ARPE-19,6 or D40714 among others. Such defined cell lines offer advantages for biochemical and molecular biological studies because they 10.1021/pr050420t CCC: $33.50

 2006 American Chemical Society

Differential Profiling of Human RPE Cell Lines

allow the investigation of a pure, single cell type under experimental conditions and provide a limitless and homogeneous supply for in vitro experiments. However, the RPE cell lines may differ from each other and from primary RPE cells in various properties. hTERT-RPE is a human RPE cell line that stably expresses human telomerase reverse transcriptase (hTERT). Telomerase, a ribonucleoprotein complex, has been shown to regulate the replicative lifespan of most human somatic cells by maintaining the telomeres at the ends of chromosomes.15 hTERT-RPE have been reported to maintain a primary RPE cell morphology and exhibit a nontransformed cell phenotype.13 Although genetically modified, these cells retain normal growth control in response to serum deprivation and high cell density and can acquire differentiation characteristics in long-term culture.16 ARPE-19 is a human RPE cell line which arose spontaneously from a primary RPE cell culture. Despite the unknown nature of the event conferring immortality, ARPE-19 is a valuable source of human RPE cells. These cells display a highly epithelial morphology forming a hexagonal cobblestone layer, exhibit morphological polarization and achieve biochemical differentiation when placed in long-term culture.6 Although commonly used in RPE cell research, it remains to be determined to what extent these RPE cell lines and epRPE cell cultures are comparable in view of the overall protein expression profile or functional properties. In an attempt to acquire information about distinct differences between human epRPE cells, ARPE-19, and hTERT-RPE, we conducted a systematic analysis of the protein expression patterns at the global proteomic level. Thus, we compared steady state and metabolic protein expression profiles of human epRPE cells, and the commercially available immortalized human RPE cell lines ARPE-19 and hTERT-RPE. Resultant protein expression patterns were taken as a fingerprint of the physiological state of the RPE cells under a defined cell culture condition, with the aim of first identifying individual differences, followed by clustering protein sets attributable to specific functional features.

Materials and Methods Isolation of Human RPE Cells (epRPE). Eyes from three human donors were obtained from the Munich University Hospital Eye Bank and processed within 4 to 16 h after death. The donors ranged in age between 18 and 79 years. None of the donors had a known history of eye disease. Methods for securing human tissue were humane, included proper consent and approval, complied with the Declaration of Helsinki, and were approved by the local ethic committee. Human retinal pigment epithelium cells were harvested following the procedure as described previously in much detail.3,17,18 In brief, after the anterior segment from each donor eye was removed, the neural retinas were carefully peeled away from the RPEchoroid-sclera using fine forceps. The eye cup then was rinsed with Ca2+ and Mg2+ -free Hank’s balanced salt solution (GibcoBRL, Karlsruhe, Germany), and filled with 0.25% trypsin (Gibco) for 30 min at 37 °C. The trypsin was then replaced with Dulbecco’s modified Eagles medium (DMEM, Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS, Biochrom). Using a pipet, the media was gently agitated, releasing the RPE into the media by avoiding damage to Bruch’s membrane. The RPE cell suspension was checked for cross contamination using a microscope, transferred to a 2 mL Eppendorf tube and centrifuged for 5 min at 800 rpm in a microfuge (Centrifuge 5415 D, Eppendorf, Hamburg, Germany).

research articles Human RPE Cell Culture and Metabolic Labeling. For establishment of RPE cell cultures the suspension of harvested RPE cells was transferred to a 50-mL flask (Falcon, Wiesbaden, Germany) containing 20 mL of DMEM (Biochrom) supplemented with 20% FCS (Biochrom) and maintained at 37 °C and 5% CO2. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin using a pan-CK antibody (Sigma-Aldrich, Deisenhofen, Germany).19 The cells were tested and found free of contaminating macrophages (anti-CD11; Sigma-Aldrich) and endothelial cells (anti-von Willebrand factor, Sigma-Aldrich; data not shown). After reaching confluence, primary RPE cells (epRPE) were subcultured to passage 3 and maintained in DMEM (Biochrom) supplemented with 10% FCS (Biochrom) at 37 °C and 5% CO2. ARPE-19 cells were obtained from ATTC (Rockville, MD) and telomerase immortalized human RPE cells (hTERT-RPE) were purchased from Clontech (Heidelberg, Germany). Both, ARPE-19 and hTERTRPE were routinely passaged once a week and maintained in DMEM/Ham’s F12 (Biochrom) supplemented with 10% FCS at 37 °C and 5% CO2, as recommended in the vendors data sheet. epRPE cells, ARPE-19, and hTERT-RPE were grown on plastic 10 cm tissue culture dishes until they had reached 90% to 100% confluence. Cells were then preconditioned in methionine-free, cysteine-free medium (Gibco-BRL, Cat.-No. 11963022) without serum for 4 h. After two washes in conditioning medium 500 µCi Trans-35S-Label (ICN Biomedicals, Cat.-No. 51006, Irvine, California) was added to the conditioning medium for 17 h. Protein Extract Preparation and 2D Electrophoresis (2-DE). For isolation of cellular protein extracts from cultured RPE cells, cells were washed twice with serum-free medium, followed by a wash in Ca2+ and Mg2+-free 1× phosphate buffered saline (PBS; 3mM Na2HPO4, 4mM KH2PO4, 160mM NaCl), pH 7.4, and a third wash in 0.5× PBS to reduce contamination with salts. Subsequently, cells were collected and lysed in denaturing lysis buffer containing 9 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Merck), 4% CHAPS (Sigma), 1% DTE (Merck), 2.5 µM EGTA (Sigma), 2.5 µM EDTA (Sigma) and an appropriate amount of protease inhibitors (Complete Mini; Roche, Mannheim, Germany) for 10 min at room temperature (rt) and centrifuged through a QIAshredder Mini Column (Qiagen, Hilden, Germany). Lysates were then further cleared by centrifugation at 22 000 × g for 45 min at room temperature. Protein concentrations were determined by the Bradford protein assay reagent (Biorad, Munich, Germany). Freshly prepared lysate containing 150 µg total protein was loaded onto each gel. First dimension isoelectric focusing was performed using precast 24 cm immobilized pH gradient (IPG) strips (24 cm Immobiline DryStrip pH 4-7; Amersham Pharmacia Biotech, Braunschweig, Germany). IPG strips were rehydrated overnight with 150 µg protein diluted to 460 µL with reswelling solution (9 M urea, 2 M thiourea, 4% CHAPS, 2.5 µM EGTA, 2.5 µM EDTA, 1% DTE, 4 mM Tris, 0.25% (w/v) bromophenolblue (BPB), and 0.7% (v/v) Pharmalytes, pH 3-10, Amersham Pharmacia Biotech). Isoelectric focusing was performed at 20 °C with a Multiphore II system (Amersham Pharmacia Biotech) as desribed before.20 Equilibrated IPG strips were then placed on top of 9% to15% linear gradient polyacrylamide gels (PAGE) and electrophoresed as described.20 All gels were silver stained21 and dried between cellophane sheets, followed by exposure to Kodak X-omat AR film (Kodak, Rochester, NY) at -70 °C for 1 week. Journal of Proteome Research • Vol. 5, No. 4, 2006 863

research articles

Alge et al.

Figure 1. Phase contrast images showing the morphological phenotype of primary human RPE cells (epRPE) of passage 3 (A), hTERTRPE cells (B), and ARPE-19 cells (C) grown on 10 cm tissue culture plastic plates until they reached 90% to 100% confluence.

Image Analysis. Image analysis was performed as described before.22 In brief, the three best focusing results were selected from 8 silver-stained gels per cell type (epRPE; ARPE-19; hTERT-RPE), scanned on a transmission scanner (Epson GT9600) with 12 bit/300 dpi resolution and resultant gel images were then imported to a 2-DE analysis software (Proteom Weaver; release 2.1.; BioRad, Munich, Germany). The following parameters for protein spot detection were used: minimum spot radius 4, minimum spot intensity (volume above base level) of 2000 and minimum contrast (height above base level) of 10. Gels from each experiment were processed by the pairmatch-based normalization, which erases intensity differences of similar spots in different gels not due to regulation but experimental variability of the method (e.g., protein load, silver stain intensity). Subsequently, protein spots from respective gel images (epRPE, ARPE-19, and hTERT-RPE) were matched and filtered to find significant differences within the detection limit as follows: only spots matched in all gel images or autoradiographs were considered and only spots exceeding an intensity threshold of 0.1 were taken for further analysis. The remaining spots were sorted according to the respective regulation factor. The threshold regulation factor depends on the experimental reproducibility and was determined by the Proteom Weaver software. Only those spots regulated more than the factor required for significance were further considered as candidate spots and subsequently subjected to manual verification for matching accuracy to avoid assigning false positives as follows: (i) a discrete spot had to be matched across all nine gels or autoradiographs in the three RPE cell lines to be included in the analysis; (ii) an intercell-linevariation was only taken into account if it occurred in not less but two of three gels per cell type, and if it was confirmed by manual comparison of the respective images. In-Gel Digestion and MALDI-TOF Analysis. Selected spots were excised from dried 2-DE gels, transferred to 96 well plates (Nunc 22-260; Nunc, Wiesbaden, Germany), destained23 and tryptically digested as described before.3,20 Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass fingerprints were obtained on a Bruker Reflex III mass spectrometer (Bruker-Daltonik, Bremen, Germany) equipped with a Scout 384 inlet. Aliquots of the digests (0.5 µL) were mixed with 0.5 µL matrix consisting of 2,5-dihydroxybenzoic acid (Sigma) (20 mg/mL in 20% acetonitrile, 0.1% trifluoroacetic acid (TFA)) and 2-hydroxy-5-methoxybenzoic acid (Fluka, Buchs, CH) (20 mg/mL in 20% acetonitrile, 0.1% TFA) at a 9:1 ratio (v/v) and spotted onto a 400µm anchor steel target (Bruker-Daltronik, Bremen, Germany). Mass analyses obtained in the positive ion reflector mode were run manually. For calibration angiotensin-2-acetate (1046.54 864

Journal of Proteome Research • Vol. 5, No. 4, 2006

Da), substance P (1347.74 Da), bombesin (1619.82 Da), and ACTH 18-3 (2465.20 Da) were recorded. Internal calibration using peptides resulting from autodigestion of trypsin fragments (1045.564 Da; 2211.10 Da) was also performed. Database Search. Database searches were performed using the Mascot software package (Version 1.9.05, Matrix Science, London, UK).24 Parameter settings were: 100 ppm mass accuracy, one miscleavage allowed, the search was done in all available mammal and, in assorted cases, in eukarya sequences. Peptide mass fingerprints were searched for matches with the virtually generated tryptic protein masses of the protein databases NCBInr, MSDB, and EnsemblC. A protein was regarded as identified, if the following four criteria were fulfilled: (i) the probability based MOWSE score25 was above the 5% significance threshold for the respective database, (ii) the matched peptide masses were abundant in the spectrum, (iii) the theoretical isoelectric point (pI) and the molecular weight (Mr) of the search result could be correlated with the 2-DE position of the corresponding spot, and (iv) the matched sequence did not contain more than 20% uncleaved peptides. Functional classification was based on the classification provided in the Swiss-Prot protein knowledge database (http:// www.expasy.org).

Results Morphology of Human RPE Cells in Culture. RPE cell cultures were grown to 90% to 100% confluence with 10% FCS in the medium, followed by serum starvation for 21 h. Under this cell culture condition, all three RPE cell types developed into a variety of morphological types. Whereas early passage RPE (epRPE) cell cultures (Figure 1A) and ARPE-19 (Figure 1C) appeared to be more homogeneous in shape with a predominance of cuboidal cells, hTERT-RPE cell cultures (Figure 1B) were populated with elongated fibroblastic and flattened stellate shapes. With the exception of a little perinuclear pigmentation, the epRPE cells were devoid of pigment. ARPE19 cells also showed features characteristic of RPE, including well defined cell borders, an overall cobblestone appearance and noticeable pigmentation. After having reached confluence, epRPE cells and ARPE-19 displayed a densly packed monolayer of polygonal cells with well-defined borders, whereas hTertRPE cells appeared to be multilaminar, as determined by focusing through the cultures at the light microscopic level (data not shown). Comparison of the epRPE Proteome with that of ARPE-19 and hTERT-RPE Cells. In an attempt to provide a comparative analysis of the global proteome expression pattern of human epRPE, ARPE-19, and hTERT-RPE, 2-DE gels of all three cell


research articles

Figure 2. Protein spots differentially expressed between hTERT-RPE cells and early passage human RPE (epRPE) cells. (A) & (B): Silver stained 2-DE pattern of whole-cellular protein extracts from hTERT-RPE cells (A) and epRPE cells (B). Images (C) & (D) represent the corresponding autoradiographs of the 2-DE patterns shown in (A) & (B), respectively. RPE cells were grown on tissue culture plastic plates until they reached 90% to 100% confluence. Cells were then depleted of serum for 21 h with 35S-pulse labeling for 17 h and processed as described in the Materials and Methods section. Cellular lysates (150 µg total protein) were separated on a 24-cm, pH 4-7 linear IPG strip, followed by 9-15% SDS-PAGE. The images represent a representative gel from two experiments performed in quadruplets. Circles and letters in roman type indicate protein spots that were up-regulated in hTERT-RPE cells. Boxes and numbers in italics indicate proteins that were down-regulated. Numbered spots were identified by MALDI-TOF MS and identifications are listed in Tables 2 and 3.

lines were prepared and resolved proteins were visualized by silver staining. To further screen for differences in de novo protein synthesis, incorporation of radiolabeled methionine and cysteine into newly synthesized proteins was visualized by autoradiography of the respective 2-DE gels. The 2-DE

protein pattern of epRPE cell lysates was monitored, and compared to that APRE-19 and hTERT-RPE cells. As illustrated in Figures 2 and 3 the overall protein expression patterns displayed a high degree of similarity in both, silver stained gels (Figure 2A,B; Figure 3A,B) as well as in the respective autoraJournal of Proteome Research • Vol. 5, No. 4, 2006 865

research articles

Alge et al.

Figure 3. Protein spots differentially expressed between ARPE-19 cells and early passage human RPE (epRPE) cells. (A) & (B): Silver stained 2-DE pattern of whole-cellular protein extracts from ARPE-19 cells (A) and epRPE cells (B). Images (C) & (D) represent the corresponding autoradiographs of the 2-DE patterns shown in (A) & (B), respectively. Cells were grown on tissue culture plastic plates until they reached 90% to 100% confluence. Cells were then depleted of serum for 21 h with 35S-pulse labeling for 17 h and processed as described in the Materials and Methods section. Cellular lysates (150 µg total protein) were separated on a 24-cm, pH 4-7 linear IPG strip, followed by 9-15% SDS-PAGE. The images represent a representative gel from two experiments performed in quadruplets. Circles and letters in roman type indicate protein spots that were up-regulated in ARPE-19 cells. Boxes and numbers in italic indicate proteins that were down-regulated. Numbered spots were identified by MALDI-TOF MS and identifications are listed in Tables 4 and 5.

diographs (Figure 2C,D; Figure 3C,D). The gel images and autoradiographs were analyzed using the Proteome Weaver software (for settings see Materials and Methods) and the statistically significant regulation factors (p < 0.01) were calculated. On silver stained gels 3266 ((357), 2791 ((62), and 3120 ((106) individual protein spots were detected for epRPE, 866


ARPE-19, and hTERT-RPE, respectively, and 2806 ((142), 2723 ((130), and 2991 ((362) spots were detected in the corresponding autoradiographs. These spots were sorted by the average intensity in the epRPE gel image (defined by an internal algorithm of the software) and all spots with an average intensity above 0.1 were further analyzed (1644 ( 102, 1612 (

research articles


Table 1. Minimal Statistical Significant Regulation Factors (MF) for Differential Protein Expression between Human Early Passage RPE Cells, hTERT-RPE and ARPE-19 Cells 35S

silver staining a

p

MF upregulation

no. of spots

< 0.001 < 0.01

> 3.29 > 2.54

13 13

< 0.001 < 0.01

> 2.56 > 2.09

16 5

a

a

MF downregulation

no. of spots

a

p

MF upregulation

Early Passage RPE Cells versus hTERT-RPE < 0.31 7 < 0.001 > 2.57 < 0.39 19 < 0.01 > 2.09 Early Passage RPE Cells versus ARPE 19 < 0.39 9 < 0.001 > 2.74 < 0.47 4 < 0.01 > 2.02

labelling no. of spots

a

MF downregulation

no. of spots

19 11

< 0.39 < 0.47

18 9

15 8

< 0.36 < 0.49

5 5

MF, minimal statistical significant regulation factor.

80, and 1635 ( 120 spots in autoradiographs of epRPE, ARPE19, and hTERT-RPE, respectively, and 1651 (146, 1486 ( 19, and 1505 ( 91 spots in silver stained gels of epRPE, ARPE-19, and hTERT-RPE, respectively). Differences found in not less but two gels from each cell type were compared and the data were filtered to reveal statistically significant changes in protein abundance (p < 0.01), resulting in an overall cutoff at a >2.02fold difference for up-regulation and a factor 3.29; down-regulation, 7 spots: 2.54; down-regulation, 19 spots: 2.57; down-regulation, 18 spots: 2.09; down-regulation, 9 spots: 2.56; downregulation, 9 spots: 2.09; down-regulation, 4 spots: 2.74; down-regulation, 5 spots: 2.02; down-regulation, 5 spots: < 0.49). Identification of Differentially Expressed Proteins by MALDITOF. After comparison of protein expression patterns a total of 134 spots, which were regarded to be significantly up- or down-regulated in comparison to epRPE cells, were excised from the gels. Peptide mass fingerprint spectra were recorded on a MALDI-TOF mass spectrometer and then identified by comparison with public databases (for details, see Materials and Methods). Proteins were identified from 116 spots excised from 2-DE gels of epRPE, hTERT-RPE and ARPE-19 cell samples, for an overall identification yield of 86%. For 6.7% of the remainder, MS data were of insufficient quality, and protein identification was not possible. Another 3.6% could not be identified because of contamination of the tryptic digests with keratins, and despite good MS data, further 3.7% showed no significant homology to known mammalian protein sequences

presented in the database or matched with hypothetical proteins, suggesting that they might be yet unknown proteins. Identified proteins, accession numbers, and Swiss-Prot entry names are listed in Tables 2-5. Theoretical Mr, pI, percentage of sequence coverage, probability based MOWSE score, and specific functions are also included. Spot numbers are assigned on the gel images shown in Figures 2 (hTERT-RPE vs epRPE) and 3 (ARPE-19 vs epRPE). A summary of differentially expressed proteins classified by broad functional criteria is provided in Table 6. As observed often in 2-DE analysis, eight proteins identified here were found in multiple spots on several positions on the gel. For example, superoxide dismutase (3578, 3703) and cathepsin D (3560, 3559, 3518, 3519) were found at different Mr’s, but with the same pI. Furthermore, in all spots identified as Cathepsin D investigation of the sequence coverage revealed that PMF consistently only covered the portion of the protein named “heavy chain” (residues 151-412) with a predicted Mr of 26629 Da. The proteins tubulin β-1 chain (3495, 3496, 3497, 3713), similar to tubulin β-5 (3505, 3508), tropomyosin 1 alpha (3548, 3549), myosin regulatory light chain 2 (3552, 3553), annexin A3 (3517, 3528), dihydropyiminidase related protein 3 (3603, 3604) and calumenin precursor (3543, 3544) were identified in several spots with slightly different Mr’s and pI’s, suggesting the presence of different isoforms or proteins with differential modification. A distinct configuration was observed for integrin alpha 3 (3618, 3574, 3598), which was represented with a series of 3 to 7 spots, closely aligned with an interspot pI difference of 0.05 to 0.06. Proteins Differentially Expressed between Human epRPE Cells and hTERT-RPE. 1. Proteins Expressed with Higher Abundance in hTERTRPE Cells. Twenty-six spots were recorded as expressed with a higher abundance in the hTERT-RPE baseline proteome, representing 20 distinct proteins (Figure 2A,B). Thirty spots were found to be synthesized at a higher rate, yielding an identification of 21 distinct proteins (Figure 2C,D). Overall, comparative protein expression analysis of the baseline and de novo synthesized hTERT-RPE proteome displayed a high degree of similarity with a total of 22 protein spots expressed with a higher abundance under both analytical conditions. A summary of up-regulated proteins is provided in Table 2, for an overview of functional grouping see Table 6. In addition, the spot numbers are assigned on the individual gel images in Figure 2A,B for silver stained gels and in Figure 2C,D for the corresponding autoradiographs. A markedly increased abundance in the baseline as well as the in de novo synthesized hTERT-RPE proteome was monitored for tropomyosin 1 alpha (3548, 3549) and talin 1 (3599), all of which are involved in stress fiber formation and cell Journal of Proteome Research • Vol. 5, No. 4, 2006 867

research articles

Alge et al.

Table 2. Identification Results of Proteins Expressed with Higher Abundance in hTERT-RPE Cells but in Human Early Passage RPE Cellsd Sia Arb

spot top no. MOWSE score

Swiss-Prot entry name

Swiss-Prot/ Trembl acc. no.

x

3488

190

OAT_HUMAN

P04181

x

x 3492 x 3507

116 92

TPM2_HUMAN Q9N5S5_CAEEL

P07951 Q9N5S5

x

x 3543

222

CALU_HUMAN

x

x 3544

202

x x

3547 x 3548

x

protein name

Mr (Da)

c seq. cov. (%)

pI

biological process

48846

37%

29980 17015

18% 26%

O43852

Ornithine aminotransferase Tropomyosin beta chain Hypothetical protein F49H12.3 Calumenin [Precursor]

37198

56%

CALU_HUMAN

O43852

Calumenin [Precursor]

37198

51%

53 262

EF1B_HUMAN TPM1_HUMAN

P24534 P09493

Elongation factor 1-beta Tropomyosin 1, alpha chain

24788 32746

19% 59%

x 3549

222

TPM1_HUMAN

P09493

Tropomyosin 1, alpha chain

32746

50%

x

x 3500

120

CATD_HUMAN

P07339

Cathepsin D, heavy chain, EC 3.4.23.5

26629

31%

x

3551

99

ELOB_HUMAN

Q15370

Elongin b residues 1-120, chain A

13113

36%

x 3552

86

MLRN_HUMAN

P24844

19871

41%

x 3553

91

MLRN_HUMAN

P24844

19740

52%

x

x 3558

117

CATD_HUMAN

P07339

26629

44%

x

x 3559

90

CATD_HUMAN

P07339

26629

31%

x

x 3560

184

CATD_HUMAN

P07339

26629

60%

x

x 3561

80

NNMT_HUMAN

P40261

30011

26%

x

x 3563

127

NPM_HUMAN

P06748

Myosin regulatory light chain 2, smooth muscle isoform Myosin regulatory light chain 2, smooth muscle isoform Cathepsin D, heavy chain, EC 3.4.23.5 Cathepsin D, heavy chain, EC 3.4.23.5 Cathepsin D, heavy chain, EC 3.4.23.5 Nicotinamide Nmethyltransferase, EC 2.1.1.1 Nucleophosmin

31090

27%

x

3566

123

PDIA6_HUMAN

Q15084

48490

32%

x

x 3570

189

TGM2_HUMAN

P21980

Protein disulfide isomerase A6, EC 5.3.4.1 Tissue transglutaminase, EC 2.3.2.13

78420

31%

x

x 3572

84

RB6I2_HUMAN

Q9UIK7

RAB6 interacting protein 2

108840

11%

x

x 3573

83

LAMC1_HUMAN

P11047

Laminin gamma-1 chain

183195

12%

x

x 3574 x 3575

118 137

ITA3_HUMAN PTRF_HUMAN

P26006 Q9HAP4

Integrin alpha-3 Leucine-zipper protein FKSG13

119820 43449

14% 28%

x

x 3577

127

GSTO1_HUMAN

P78417

27833

39%

x

x 3578

86

SODC_HUMAN

P00441

16023

50%

5.70 Antioxidant activity

x x

x 3598 x 3599

113 101

ITA3_HUMAN TLN1_HUMAN

A40021 Q9Y490

Glutathione transferase omega 1, EC 2.5.1.18 Superoxide dismutase [Cu-Zn] Integrin alpha-3 Talin 1

5.11 Protein modification; catalyzes the cross-linking of proteins. Positive regulation of cell adhesion 6.19 IkappaB kinase regulatory subunit; involved in endosome trafficking to the Golgi apparatus 5.01 Binds to cells via a high affinity receptor; mediates attachment, migration and organization of cells into tissues 6.60 Cell-matrix-adhesion 5.67 Termination of transcription by RNA polymerase I; required for dissociation of the ternary transcription complex. 6.23 Participates in redox reactions

119820 271653

13% 12%

x

x 3601

169

PLOD2_HUMANW

Q8N170

Lysine hydroxylase 2

87784

15%

x

x 3603

111

DPYL3_HUMAN

Q14195

Dihydropyrimidinase related protein 3

62323

26%

x

x 3604

148

DPYL3_HUMAN

Q14195

Dihydropyrimidinase related protein 3

62323

32%

6.6 Cell-matrix-adhesion 5.77 Participates in cytoskeletal anchoring in focal adhesions, contributes to cell motility 6.24 Protein modification; forms hydroxlysine residues in collagens, which are essential for the stability of intermolecular collagen crosslinks 6.04 Involved in the signalling pathway of collapsin/semaphorin; neuronal differentiation 6.04 Involved in the signalling pathway of collapsin/semaphorin; neuronal differentiation

868


6.57 Amino acid metabolism; urea cycle. 4.70 Stress fiber component. 4.89 Voltage-gated potassium channel activity 4.47 Ca2+ -binding; regulation of vitamin K-dependent carboxylation of aminoterminal glutamate residues 4.47 Ca2+ -binding; regulation of vitamin K-dependent carboxylation of aminoterminal glutamate residues 4.5 Translational elongation 4.69 Stress fiber component; in nonmuscle cells is implicated in stabilizing cytoskeleton actin filaments 4.69 Stress fiber component; in nonmuscle cells is implicated in stabilizing cytoskeleton actin filaments 5.56 Acid protease active in intracellular protein breakdown 4.99 Protein complex assembly; RNA elongation from Pol II promoter 4.80 Regulation of both smooth muscle and nonmuscle cell contractile activity 4.8 Regulation of both smooth muscle and nonmuscle cell contractile activity 5.56 Acid protease; intracellular protein breakdown 5.56 Acid protease; intracellular protein breakdown 5.56 Acid protease; intracellular protein breakdown 5.56 Catalyzes the N-methylation of nicotinamide and other pyridines 4.71 Transcription coactivator activity (activation of NF-kappaB) 4.95 Protein folding

research articles

Differential Profiling of Human RPE Cell Lines Table 2. (Continued) spot no.

top MOWSE score


x

3605

110

Q8WVY7_HUMAN

Q8WVY7

x

3607

95

BIEA_HUMAN

P53004

x

3608

145

TPIS_HUMAN

P60174

x

3710

105

LEG1_HUMAN

P09382

Sia Arb


protein name

Mr (Da)

CTD-like phosphatase 36838 domain-containing protein Biliverdin reductase 33722 (EC 1.3.1.24) Triosephosphate isomerase (EC 5.3.1.1) Galectin-1

c seq. cov. (%)

28% 30%

26807

56%

14917

50

pI

biological process

6.07 Protein modification; peptidase activity 6.06 Involved in the bilirubin-biliverdin redox system. Heme metabolism (first step); 6.51 Glycolysis 5.34 β-galactoside binding matricellular protein, implicated in cell adhesion, migration, apoptosis

a Si, up-regulated on silver stained gels. b Ar, up-regulated on autoradiographs of the respective gels as determined by 35S-pulse labelling. c Seq. Cov., sequence coverage; Top MOWSE score, Mascot score. d Spot numbers are indicated in Figure 2.

adhesion. For these proteins the most striking difference was seen in the protein synthesis rate of with a 3 to 9-fold higher abundance in hTERT-RPE autoradiographs. Myosin regulatory light chain 2 (3552, 3553) and tropomyosin beta chain (3492) are further elements of the contractile cellular cytoskeleton, however, the latter ones were recorded as significantly upregulated in autoradiographs exclusively. A significantly higher abundance in silver stained gels and autoradiographs was further noted for proteins attributed to protein modification (3570 tissue transglutaminase; 3601 lysine hydroxylase) and the extracellular matrix (3573 laminin gamma-1 chain). Other identified proteins up-regulated under both analytical conditions were distributed over several functional groups, including intracellular protein breakdown (3500, 3558, 3559, 3560 cathepsin D), signaling (3603, 3604 dihydropyriminidase related protein-3), transcriptional control (3563 nucleophosmin), Ca2+binding (3543, 3544 calumenin precursor), and endosomal trafficking (3572 RAB6 interacting protein 2). One hypothetical protein (3507 hypothetical protein F49H12.3), which encodes for a presumable voltage gated potassium channel, and two detoxification enzymes (3577 glutathione transferase omega 1, 3561 nicotinamide N-methyltransferase) were also identified as up-regulated in baseline and de novo synthesized hTERTRPE proteome. The localization of superoxide dismutase (3578, 3703) in 2-DE gels from epRPE cells and hTERT-RPE differed slightly in the Mr and was therefore monitored as differentially expressed, most notably due to the presence of different isoforms. As described above a similar observation was made for cathepsin D. Integrin alpha 3 (3598, 3574, 3618) displayed a characteristic configuration of multiple spots close together in a row with a basic shift of the whole configuration of approximately 0.2 to 0.27 pH units but equal spot intensity as in epRPE cells. A higher abundance of protein expression in the steady-state hTERT-RPE proteome alone was observed for protein disulfide isomerase A6 (3566), furthermore for ornithine aminotransferase (3488), a key enzyme of the urea cycle, and for two proteins related to translational and transcriptional elongation (3547 elongation factor 1-β; 3551 elongin B residues 1-120); up-regulation at the level of protein synthesis exclusively was found for biliverdin reductase (3607), a key enzyme in recycling the major physiologic antioxidant cytoprotectant bilirubin and CDT-like phosphatase containing protein (3605), which encodes for a dipeptidase that regenerates free ubiquitin for reuse during protein degradation. Finally, leucine-zipper protein FKSG13 (3575), which is required for dissociation of the ternary transcription complex, and triosephosphate isomerase (3608) were found to be synthesized at a higher rate.

2. Proteins Expressed with Lower Abundance in hTERTRPE Cells. Suppressed expression at the steady-state proteomic level when compared to epRPE was noted for 25 protein spots with 23 distinct proteins identified (Figure 2A,B). In autoradiographs a total of 27 spots representing 24 distinct proteins were regarded as down-regulated (Figure 2C,D). Out of these, sixteen distinct proteins were found at a lower abundance in both, silver stained gels and autoradiographs (Tables 3 and 6). These included a group of four structural components (3508 similar to tubulin-β5; 3525 calponin-3; 3655 ezrin-radixinmoesin binding phophoprotein-50; 3660 moesin; 3525 calponin), whereas two further structural elements were found to be synthesized at a significantly lower rate exclusively (3713 tubulin beta-1 chain; 3639 ezrin). Other proteins strongly downregulated at the steady state and de novo synthesized protein expression level of hTERT-RPE cells were involved in detoxification processes (3688 lactoylglutathione lyase; 3703 superoxide dismutase), protein catabolism and modification (3690 cathepsin D; 3676 WW domain binding protein 2; 3642 plasminogen activator inhibitor 1), cell proliferation (3508 c-mycresponsive protein RCL), translational control (3629 similar to eukaryotic translation termination factor 1), and Ca2+-binding (3680 annexin A3). Proteins related to signaling processes were either expressed with lower abundance in the baseline as well as 35S-pulse labeled hTERT-RPE proteome (3640 adenylyl cyclase-associated protein-2; 3677 NG,NG-dimethylarginine dimethylaminohydrolase 1), in silver stained gels alone (3672 guanine nucleotide-binding protein G(i) alpha-2 subunit; 3634 zyxin), or in autoradiographs exclusively (3661 guanine nucleotide-binding protein G(i) alpha-1 subunit). Lower abundance in the steady-state protein expression profile alone included the following biological activities: DNA replication (3632 DNA replication licensing factor MCM7), selenium binding (3668 similar to selenium binding protein 1), Ca2+-binding (3702 calmyrin), and distinct functions in protein metabolism, including lysosomal and nonlysosomal proteolysis (3689 cathepsin B), protein folding (3652 FK506-binding protein 4), and mitochondrial protein synthesis (3706, 39S ribosomal protein L12). Suppressed de novo protein synthesis was recorded for ribosomal RNA processing protein 41 (3537), cathepsin D (3692), the molecular chaperone endoplasmin (3670), interleukin-18 (3503), and inositol 1-phosphate synthase (3650). Last SNAP-23 (3686), which encodes for a protein regulating vesiclemembrane fusion, was identified in a spot with lower de novo protein synthesis rate in hTERT-RPE cells. Proteins Differentially Expressed between Human epRPE Cells and ARPE-19. Journal of Proteome Research • Vol. 5, No. 4, 2006 869

research articles

Alge et al.

Table 3. Identification Results of Proteins Expressed with Lower Abundance in hTERT-RPE Cells but in Human Early Passage RPE Cellsd

Sia

Arb

top spot MOWSE score no.

x 3503

149

x

x 3508

84

x

x 3525

x



protein name

Mr (Da)

c seq. cov. (%)

IL18_HUMAN

Q14116

Interleukin-18 [Precursor]

22597

66%

Q96B85_HUMAN

Q96B85

21554

32%

110

CNN3_HUMAN

Q15417

Similar to tubulin, beta 5 [Fragment] Calponin 3

36562

34%

3526

208

CNN3_HUMAN

Q15417

Calponin 3

36562

58%

x 3537

105

EXOS4_HUMAN

Q9NPD3

35%

x

x 3508

70

RCL_HUMAN

O43598

x x

x 3618 x 3629

70 99

ITA3_HUMAN Q96CG1_HUMAN

P26006 Q96CG1

x

3632

146

MCM7_HUMAN

P33993

x

x 3634

145

ZYX_HUMAN

Q15942

Ribosomal RNA 26652 processing protein 41, EC 3.1.13 c-Myc-responsive protein 19211 RCL Integrin alpha-3 119820 Similar to eukaryotic 45662 translation termination factor 1 DNA replication licensing 81857 factor MCM7 Zyxin 2 62436

31%

x 3639

153

EZRI_HUMAN

P15311

Ezrin

69484

24%

x 3640

114

CAP2_HUMAN

P40123

53076

23%

x 3642

94

PAI1_HUMAN

P05121

Adenylyl cyclaseassociated protein 2, CAP 2 Plasminogen activator inhibitor-1

43389

28%

x

x 3650 3652

60 163

Q9H2Y2_HUMAN FKBP4_HUMAN

Q9H2Y2 Q02790

Inositol 1-phosphate synthase FK506-binding protein 4, EC 5.2.1.8

61528 52057

13% 36%

x

x 3655

253

NHERF_HUMAN

O14745

Ezrin-radixin-moesin binding phosphoprotein-50

39130

60%

x

x 3660

110

MOES_HUMAN

P26038

Moesin

67892

23%

x 3661

94

GNAI1_HUMAN

P04898

40774

28%

3668

102

Q96GX7_HUMAN

Q96GX7

52928

25%

x 3670

90

ENPL_HUMAN

P14625

Guanine nucleotide-binding protein G(i), alpha-1 subunit Similar to selenium binding protein 1 Endoplasmin

90309

12%

x

3672

111

GNAI2_HUMAN

P04899

40864

39%

x

x 3676

88

WBP2_HUMAN

Q969T9

28182

25%

x

x 3677

81

DDAH1_HUMAN

O94760

31313

27%

x

x 3680

195

ANXA3_HUMAN

P12429

36524

48%

x 3686

85

SNP23_HUMAN

O00161

23682

40%

x

x 3688

91

LGUL_HUMAN

Q04760

20803

31%

x

3689

61

CATB_HUMAN

P07858

22972

34%

x xx 3690

122

CATD_HUMAN

P07339

26629

44%

xx 3692

189

CATD_HUMAN

P07339

26629

69%

x 3701

125

PRDX3_HUMAN

P30048

28017

43%

x

x

870


Guanine nucleotidebinding protein G(i), alpha-2 subunit WW domain binding protein 2, WBP-2 NG,NG-dimethylarginine dimethylaminohydrolase 1 EC 3.5.3.18 Annexin A3 Synaptosomalassociated protein 23, SNAP-23 Lactoylglutathione lyase EC 4.4.1.5 Cathepsin B (EC 3.4.22.1), chain B Cathepsin D, heavy chain, EC 3.4.23.5 Cathepsin D, heavy chain, EC 3.4.23.5 Antioxidant protein 1, EC, 1.11.1.

32% 14% 30% 26%

pI

biological process

4.54 Cell-cell signaling, apoptosis activator activity 4.77 Microtubule polymerization, microtubule based movement 5.69 Implicated in the regulation and modulation of smooth muscle contraction 5.69 Implicated in the regulation and modulation of smooth muscle contraction 6.07 rRNA processing; component of the exosome multienzyme ribonuclease complex 4.97 Cell proliferation; induces anchorage-independent growth. 6.60 Cell-matrix-adhesion 5.4 Translation termination 6.08 Required for cell proliferation and DNA replication 6.22 Associates with actin cytoskeleton near adhesion plaques. Component of signal transduction pathways that mediate adhesion-stimulated changes in gene expression 5.94 Microvillar membrane protein (cytoplasmic side); involved in connections of cytoskeletal structures to the plasma membrane 5.95 Establishment and/or maintenance of cell polarity, signal transduction 6.71 Inhibitor of the fibrinolytic system. Regulates adhesion, detachment and migration of cells 5.54 Phospholipid biosynthesis 5.35 Protein folding; may play a role in the intracellular trafficking of hetero-oligomeric steroid hormone receptors 5.50 Scaffold protein that connects plasma membrane proteins with members of the ezrin/moesin/ radixin family. 6.08 Connects cytoskeletal structures to the plasma membrane 5.7 G-protein coupled receptor protein signaling pathway 5.93 Selenium binding 4.73 Molecular chaperone; functions in processing and transport of secreted proteins 5.34 G-protein coupled receptor protein signaling pathway 5.65 Presumably involved in protein-protein interaction, perhaps with the protein target to be ubiquitinylated 5.53 Arginine catabolism, nitric oxide mediated signal transduction 5.63 Ca2+-dependent phospholipid binding 4.89 Regulators of secretory granule/ plasma membrane fusion events 5.25 Detoxification; catalyzes conversion of cytosolic methylglyoxal to lactoylglutathione 5.2 Proteolysis and peptidolysis 5.56 Acid protease; intracellular protein breakdown 5.56 Acid protease; intracellular protein breakdown 7.67 Protects radical-sensitive enzymes from oxidative damage by a radical-generating system

research articles

Differential Profiling of Human RPE Cell Lines Table 3. (Continued)

Sia Arb

x x x

x

x x

spot no.

top MOWSE score

3702

72

C1IB_HUMAN

Q96IU4

3703 3706

122 84

SODC_HUMAN RM12_HUMAN

P00441 P52815

3708

91

TBCA_HUMAN

O75347

3713

113

TBB1_HUMAN

P07437



Mr (Da)

c seq. cov. (%)

Calmyrin, CIB-1

22446

33%

Superoxide dismutase [Cu-Zn] 39S ribosomal protein L12, mitochondrial Tubulin-specific chaperone A Tubulin beta-1 chain

16023 10540

65% 53%

12773

43%

50240

27%

protein name

pI

biological process

5.94 Ca2+-binding, probably acts as a sensor transferring Ca2+ signals 5.7 Antioxidant activity 6.6 Protein synthesis; mitochondrial 5.52 Co-chaperone activity in betatubulin folding 4.75 Microtubule-based processes, chaperone activity

a Si, down-regulated on silver stained gels. b Ar, down-regulated on autoradiographs of the respective gels as determined by 35S-pulse labelling. c Seq. Cov., sequence coverage; Top MOWSE score, Mascot score. d Spot numbers are indicated in Figure 2.

1. Proteins Expressed with Higher Abundance in ARPE-19 Cells. Twenty-one spots were monitored as up-regulated in silver stained gels (Figure 3A,B), and 19 distinct proteins were identified. An increased rate of de novo protein synthesis was observed for 23 spots representing 22 distinct proteins (Figure 3C,D). Numbered spots are identified in Table 4 and functionally grouped in Table 6. Identified proteins exhibiting the strongest increase in abundance in ARPE-19 samples, both in baseline cellular protein content and in de novo protein synthesis, were the precursor of the proinflammatory cytokine interleukin-18 (3503) (up-regulation 18-fold and 8-fold, respectively) and components of the microtubule cytoskeleton (3495, 3496, 3497 tubulin β-1 chain; 3505, 3508 similar to tubulin β-5; 3516 tubulin β-5 chain). A further markedly up-regulated cytoskeletal element in silver stained gels and autoradiographs was F-actin capping protein alpha-1 subunit (3679), whereas tubulin β-6 chain (3527), the actin-binding protein calponin 3 (3526), and the nuclear envelope protein lamin B2 (3510) were found upregulated in the ARPE-19 baseline proteome exclusively. The stress fiber components tropomyosin beta chain (3492) and tropomyosin 1 alpha (3549) were found at a significantly higher abundance in ARPE-19 autoradiographs alone. Markedly higher baseline expression levels and synthesis rates were also noted for the proteolytic enzymes cathepsin D (3500, 3519) and cathepsin H (3536), furthermore for two proteins involved in growth control (3516 serine/threonine protein phosphatase PP2A-alpha; 3508 c-myc-responsive protein RCL), KIAA0193 (3484), a homologue to the exocytosis-related protein secernin 1, ALG-2 interacting protein (3538), hypothetical protein F49H12.3 (3705), and annexin V mutant (3499). An increase in abundance in silver stained gels exclusively was noted for single proteins attributed to diverse functional groups. These comprised Ca2+-binding (3515 Annexin A2), protein metabolism (3518 cathepsin D; 3498 protein disulfide isomerase), energy metabolism (3541 phosphoglycomutase), and transcriptional control (3530 TDP-43). Likewise increased de novo protein synthesis exclusively was observed for single proteins attributed to diverse functional groups, including Ca2+-binding (3517, 3528 annexin A3), ribosomal RNA processing (3527 ribosomal RNA processing protein 41), polyamine metabolism (3490 spermine synthase), and DNA replication and proliferation (3493 proliferating cell nuclear antigen). Last, an up-regulation of follistatin-related protein 1 (3514) and caspase 3 (3533) was observed only in autoradiographs. 2. Proteins Expressed with Lower Abundance in ARPE-19 Cells. Lower abundance in protein expression was recorded for 13 spots in the baseline (Figure 3A,B) and 10 spots in the

de novo synthesized ARPE-19 proteome (Figure 3C,D), resulting in the identification of 15 distinct proteins. For a comprehensive list of down-regulated identified proteins see Tables 5 and 6 for classification by broad functional criteria. The most pronounced down-regulation in both, silver stained gels as well as in autoradiographs, was found for the cytosolic dipeptidase carnosinase 2 (3664) and the detoxification enzyme lactoylgluthatione lyase (3688), which were down-regulated with a factor 0.20 and 0.18, respectively. Suppressed expression in the baseline and de novo protein synthesis protein expression profile of ARPE-19 cells was also recorded for three structural components (3631 gelsolin; 3615 alpha-actinin-4; 3553 myosin regulatory light chain 2), the adhesion plaqueassociated mechanotransducer zyxin (3634), an isoform of cathepsin D (3690) and cellular retinol-binding protein 1 (3709). Lower abundance in silver stained gels alone was observed alpha-actinin-1 (3620), the signal transducer guanine nucleotide-binding protein G(i) alpha-2 (3672), tissue transglutaminase (3672), thioredoxin-like protein 2 (3674), and thymidine kinase (3698); a lower protein synthesis rate was found for ubiquilin-1 (3644), an ubiquitin-related protein that colocalizes with G proteins in pseudopods,26 and the pro-apoptotic protein prostate apoptosis response protein-4 (3659 Par-4). Similarities and Differences between ARPE-19 and hTERTRPE in Relation to epRPE. Several protein spots attracted attention because they were differentially regulated in both cell lines. Whereas some proteins were similarly up- or downregulated in both cell lines, others exhibited controversial regulation. Both immortalized RPE cell lines expressed a higher abundance of hypothetical protein F49H12.3 (3507) in silver stained gel images and autoradiographs, of guanine nucleotidebinding protein G(i) alpha-2 (3672) in silver stained gels, and of tropomyosin beta chain (3492) and tropomyosin 1 alpha (3459) in autoradiographs. Lactoylglutathione lyase (3688) and zyxin (3634) were down-regulated in both cell lines in silver stained gels and autoradiographs. Contrarily regulated in the steady state and de novo synthesized proteome were RCL (3508), and similar to tubulin β-5 (3508), as well as calponin-3 (3526) in silver stained gels, and interleukin 18 precursor (3503), ribosomal RNA processing protein 41 (3527) and tubulin β-1 chain (3713) in autoradiographss all of which were up-regulated in ARPE-19 cells and downregulated in hTERT-RPE cells. Furthermore, as opposed to the findings made for hTERT-RPE cells, tissue transglutaminase (3570, 3627) was down-regulated in silver stained gels derived from ARPE-19, and myosin regulatory light chain-2 (3553) was synthesized at a lower rate. Finally, tubulin β-1 chain was identified in spots allocated to different positions in the 2-DE Journal of Proteome Research • Vol. 5, No. 4, 2006 871

research articles

Alge et al.

Table 4. Identification Results of Proteins Expressed with Higher Abundance in ARPE-19 Cells but in Human Early Passage RPE Cellsd

Sia

x

Arb

top spot MOWSE score no.



x 3484

102

SCRN1_HUMAN

Q12765

x 3490

113

SPSY_HUMAN

Q9UQS1

x 3492

116

TPM2_HUMAN

P07951

x 3493

137

PCNA_HUMAN

P12004

x 3495

118

TBB1_HUMAN

x 3496

110

x

x 3497

x

protein name

biological process

49997

21%

4.83 Exocytosis

39926

21%

4.78 Polyamine metabolism

29980

18%

Proliferating cell nuclear antigen [Fragment]

28769

73%

P07437

Tubulin beta-1 chain

49362

20%

TBB1_HUMAN

P07437


49362

20%

110

TBB1_HUMAN

P07437


49362

18%

3498

84

PDIA1_HUMAN

P07237

57480

19%

x

3499

155

none

1SAV

35609

37%

x

x 3500

103

CATD_HUMAN

P07339

26629

31%

x

x 3503

149

IL18_HUMAN

Q14116

Protein disulfide isomerase, EC 5.3.4.1 annexin v mutant P13, P87, P119, P163, and P248 substituted with thioproline (prs) Cathepsin D, heavy chain, EC 3.4.23.5 Interleukin-18 [Precursor]

4.70 Stress fiber component; in nonmuscle cells is implicated in stabilizing cytoskeleton actin filaments 4.43 Regulation of DNA replication, cell proliferation, progression through cell cycle. 4.71 Microtubule-based processes, chaperone activity 4.71 Microtubule-based processes, chaperone activity 4.71 Microtubule-based processes, chaperone activity 4.76 Catalyzes protein folding and thiol-disulfide interchange reactions 5.05 unclassified

22597

66%

x

3505

94

Q96B85_HUMAN

Q96B85

21554

26%

x

x 3507 x 3508

92 84

Q9N5S5_CAEEL Q96B85_HUMAN

Q9N5S5 Q96B85

17015 21554

26% 32%

x 3508

70

RCL_HUMAN

O43598

19211

32%

3510

165

LAM2_HUMAN

Q03252

67762

28%

x 3514

122

FSTL1_HUMAN

Q12841

Follistatin-related protein 1 [Precursor]

36103

29%

x

3515

275

ANXA2_HUMAN

P07355

Annexin A2

38677

56%

x

x 3516

112

TBB4_HUMAN

P05218


50096

23

x

x 3516

84

P2AA_HUMAN

P05323

Serine/threonine protein phosphatase 2A (PP2A-alpha)

36142

28%

x 3517

135

ANXA3_HUMAN

P12429

Annexin A3

36524

34%

x

3518

193

CATD_HUMAN

P07339

26629

33%

x

x 3519

193

CATD_HUMAN

P07339

26629

33%

x

3526

208

CNN3_HUMAN

Q15417

Cathepsin D, heavy chain, EC 3.4.23.5 Cathepsin D, heavy chain, EC 3.4.23.5 Calponin 3

36562

58%

x

x

x

Protein KIAA0193 (homologue to secernin 1, NP_055581) Spermidine aminopropyltransferase Tropomyosin beta chain

c Mr seq. (Da) cov. (%) pI

Similar to tubulin, beta 5 [Fragment] hypothet. 16.8 kDa protein Similar to tubulin, beta 5 [Fragment] c-Myc-responsive protein RCL Lamin B2

x 3527

138

TBB6_HUMAN

Q9BUF5


50281

28%

x 3528

275

ANXA3_HUMAN

P12429

Annexin A3

36524

68%

3530

87

TADBP_HUMAN

Q13148

45053

32%

x 3533

82

CASP3_HUMAN

P42574

TAR DNA-binding protein43 (TDP-43) Caspase-3

32030

21%

x

x 3536 x 3537

84 105

Q96NY6_HUMAN EXOS4_HUMAN

Q96NY6 Q9NPD3

38079 26652

19% 35%

x

x 3538

367

PDC6I_HUMAN

Q8WUM4

Cathepsin H Exosome complex exonuclease RRP41, EC 3.1.13 ALG-2 interacting protein 1

96563

43%

x

3541 x 3549

150 222

PGMU_HUMAN TPM1_HUMAN

P36871 P09493

Phosphoglucomutase, EC 5.4.2.2 61565 Tropomyosin 1, alpha 32746 chain

30% 50%

x

x 3679

182

CAZ1_HUMAN

P52907

F-actin capping protein alpha-1 subunit

66%

x

33073

5.56 Acid protease; active in intracellular protein breakdown 4.54 Cell-cell signaling, apoptosis activator activity 4.77 Microtubule polymerization, microtubule based movement 4.89 Voltage-gated potassium channel 4.77 Microtubule polymerization, microtubule based movement 4.97 Cell proliferation; induces anchorage-independent growth. 5.29 Component of the nucleoplasmic side of the inner nuclear membrane 5.39 Modulates the action of some growth factors on cell proliferation and differentiation 7.56 Ca2+ -binding; membrane organization; may cross-link plasma membrane phospholipids with actin and the cytoskeleton; exocytosis 4.75 Microtubule-based processes, chaperone activity 5.30 Negative regulation of cell growth; regulation of adhesion, apoptosis, translation and transcription 5.63 Ca2+-dependent phospholipid binding 5.56 Acid protease; intracellular protein breakdown 5.56 Acid protease; intracellular protein breakdown 5.69 Thin filament-associated protein that is implicated in the modulation of smooth muscle contraction 4.77 Major constituent of microtubules 5.63 Ca2+-dependent phospholipid binding 5.27 Transcription factor activity; involved in mitosis; binds microtubules 6.09 Induction of apoptosis by intraand extracellular signals 8.34 Proteolysis and peptidolysis 6.07 rRNA processing 6.13 Interacts with proteins associated with apoptosis 6.32 Glycolysis 4.69 Stress fiber component; in nonmuscle cells is implicated in stabilizing cytoskeleton actin filaments 5.45 Binds in a Ca2+-independent manner to the fast growing ends of actin filaments.

a Si, up-regulated on silver stained gels. b Ar, up-regulated on autoradiographs of the respective gels as determined by 35S-pulse labelling. c Seq. Cov., sequence coverage; Top MOWSE score, Mascot score. d Spot numbers are indicated in Figure 3.

872


research articles


Table 5. Identification Results of Proteins Expressed with Lower Abundance in ARPE-19 Cells but in Human Early Passage RPE Cellsd

Sia

Arb

spot no.

top MOWSE score


x

x

3615

209

ACTN4_HUMAN

O43707

x

3620

165

ACTN1_HUMAN

x

3627

155

Mr (Da)

c seq. cov. (%)

Non-muscle alpha-actinin 4

105245

24%

P12814

Alpha-actinin 1

103480

19%

TGM2_HUMAN

P21980

78420

28%

86043

17%


protein name

x

x

3631

109

GELS_HUMAN

P06396

Tissue transglutaminase, EC 2.3.2.13 Gelsolin

x

x

3634

145

ZYX_HUMAN

Q15942

Zyxin 2

62436

31%

x

3644

105

UBQL1_HUMAN

Q9UMX0

Ubiquilin (PLIC-1)

62479

24%

x

3659

98

PAWR_HUMAN

Q96IZ0

prostate apoptosis response protein 4 (par4)

36659

27%

x

3664 3672

92 111

CPGL1_HUMAN GNAI2_HUMAN

Q96KP4 P04899

53187 40864

21% 39%

3674

113

TXNL2_HUMAN

O76003

37692

29%

3688

91

LGUL_HUMAN

Q04760

Carnosinase 2 Guanine nucleotidebinding protein G(i), alpha-2 subunit Thioredoxin-like protein 2 Lactoylglutathione lyase EC 4.4.1.5

20803

31%

x x x x

x

x

x

x

3690

122

CATD_HUMAN

P07339

3698

63

KITM_HUMAN

O00142

x

x

3709

110

RET1_HUMAN

P09455

x

x

3553

91

MLRN_HUMAN

P24844

Cathepsin D, heavy chain, EC 3.4.23.5 Thymidine kinase 2, [Precursor], EC 2.7.1.21 Cellular retinol-binding protein I (CRBP) Myosin regulatory light chain 2, smooth muscle isoform

pI

biological process

5.27 Actin binding protein; anchors actin to intracellular structures, colocalizes with actin stress fibers 5.22 Bundling protein; anchors actin to intracellular structures 5.11 Protein modification; catalyzes the cross-linking of proteins. Positive regulation of cell adhesion 5.9 Actin-modulating protein: promotes assembly of actin monomers into filaments 6.22 Associates with actin cytoskeleton near adhesion plaques. Component of signal transduction pathways that mediate adhesionstimulated changes in gene expression 5.02 Links CD47 to the cytoskeleton. Promotes the surface expression of GABA-A receptors 5.35 Pro-apoptotic protein by activation of the Fas prodeath pathway and coparallel inhibition of NF-kappaB transcriptional activity 5.66 Nonspecific dipeptidase 5.34 G-protein coupled receptor protein signaling

26629

44%

27856

22%

5.37 May regulate the function of the thioredoxin system 5.25 Detoxification; catalyzes conversion of cytosolic methylglyoxal to lactoylglutathione 5.56 Acid protease active in intracellular protein breakdown 6.37 Nucleic acid metabolism

15880

55%

4.99 Vitamin A metabolism

19740

52%

4.8

Regulation of both smooth muscle and nonmuscle cell contractile activity

a Si, down-regulated on silver stained gels. b Ar, down-regulated on autoradiographs of the respective gels as determined by 35S-pulse labelling. c Seq. Cov., sequence coverage; Top MOWSE score, Mascot score. d Spot numbers are indicated in Figure 3.

gels of the two cell lines (3713 in hTERT-RPE; 3495, 3496, 3497 in ARPE-19).

Discussion The present study for the first time gives a representative overview of the most striking differences in protein expression patterns between human early passage RPE cells (epRPE) and the two immortalized human RPE cell lines under a cell culture condition frequently used for in vitro RPE research. The cellular proteome of ARPE-19 and hTERT-RPE was compared to that of epRPE cells, and proteins were first grouped based on their expression patterns (Figures 2 and 3; Tables 2 to 5), followed by functional classification to illustrate the underlying biological processes (Table 6). Experimental artifacts and major proteolysis were avoided as demonstrated by the unchanged expression levels of the majority of proteins. Notably, there was a high correlation between proteins differentially expressed in both, steady state and de novo synthesized RPE proteome, suggesting that the baseline protein expression pattern of cultured RPE cells well reflects the metabolic activity in the observation period (Tables 2-5). None of the investigated cell lines exhibited the protein expression pattern found in native RPE,3 and they all contained features typically ascribed to a dedifferentiated molecular RPE phenotype.3,5,27 Although under the given cell culture conditions

the three cell lines appeared to differ to a great extent in terms of cellular morphology (Figure 1), the overall protein expression patterns displayed a high degree of similarity. At the same time intercell line comparison revealed that several biological processes differed on the translational level and a few of these are discussed below. Functional Implications of Differentially Expressed Proteins. 1. Proteins Differentially Expressed between hTERT-RPE and epRPE Cells. 1.1. Higher Abundance of Proteins Involved in Cell Contraction, Adhesion and Migration. Although also found upregulated in ARPE-19 autoradiographs, the most pronounced up-regulation of three tropomyosin isoforms and related proteins (3548, 3549 tropomyosin 1 alpha chain; 3492 tropomyosin 2 beta chain; 3552, 3553 myosin regulatory light chain 2; 3599 talin) was observed in hTERT-RPE cells. This difference was even more emphasized in autoradigraphs with an 8-fold higher synthesis rate of tropomyosin 1 compared to epRPE cells. Tropomyosin and myosin are main components of stress fibers, which are prominent components of the cytoskeleton of fibroblasts and undifferentiated RPE cells in culture.28 Stress fibers are thought to contract and thus allow the cells to exert tension on the extracellular matrix (ECM), an essential process in cell migration and wound healing. At one Journal of Proteome Research • Vol. 5, No. 4, 2006 873

research articles

Alge et al.

Table 6. Functional Grouping of Proteins Differentially Expressed Between Human Early Passage and Immoratlized Human RPE Cell Lines up-regulated ARPE 19

signaling

ALG-2 interacting protein 3538a,b

structural components

Tropomyosin 1, alpha 3549b Tropomyosin beta chain 3492b F-actin capping protein alpha subunit 3679a,b

down-regulated hTert-RPE

Ca2+ - binding

hTert-RPE

Zyxin 3634a,b

Zyxin 3634a

Guanine nucleotide-binding protein G(i), alpha-2 subunit 3672a Ubiquilin 1 3644b

Tropomyosin 1, alpha 3549a,b Tropomyosin beta chain 3492b

Gelsolin 3631a,b Alpha-actinin 1 3620a

Guanine nucleotide-binding protein G(i), alpha-2 subunit 3672a Guanine nucleotide-binding protein G(i), alpha-1 3661b Adenylyl cyclase-associated protein CAP2 3640a,b Moesin 3660a,b Ezrin 3639b

Tropomyosin 1, fibroblast and

Alpha-actinin 4 3615a,b

Ezrin-radixin-moesin binding

Myosin regulatory light chain 2 3553a,b

phosphoprotein-50 3655a,b

epithelial cell

3548a,b

Tubulin beta-6 chain 3527a Myosin regulatory light chain 2 Tubulin beta-5 chain 3516a,b Tubulin beta-1 chain 3495,a,b 3496,b 3497a,b Similar to tubulin, beta 5 3505,a,b 3508b Lamin B2 3510a Calponin 3 3526a Annexin V mutant 3499a,b Annexin A2 3515a Annexin A3 3517b 3528b

ARPE 19

Dihydropyrimidinase related protein 3 3604a,b, 3603a,b

3552,b 3553b

Tubulin-specific chaperone 3708a Tubulin beta-1 chain 3713b

Talin 1 3599a,b

Similar to tubulin, beta 5 3508a,b Calponin 3 3525,a,b 3526a

Calumenin (Prec) 3544,a,b 3543a,b

Calmyrin 3702a Annexin A3 3680a,b

detoxification

Glutathione transferase omega 1 Lactoylglutathione lyase 3688a,b 3577a,b Thioredoxin-like protein 2 3674a Superoxide dismutase 3578a,b Biliverdin reductase 3607b Nicotinamide Nmethyltransferase 3561a,b protein metabolism Cathepsin D 3500,a,b 3518,a Cathepsin D 3500,a,b 3560a,b Cathepsin D 3690a,b and modification 3519a,b 3558,a,b 3559a,b Carnosinase 2 3664a,b Cathepsin H 3536a,b CTD-like phosphatase domain- Tissue transglutaminase 3627a Protein disulfide isomerase containing protein 3605b 3498a Protein disulfide isomerase A6 Ribosomal RNA processing 3566a protein 41 3537b Tissue transglutaminase 3570a,b Lysine hydroxylase 3601a,b miscellaneous Phosphoglucomutase 3541a Ornithine aminotransferase CRBP 3709a,b 3488a metabolic pathways Spermine synthase 3490b Triosephosphate isomerase 3608b a,b transcriptional and PP2A-alpha 3516 Nucleophosmin 3563a,b Par-4 3659b translational control TDP-43 3530a Elongin b residues 1-120 3551a Elongation factor 1-β 3547a Leucine-zipper protein FKSG13 3575b DNA replication Proliferating cell nuclear Thymidine kinase 2 and antigen (Prec, short b proliferation 3493 splice variant) 36981 a,b RCL 3508 mitochondrial processes unclassified Secernin 1 3484a,b Integrin alpha-3 3598,a,b 3574a,b Annexin V mutant 3499a,b Laminin gamma-1 chain 3573a,b Caspase 3 3533b RAB6 interacting protein 3650b 2 3572a,b Hypothetical protein Hypothetical protein F49H12.3 F49H12.3 3507a,b 3507a,b Follistatin-related protein 1 Galectin-1 3710b 3514b Interleukin-18 (Prec) 3503a,b

Lactoylglutathione lyase 3688a,b Antioxidant protein 1 3701b Superoxide dismutase 3703a,b

Cathepsin D 3692,b 3690a,b Cathepsin B 3689a Proteasome subunit beta type 4 3695a FK506-binding protein 4 3652a Ribosomal RNA processing protein 41 3537b PAI-1 3642a,b NG,NG-dimethylarginine dimethylaminohydrolase 1 3677a,b Similar to eukaryotic translation termination factor 1 3629a,b

DNA replication licensing factor MCM7 3632a RCL 3508a,b 39S ribosomal protein L12 3706a Integrin alpha-3 3618a,b Inositol 1-phosphate synthase WW domain binding protein 2 3676a,b Endoplasmin 3670b Interleukin 18 3503b SNAP-23 3686b Similar to selenium binding protein 1 3668a

Spot numbers are indicated in Figures 2 and 3. a Regulated in silver stained gels. b Regulated in autoradiographs; Par4, prostate apoptosis response protein 4; PAI-1, plasminogen activator inhibitor 1; CRBP, cellular retinol binding protein; PP2A-alpha, serine/threonine protein phosphatase 2A; TDP-43, TAR DNAbinding protein-43; RCL, c-Myc-responsive protein RCL; Prec, Precursor.

874



end, stress fibers insert into a cytoplasmic network of intermediate filaments, at the other end they insert into focal adhesion complexes. Here, the cell cortex is attached to components of the ECM by transmembrane integrin molecules, which are linked to the actin cytoskeleton via a web of membrane-associated anchor proteins, such as talin.29,30 Focal adhesions are prerequisites for establishing cell contacts, adhesion to ECM, morphological changes and migration. Furthermore, they promote cell survival and cell proliferation signals.31,32 The up-regulation of tropomyosins, myosin regulatory light chain and talin in hTERT-RPE can thus be linked the reestablishment of cell-substratum adhesion, cell contraction, as well as an increased ability to migrate and generate tractional forces in culture, an observation which correlates well with the dedifferentiated, myo-fibroblastic phenotype of the hTERT-RPE cells. 1.2. Higher Abundance of Proteins Linked to Extracellular Matrix Formation and Modification. These included the ECM component laminin gamma-1 chain (3573), lysine hydroxylase (3601), an enzyme catalyzing the formation of stable intermolecular cross-links in collagens,33 the matricellular protein galectin-1 (3555), which promotes RPE cell migration,34 and tissue transglutaminase (tTG2) (3570), a Ca2+-dependent peptide-cross-linking enzyme, which has been shown to irreversibly cross-link the ECM deposited by RPE cells in proliferative vitreoretinopathy.18 Interestingly tTG2 appeared be constitutively down-regulated in ARPE-19 cells, and no differential expression of the other components was evident between epRPE and ARPE-19 cells, a finding that further supports the notion that hTERT-RPE cells and the latter cell lines may exhibit considerable differences in terms of cell-substratum-adhesion, but also ECM deposition and modification. 2-DE based comparison furthermore revealed a relatively lower abundance of plasminogen activator inhibitor-1 (PAI-1) (3642), a serine proteinase inhibitor, which is one of the main regulators of the fibrinolytic system. Independent of its antiproteolytic activity it appears to interfere with cellular migration or matrix binding, and can detach cells from vitronectin, fibronectin and type-1 collagen by disrupting ECM-integrin and urokinase plasminogen activator-ECM interactions.35 Down-regulation of this “deadhesive” protein may thus further be seen in the context of an increase in adhesive strength in the establishment of cellsubstratum contacts. 1.3. Expression of Proteins Related to Cellular Polarization are Decreased in hTERT-RPE. These included components of the microtubule cytoskeleton (3713 tubulin beta-1 chain; 3508 tubulin-beta 5), further ezrin-radixin-moesin binding phosphoprotein 50 (EBP50) (3655), ezrin (3633, 3639 in autoradiographs) and moesin (3660), as well as adenylyl cyclase associated protein CAP2 (3640). Because of the vectorial nature of the processes carried out by RPE cells, they are highly polarized in vivo.36,37 Ezrin-radixin-moesin (ERM) proteins regulate the organization and function of specific cortical structures in polarized epithelial cells by connecting F-actin to plasma membrane proteins.37 EBP50 is required for the maintenance of ERM proteins at the apical membrane of polarized epithelia and is thought to segregate membrane proteins to specific membrane surfaces.38 A recent study demonstrated that in the RPE EBP50 interacts with cellular retinaldehyde-binding protein (CRALBP) and that this interaction may provide a structural basis for the apical localization of a retinoid-processing complex in the RPE.38 Ezrin, is a polarization marker of the RPE located in the apical microvilli, and like moesin it tethers the

research articles actin cytoskeleton to specific plasma membrane proteins.39 CAP2 belongs to a family of proteins that play a role in regulating actin remodeling in response to cellular signals such as nutritional responses or environmental changes. CAP2 binds actin, associates with adenylyl cyclase, and controls the polarized distribution of actin filaments.40 As RPE cells become confluent and regain a cuboidal shape, F-actin redistributes to the apical face of the cells.41 Unfortunately, the role of mammalian CAP2 has not been studied extensively and no data exist regarding CAP2 expression in the RPE. However, findings obtained from Drosophila studies support a role of CAP in eye development and maintaining polarity during early cell differentiation.42 In a Dictyostelium CAP knockout mutant loss of the cyclase-associated protein homologue caused a drastically lowered sensitivity to external signals resulting in reduced cell polarity.43 The lower abundance of three members of the ERM-complex and CAP2 in hTERT-RPE cells may point out that under the cell culture conditions employed hTERT-RPE cells exhibit a lower degree of polarization but ARPE-19 cells or epRPE cells. 1.4. Pattern Shifts of Proteins Responsible for Detoxification Processes. Two antioxidative enzymes were found with an up-regulated (3607 biliverdin reductase; 3577 glutathione transferase omega 1) and one with a down-regulated (3701 antioxidant protein 1) protein synthesis rate in hTERT-RPE compared to epRPE. Biliverdin reductase occurs ubiquitously in human tissues, and catalyzes the conversion of biliverdin to bilirubin, a potent cellular antioxidant which can protect cells effectively from excess of H2O2.44 Also up-regulated was glutathione transferase omega 1 (3577 GSTO1), another antioxidant enzyme that catalyzes the conjugation of glutathione to a wide variety of endogenous and exogenous electrophilic compounds. Although its exact function is not known, recent data suggest that GSTO1 maybe involved in the etiology of neurodegenerative disease45 and play a role in anti-cancer drug resistance.46 This might be of some interest in view of a concomitant higher abundance of nicotinamide N-methyltransferase (3561), another detoxification enzyme which is important for biotransformation of many drugs and xenobiotic compounds. A distinct observation from this study was that a protein spot identified as lactoylglutathione lyase (3688) appeared strongly down-regulated in both immortalized RPE cell lines. This markedly suppressed expression was noted in the baseline cellular protein content as well as in the newly synthesized proteome suggesting a decreased requirement for this enzyme under long-term in vitro conditions. Lactoylglutathione lyase is a member of the metalloglutathione transferase superfamily and plays a critical role in cells by providing an enzymatic defense against glycation.47 Although to the best of our knowledge no data regarding lactoylgutathione lyase expression and function in the RPE exist, the almost complete absence of this enzyme in both immortalized RPE cell lines as opposed to epRPE cells may result from an adaptive response to prolonged cell culture conditions lacking its physiological substrates. The apparent down-regulation of CRBP-1 (3709) in ARPE-19 cells and hTERT-RPE, a key enzyme in visual pigment recycling, further substantiates this consideration. 2. Proteins Differentially Expressed between ARPE-19 and epRPE Cells. 2.1. Higher Abundance of Components of the Microtubule Cytoskeleton. The most evident difference between epRPE and ARPE-19 could be attributed to proteins associated with the microtubule cytoskeleton. Higher abundance of tubulins in ARPE-19 could be allocated to a total of Journal of Proteome Research • Vol. 5, No. 4, 2006 875

research articles seven spots on both, silver stained and 35S pulse labeled gels, and resulted in the identification of four different isoforms (3495, 3496, 3497 tubulin beta-1 chain; 3516 tubulin beta-5 chain; 3508 similar to tubulin beta 5; 3505 tubulin beta 6 chain). Microtubules are ubiquitous cytoskeletal components essential for RPE function. The microtubule cytoskeleton is required for several cellular activities, including intracellular protein transport, exocytosis, microtubule based movement and maintenance of the polarized state.48,49 Although tropomyosin 1 alpha (3549) and tropomyosin beta chain (3492) were synthesized at a higher rate than in epRPE cells, the recorded relative lower abundance of proteins attributed to cell migration, contraction, and transmission of adhesion-generated signaling (3631 gelsolin; 3553 myosin regulatory light chain 2; 3620 alpha actinin-4; 3634 zyxin)50,51 together with the higher abundance of components of the microtubule cytoskeleton may correspond well to the epitheloid, presumably stationary phenotype of the ARPE-19 cells. This is of particular interest in view of the findings made for hTERT-RPE cells, which exhibit a more dedifferentiated, fibroblast-like morphology together with a relative down-regulation of tubulin beta-1 chain (3713), tubulin beta-5 (3508), tubulin specific chaperone A (3708), as well as several polarization markers as described above. 2.2. Proteins Attributed to Cell Growth are Differentially Expressed between ARPE-19 and epRPE Cells. From the results of 2-DE we found a relative higher abundance of two pro-proliferative proteins (3493 proliferating cell nuclear antigen (PCNA); 3508 c-myc responsive element RCL) and a decreased protein synthesis rate of the anti-proliferative Par-4 (3659). However, concomitantly intercell line comparison revealed an up-regulation of proteins attributed to suppression of cell growth, including serine/threonine protein phosphatase 2A (PP2A-alpha) (3516) and follistatin-related protein 1 (FRP1) (3514), together with a down-regulation of the cell proliferation marker thymidine kinase 2 (3698). PP2A-alpha is a multitask enzyme system that appears to be critically involved in a wide array of cellular functions including metabolism, transcription, apoptosis and negative regulation of cellular growth.52-56 Similar functional implications are ascribed to Par-4, which is thought to have a suppressive effect on cell growth.57 FRP1, which was detected only in the autoradiographs derived from ARPE-19 cells, but not in epRPE cells or hTERT-RPE, is a secreted protein containing a similar amino acid sequence to follistatin, and is believed to have functional similarities to the anti-proliferative protein follistatin.58 Follistatin leads to a delay in TGF-β mediated wound healing of the skin,59,60 induces angiogenesis in the cornea,61 and has an important role in retinal development.62 A functional interrelation to the up-regulation of proproliferative proteins may be represented by the up-regulation of spermine synthase (3490) in autoradiographs from ARPE19 cells. Spermine synthase is a key enzyme in polyamine (putrescine, spermidine, and spermine) synthesis. A number of studies with various inhibitors of the enzymes of polyamine metabolism have established that polyamines are indispensable for cell growth,63,64 an effect that is presumed to be primarily due to the stimulation of protein synthesis.64 Although the overall functional implication of the presumably controversial regulation of proteins related to cell growth appears complicated, it is noteworthy that different pathways of growth control may be activated in the three cell types. 876


Alge et al.

2.3. Four Proteins Found Attributed to Programmed Cell Death. Another group of four proteins found expressed at significantly higher levels in ARPE-19 cells can be attributed to programmed cell death. These comprised serine/threonine protein phosphatase 2A (PP2A-alpha) (3516), interleukin-18 precursor (IL-18) (3503), programmed cell death 6-interacting protein (AIP1) (3538), and caspase-3 (3533), a main downstream executioner of both, the intrinsic and extrinsic apoptotic pathway. Furthermore, the increased synthesis of spermine synthase (3490) in ARPE-19 cells may also be related to the regulation of apoptosis. Studies in various cellular systems evidenced that polyamine depletion inhibits apoptosis.65,66 Although contradictory with respect to the presumable functions in regulation of cell growth, several reports demonstrated that spermine is the most potent activator of PP2A, 67,68 whichs in addition to its function in suppression of cell growthsexerts a positive regulatory function in apoptosis.53,55 A pro-apototic activity has furthermore been reported for IL-18 (3508): studies in transgenic mice revealed that IL-18 differentially regulates apoptosis by the death inducing factors Fas and TNF-alpha.69,70 AIP1 also exerts a proapoptotic activity in executing the calcium-dependent requirements along the cell death pathway. Overexpression of a truncated form of AIP1 protected HeLa and COS cells from death induced by trophic factors withdrawal.71 However, it is noteworthy that we also found a suppressed protein synthesis rate of Par-4 (3659), another proapoptotic protein.57 In the present study cells had been serum starved for 21 h prior to harvesting, which may have initiated the apoptotic machinery. Thus, the up-regulation of proteins related to programed cell death may be a consequence of nutrient depletion. 2.4. Up-Regulation of IL-18. An interesting observation was the high abundance of IL-18 in ARPE-19 cellular proteomes. The respective protein spot was up-regulated with high significance in both silver (18-fold) and 35S pulse-labeled images (8-fold), whereas it was hardly detectable in epRPE cells and invisible in hTERT-RPE cells. IL-18 plays an important role in positive regulation of activated T-cell proliferation, T-helper 2 type immune response, interleukin-13, and interleukin-2 biosynthesis,72,73 but its role in the eye remains to be determined. Only recently has it been reported that IL-18 is constitutively expressed by RPE cell lines,73 but unfortunately no data regarding IL-18 expression in native RPE under normal or pathologic conditions exist in the literature. A study in IL-18 knock out mice found an abnormal retinal vascular formation during early development in the absence of IL-18, suggesting anti-angiogenic properties of IL-18 in the retina. At the same time, several angiogenesis-related factors were overexpressed in the retinas of IL-18 deficient mice.74 With respect to these observations caution should be applied when using epRPE, hTERT-RPE or ARPE-19 cells for investigation of T-cell responses or expression of angiogenesis-related factors, since the predominance of IL-18 in ARPE-19 cells may entail different experimental outcomes. Concluding Remarks. In conclusion proteomic analysis identified a high degree of similarity between hTERT-RPE cells, ARPE-19 cells, and epRPE cells. Overall, under the respective in vitro conditions, hTERT-RPE cells appeared to exhibit stronger differences when compared to epRPE cells but ARPE19 cells. This was evident both with respect to number of differentially expressed proteins as well as in classification of the respective proteins to certain functional groups. In both immortalized cell lines the most pronounced difference in


protein expression and synthesis rate was evident with respect to a group of proteins attributed to cytoskeletal remodeling. Whereas ARPE-19 cells displayed major differences in the microtubule-associated cytoskeleton, hTERT-RPE cells showed a high abundance of components of the contractile cellular cytoskeleton together with a down-regulation of cell polarization markers. The high abundance of cytoskeletal and related elements in the RPE proteome of cultured RPE cells may reflect the importance of these components under the chosen in vitro conditions, suggesting that the three cell types may respond differently, when plated on plastic. In summary, in hTERT-RPE cells the main proportion of proteins found differentially expressed at the proteomic level could be attributed to an increase in adhesional strength, cell contraction, cell-matrix interactions, ECM deposition and modification, as well as a loss of polarizationsfindings which well correlate with the myo-fibroblastic phenotype of these cells. Functional grouping of proteins differentially expressed between ARPE-19 cells and epRPE cells remained less conclusive. In ARPE-19 cells several not necessarily interrelated functional groups were found to be differentially expressed. Interestingly, we noted a marked up-regulation of IL-18 in ARPE-19 cells and identified FRP1 in autoradiographs from ARPE-19 cells, exclusively. A finding, that implies that a defined cell culture condition may elicit cell-type dependent responses with respect to certain functional groups. While the data generated in this study certifies to the sensitivity and value of differential proteomics in screening for intercell type variations, there are a number of cautionary considerations. First, one single cell culture condition was tested, and the findings may therefore not be intrinsic properties of the three cell lines, but perhaps a consequence of the cell culture conditions. Present data may therefore only to a certain extent be extrapolated to other in vitro conditions. Second, for technical reasons, low abundance proteins were excluded from the comparative analysis. Third, narrow range immobilized pH gradient strips were used during isoelectric focusing to increase the number of proteins resolved. Thus a number of proteins may not have been detected due to the pI of the protein being outside the experimentally chosen analytical window. Although this may limit the interpretation of the present findings, we do believe that the reported observations should be considered in further studies that evaluate the functional groups found differentially expressed in the three cell types.

Acknowledgment. The authors thank Katja Obholzer and Stefanie Scho¨ffmann for expert technical assistance and Ulrich Welge-Luessen, MD for providing the early passage RPE cell lines. Work was funded by EU Grant Nos.: PRO-AGE-RET QLK6-CT-2001-00385, RETNET MRTN-CT-2003-504003, EVIGENORET LSHG-CT-2005-512036, and by funding from the German Federal Ministry of Education and Research: BMBFProteomics 031U108A/031U208A. References (1) Strauss, O. Physiol. Rev. 2005, 85, 845-881. (2) Schlingemann, R. O. Graefes Arch. Clin. Exp. Ophthalmol. 2004, 242, 91-101. (3) Alge, C. S.; Suppmann, S.; Priglinger, S. G.; Neubauer, A. S.; May, C. A.; Hauck, S.; Welge-Lussen, U.; Ueffing, M.; Kampik, A. Invest. Ophthalmol. Vis. Sci. 2003, 44, 3629-3641. (4) Hunt, R. C.; Davis, A. A. J. Cell. Physiol. 1990, 145, 187-199. (5) Grisanti, S.; Guidry, C. Invest. Ophthalmol. Vis. Sci. 1995, 36, 391405.

research articles (6) Dunn, K. C.; Aotaki-Keen, A. E.; Putkey, F. R.; Hjelmeland, L. M. Exp. Eye Res. 1996, 62, 155-169. (7) Pfeffer, B. A.; Clark, V. M.; Flannery, J. G.; Bok, D. Invest. Ophthalmol. Vis. Sci. 1986, 27, 1031-1040. (8) Hall, M. O.; Abrams, T. Exp. Eye Res. 1987, 45, 907-922. (9) Bosch, E.; Horwitz. J.; Bok, D. J. Histochem. Cytochem. 1993, 41, 253-263. (10) Zhu, M.; Provis, J. M.; Penfold, P. L. Aust. N. Z. J. Ophthalmol. 1998, 26 Suppl.1, 50-52. (11) Castillo, B. V. Jr.; Little, C. W.; del Cerro, C.; del Cerro, M. Curr. Eye Res. 1995, 14, 677-683. (12) McKay, B. S.; Burke, J. M. Exp. Cell Res. 1994, 213, 85. (13) Bodnar, A. G.; Ouellette, M.; Frolkis, M.; Holt, S. E.; Chiu, C. P.; Morin, G. B.; Harley, C. B.; Shay, J. W.; Lichtsteiner, S. Science 1998, 16 (279), 349-352. (14) Davis, A. A.; Bernstein, P. S.; Bok, D.; Turner, J.; Nachtigal, M,; Hunt, R. C. Invest. Ophthalmol. Vis. Sci. 1995, 36, 955-964. (15) Harley, C. B. Ciba Found Symp. 1997, 211, 129-145. (16) Rambhatla, L.; Chiu, C. P.; Glickman, R. D.; Rowe-Rendleman, C. Invest. Ophthalmol. Vis. Sci. 2002, 43, 1622-1630. (17) Alge, C. S.; Priglinger, S. G.; Neubauer, A. S.; Kampik, A.; Zillig, M.; Bloemendal, H.; Welge-Lussen, U. Invest. Ophthalmol. Vis. Sci. 2002, 43, 3575-3582. (18) Priglinger, S. G.; May, C. A.; Neubauer, A. S.; Alge, C. S.; Schonfeld, C. L.; Kampik, A.; Welge-Lussen, U. Invest. Ophthalmol. Vis. Sci. 2003, 44, 355-364. (19) Leschey, K. H.; Hackett, S. F.; Singer, J. H.; Campochiaro, P. A. Invest. Ophthalmol. Vis. Sci. 1990, 31, 839-846. (20) Hauck, S. M.; Schoeffmann, S.; Deeg, C. A.; Gloeckner, C. J.; Swiatek-de Lange, M.; Ueffing, M. Proteomics 2005, 5, 3623-3636. (21) Blum, H.; Beier, H.; Gross, H. J. Electrophoresis 1987, 8, 93-99. (22) Hauck, S. M.; Ekstrom, P. A.; Ahuja-Jensen, P.; Suppmann, S.; Paquet-Durand, F.; van Veen, T.; Ueffing, M. Mol. Cell. Proteomics 2005, Oct 26. (23) Gharahdaghi, F.; Weinberg, C. R.; Meagher, D. A.; Imai, B. S.; Mische, S. M. Electrophoresis 1999, 20, 601-605. (24) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (25) Pappin, D. J. Methods Mol. Biol. 1997, 64, 165-173. (26) N′Diaye, E. N.; Brown, E. J. J. Cell Biol. 2003, 163, 1157-1165. Exp. Cell. Res. 1983, 147, 379-391. (27) Casaroli-Marano, R. P.; Pagan, R.; Vilaro, S. Invest. Ophthalmol. Vis. Sci. 1999, 40, 2062-2072. (28) Opas, M.; Kalnins, V. I. Invest. Ophthalmol. Vis. Sci. 1986, 27, 1622-1633. (29) Zamir, E.; Geiger, B. J. Cell Sci. 2001, 114, 3583-3590. (30) Johnson, R. P.; Craig, S. W. J. Biol. Chem. 1994, 269, 12611-12619. (31) Frisch, S. M,. Vuori, K.; Ruoslahti, E.; Chan-Hui, P. Y. J. Cell Biol. 1996, 134, 793-799. (32) Assoian, R. K.; Schwartz, M. A. Curr. Opin. Genet. Dev. 2001, 11, 48-53. (33) Handa, J. T.; Murad, S.; Jaffe, G. J. Invest. Ophthalmol. Vis. Sci. 1994, 35, 463-469. (34) Alge, C. S.; Priglinger, S. G.; Kook, D.; Schmid, H.; Haritoglou, C.; Welge- Luessen, U.; Kampik, A. Invest. Ophthalmol. Vis. Sci. 2006, 47, 415-426. (35) Czekay, R. P.; Aertgeerts, K.; Curriden, S. A.; Loskutoff, D. J. J. Cell Biol. 2003, 160, 781-791. (36) Burnside, M. B. Exp. Eye Res. 1976, 23, 257-275. (37) Nawrot, M.; West, K.; Huang, J.; Possin, D. E.; Bretscher, A.; Crabb, J. W.; Saari, J. C. Invest. Ophthalmol. Vis. Sci. 2004, 45, 393-401. (38) Bonilha, V. L.; Bhattacharya, S. K.; West, K. A.; Crabb, J. S.; Sun, J.; Rayborn, M. E.; Nawrot, M.; Saari, J. C.; Crabb, J. W. Exp. Eye Res. 2004, 79, 419-422. (39) Zhang, M.; Bohlson, S. S.; Dy, M.; Tenner, A. J. Immunology 2005, 115, 63-73. (40) Hubberstey, A. V.; Mottillo, E. P. FASEB J. 2002, 16, 487-499. Review. (41) Matsumoto, B.; Guerin, C. J.; Anderson, D. H. Invest. Ophthalmol. Vis. Sci. 1990, 31, 879-889. (42) Baum, B.; Li, W.; Perrimon, N. A. Curr. Biol. 2000, 10, 964-973. (43) Noegel, A. A.; Blau-Wasser, R.; Sultana, H.; Muller, R.; Israel, L.; Schleicher, M.; Patel, H.; Weijer, C. J. Mol. Biol. Cell. 2004, 15, 934-945. (44) Baranano, D. E.; Rao, M.; Ferris, C. D.; Snyder, S. H. Proc Natl Acad Sci USA. 2002, 99, 16093-16098. (45) Li, Y. J.; Oliveira, S. A.; Xu, P.; Martin, E. R.; Stenger, J. E.; Scherzer, C. R.; Hauser, M. A.; Scott, W. K.; Small, G. W.; Nance, M. A.; Watts, R. L.; Hubble, J. P.; Koller, W. C.; Pahwa, R.; Stern, M. B.;

Journal of Proteome Research • Vol. 5, No. 4, 2006 877

research articles

(46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60)

878

Hiner, B. C.; Jankovic, J.; Goetz, C. G.; Mastaglia, F.; Middleton, L. T.; Roses, A. D.; Saunders: A. M.; Schmechel, D. E.; Gullans, S. R.; Haines, J. L.; Gilbert, J. R.; Vance, J. M.; Pericak-Vance, M. A.; Hulette, C.; Welsh-Bohmer, K. A. Hum. Mol. Genet. 2003, 12, 3259-3267. Townsend, D. M.; Tew, K. D. Oncogene. 2003, 22, 7369-7375. Review. Thornalley, P. J. Biochem. Soc. Trans. 2003, 31, 1343-1348. Review. Vale, R. D. Annu. Rev. Cell. Biol. 1987, 3, 347-378. Review. Beauchemin, M. L.; Leuenberger, P. M. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 1977, 203, 237-251. Arora, P. D.; Janmey, P. A.; McCulloch, C. A. Exp. Cell Res. 1999, 250, 155-167. Honda, K.; Yamada, T.; Endo, R.; Ino, Y.; Gotoh, M.; Tsuda, H.; Yamada, Y.; Chiba, H.; Hirohashi, S. J. Cell Biol. 1998, 140, 13831393. Garcia, A.; Cayla, X.; Guergnon, J.; Dessauge, F.; Hospital, V.; Rebollo, M. P.; Fleischer, A.; Rebollo, A. Biochimie 2003, 85, 721726. Review. Van Hoof, C.; Goris, J. Biochim. Biophys. Acta 2003, 1640, 97104. Review. Janssens, V.; Goris, J. Biochem J. 2001, 353, 417-439. Review. Ray, R. M.; Bhattacharya, S.; Johnson, L. R. J. Biol. Chem. 2005, 280, 31091-31100. Schonthal, A. H. Cancer Lett. 2001, 170, 1-13. Review. Garcia-Cao, I.; Duran, A.; Collado, M.; Carrascosa, M. J.; MartinCaballero, J.; Flores, J. M.; Diaz-Meco, M. T.; Moscat, J.; Serrano, M. EMBO Rep. 2005, 6, 577-583. Harrison, C. A.; Gray, P. C.; Vale, W. W.; Robertson, D. M. Trends Endocrinol. Metab. 2005, 16, 73-78. Review. Wankell, M.; Munz, B.; Hubner, G.; Hans, W.; Wolf, E.; Goppelt, A.; Werner, S. EMBO J. 2001, 20, 5361-5372. Sulyok, S.; Wankell, M.; Alzheimer, C.; Werner, S. Mol. Cell Endocrinol. 2004, 225, 127-132. Review.


Alge et al. (61) Kozian, D. H.; Ziche, M.; Augustin, H. G. Lab. Invest. 1997, 76, 267-276. (62) Belecky-Adams, T. L.; Scheurer, D.; Adler, R. Dev. Biol. 1999, 210, 107-123. (63) Nishikawa, Y.; Kar, S.; Wiest, L.; Pegg, A. E.; Carr, B. I. Biochem. J. 1997, 321, 537-543. (64) Igarashi, K.; Kashiwagi, K. Biochem. Biophys. Res. Commun. 2000, 271, 559-564. Review. (65) Schipper, R. G.; Penning, L. C.; Verhofstad, A. A. Semin. Cancer Biol. 2000, 10, 55-68. Review. (66) Penning, L. C.; Schipper, R. G.; Vercammen, D.; Verhofstad, A. A.; Denecker, T.; Beyaert, R.; Vandenabeele, P. Cytokine 1998, 10, 423-431. (67) Tung, H. Y.; Pelech, S.; Fisher, M. J.; Pogson, C. I.; Cohen, P. Eur. J. Biochem. 1985, 149, 305-313. (68) Cornwell, T.; Mehta, P.; Shenolikar, S. J. Cyclic Nucleotide Protein Phosphor. Res. 1986, 11, 373-382. (69) Marino, E.; Cardier, J. E. Cytokine 2003, 22, 142-148. (70) Finotto, S.; Siebler, J.; Hausding, M.; Schipp, M.; Wirtz, S.; Klein, S.; Protschka, M.; Doganci, A.; Lehr, H. A.; Trautwein, C.; Khosravi-Far, R.; Strand, D.; Lohse, A.; Galle, P. R.; Blessing, M.; Neurath, M. F. Gut. 2004, 53, 392-400. (71) Vito, P.; Pellegrini, L.; Guiet, C.; D’Adamio, L. J. Biol. Chem. 1999, 274, 1533-1540. (72) Tsutsui, H.; Matsui, K.; Kawada, N.; Hyodo, Y.; Hayashi, N.; Okamura, H.; Higashino, K.; Nakanishi, K. J Immunol. 1997, 159, 3961-3967. (73) Jiang, H. R.; Wei, X.; Niedbala, W.; Lumsden, L.; Liew, F. Y.; Forrester, J. V. Invest. Ophthalmol. Vis. Sci. 2001, 42, 177-182. (74) Qiao, H.; Sonoda, K. H.; Sassa, Y.; Hisatomi, T.; Yoshikawa, H.; Ikeda, Y.; Murata, T.; Akira, S.; Ishibashi, T. Lab Invest. 2004, 84, 973-980.

PR050420T

Differential Protein Profiling of Primary versus Immortalized Human

Recommend Documents