Proteomic Analysis of a Membrane Skeleton Fraction from Human Liver

Proteins of a membrane skeleton fraction from human livers were separated by 2-D gels and identified by MALDI-TOF and MALDI-TOF−TOF MS. Finally, 104...
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Proteomic Analysis of a Membrane Skeleton Fraction from Human Liver Jintang He,† Yashu Liu,† Sizhi He, Qingsong Wang, Hai Pu, and Jianguo Ji* Department of Biochemistry and Molecular Biology, College of Life Sciences, The National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, P. R. China Received April 6, 2007

The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called “membrane skeletons”. Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons. Keywords: human normal liver • membrane skeleton • proteomic • 2-DE • MALDI-TOF-TOF MS

Introduction The liver is the largest organ and the main “chemical factory” and “energy plant” in the body. It is an epithelial organ which is mainly composed of parenchymal cells (the hepatocytes) and nonparenchymal cells (for example, biliary epithelial cells, sinusoidal endothelial cells, kupffer cells, et al.). These liver cells are enriched in cytoskeletal networks around their plasma membranes.1-5 The cytoskeletal proteins connect to each other, and the interactions between them provide resistance against disruption by Triton X-100, permitting these cytoskeletal proteins and their tightly associated membrane proteins to be isolated as a “membrane skeleton”.6 Some specialized membrane domains attached to membrane skeletons have been isolated from liver. For example, membrane domains anchored to actin filaments-based skeleton include spectrin-based meshworks,7,8 adherens junctions,2 focal adhesions,9 and tight junctions;10,11 membrane domains anchored to cytokeratin-based skeleton include desmosomes12 and hemidesmosomes.13 The interactions between the membrane skeletons and plasma membranes play important roles in determining cell shape and in signal transduction pathways.14 It is possible * To whom correspondence should be addressed. P. O. Box 31, College of Life Sciences, Peking University, Beijing 100871, P. R. China; Phone, (86)10-62755470; Fax, (86)-10-62751526; E-mail, [email protected]. † These authors contributed equally to this work. 10.1021/pr070197v CCC: $37.00

 2007 American Chemical Society

that the growth and development of a cell are greatly influenced by signals received by some membrane skeleton proteins. Thus, it is necessary to comprehensively study the membrane skeletons. Proteomics provides powerful tools to separate and identify the protein components of membrane skeletons in a large scale. Generally, proteins extracted from the samples are separated by gels or liquid chromatography and then identified by mass spectrometry. So far, there have been a few proteomic studies of membrane skeletons. In an early research,4 proteins from detergent fractionated cytoskeletal compartments of rat hepatocytes were separated by two-dimensional (2-D) gels, and 8 cytoskeletal proteins were identified by immunoblotting. Recently, Nebl et al.15 applied SDS polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins from a detergentresistant membrane skeleton of neutrophil, and identified these proteins by MALDI-TOF MS. They identified some membrane skeleton proteins, including fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, alpha-actinin 1, alpha-actinin 4, vimentin, and supervillin. In addition, they also identified some lipid raft associated proteins (stomatin, flotillin 1, and flotillin 2). In some other studies, membrane skeleton proteins have also been co-purified with lipid raft.16-18 All of these studies are focused on the membrane skeleton of a specific cell, but none has systematically investigated the membrane skeletons Journal of Proteome Research 2007, 6, 3509-3518

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research articles at the level of a tissue or an organ, which reflects the actual circumstances in vivo. Despite their importance, the membrane skeletons of human liver cells are largely uncharacterized. This may be caused by the complex structure of these membrane skeletons and the low solubility of their protein components. The main purpose of the present work is to provide a representative 2-DE map of membrane skeletons from human liver, with the view of helping to elucidate the composition and function of the membrane skeletons. In this work, we prepared a Triton X-100resistant membrane skeleton fraction from human liver and separated its protein components by 2-D gels. We optimized the detergent used for the solubilization of skeleton proteins and finally selected 2% ASB-14. One-hundred and four nonredundant proteins were identified from 2-D gels by MALDITOF and MALDI-TOF-TOF MS, wherein 38 were cytoskeletal proteins. These cytoskeletal proteins were classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes, and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. All the results suggested that human liver had complex actin- and cytokeratin-based membrane skeletons.

Materials and Methods Preparation of Liver Plasma Membranes. To prepare plasma membranes, subcellular fractionation of human normal livers (provided by HLPP) was performed essentially as described.19 Briefly, after removing connective tissues, human livers were minced with scissors, added to 5 volumes of icecold homogenization buffer (HB) (0.25 M sucrose, 10 mM HEPES, 1 mM PMSF, pH 7.5), and homogenized in an ULTRATURRAX T8 homogenizer (IKA, Germany). The homogenate was filtered through four layers of nylon gauze and the filtrate was centrifuged at 1000g for 10 min in a Himac CR21 centrifuge. The resulting pellets (residue 1) were pooled and resuspended in HB. Then high-density sucrose (2.4 M sucrose, 10 mM HEPES, and 1 mM PMSF, pH 7.5) was added to obtain a concentration of 1.6 M (45.7% w/w sucrose). Twenty-five milliliters of the suspended residue 1 were transferred into each of six SW32 rotor tubes, and the suspensions were overlaid with about 13 mL of HB to fill the tube and were then centrifuged at 70 900g (Rav) for 70 min in a Beckman Optima L-80 XP ultracentrifuge. The band at the interface was collected, resuspended gently in 2 volumes of cold water and enough HB, and centrifuged at 1200 g for 10 min. The pellets (residue 2) were resuspended in HB, adjusted to a concentration of 1.45 M (42% w/w sucrose) with high-density sucrose, transferred into each of six SW32 rotor tubes, overlaid with enough HB to fill the tubes, and then centrifuged at 68 400 g (at Rav) for 60 min. The pellicle at the interface was resuspended in HB and centrifuged at 17 600 g for 10 min. The pellets (residue 3) were then resuspended in HB, and high-density sucrose was then added to bring the final concentration to 1.35 M (39.4% w/w sucrose). The suspended residue 3 were transferred into each of six P40ST tubes, overlaid with enough HB to fill the tubes, and centrifuged at 231 000g for 60 min in a Himac CP 100MX ultracentrifuge. The purified plasma membranes were collected from the interface, resuspended in enough HB, centrifuged at 14 500g, and stored in aliquots at -80 °C. Electron Microscopy. Equal volumes of a sample and 5% glutaraldehyde in PBS were mixed, incubated overnight at 3510

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4 °C, and centrifuged. The resulting pellets were postfixed in OsO4, dehydrated through a graded series of ethanol and acetone, and embedded in Epon. Then thin sections were stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM-1010; JEOL, Tokyo, Japan) at 80 KV. Western Blotting. Proteins of different fractions were separated by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). The membranes were blocked for 2 h, and then incubated with anti-flotillin-1 (Chemicon, Temecula, CA), anti-Oxphos complex I 39 kDa subunit (Molecular Probes, Eugene, OR), anti-KDEL (StressGen, Victoria, Canada), anti-Golgi 97 (Molecular Probes, Eugene, OR), anti-Na+/K+ ATPase R1 subunit (Abcam, Cambridge, MA), or anti-beta-actin (Boster Co., Wuhan, China) for 4 h or overnight. After being washed three times, the membranes were incubated with peroxidase-conjugated goat anti-rabbit or antimouse IgG (H+L) for 1 h, washed three times, and detected by Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Billerica, USA). Preparation of Detergent-Resistant Membrane Skeletons. Detergent-resistant membrane skeleton fraction was prepared as previously described15 with some modifications. Briefly, the liver plasma membranes from three human livers were pooled and resuspended to 1-2 mg of protein per mL of Triton X-100 extraction buffer (TEB: 1% Triton X-100, 25 mM Tris, 250 mM NaCl, 2.5 mM MgCl2, 1 mM EGTA, 1 mM ATP) containing 1 mM PMSF and were incubated on ice for 60 min. Then the suspension was centrifuged at 150 000g for 1 h, and the pellet was resuspended in TEB again, incubated on ice for 30 min, and centrifuged at 150 000g for 1 h. The pellet was the so-called membrane skeleton fraction. Two-Dimensional Gel Electrophoresis. Two-dimensional gel electrophoresis (2-DE) was performed as previously described18 with some modifications. Briefly, membrane skeleton fraction was solubilized in a lysis buffer containing 7 M urea, 2 M thiourea, 65 mM DTT and a detergent (4% CHAPS, 2% ASB-14 or 2% Triton X-100) at room temperature for 5 h, and replenished with 0.5% ampholytes before IEF. Samples were loaded on pH 3-10 IPG DryStrips. IEF was conducted using the IPGphorTM II (GE Healthcare, Sweden) according to the following procedure: 30 V × 8 h, 50 V × 4 h, 300 V × 1 h, 1000 V × 1 h, 3000 V × 1 h, 5000 V × 1 h, and 8000 V for a total of 65 000 V h. The second dimension was performed in a 10% SDS polyacrylamide gel. The gels were then stained by a procedure called “blue silver”20 and scanned at 300 dpi resolution. Protein spots were analyzed with ImageMaster PlatinumTM software (GE Healthcare). In Gel Protein Digestion. In gel protein digestion was performed essentially as Yang et al.21 described. Briefly, protein spots were excised with scissor-sheared pipet tips, and then transferred to V-bottom 96-well microplates loaded with 100 µL of 50% acetonitrile/25 mM ammonium bicarbonate solution per well. After being destained for 1 h, the gel pieces were dehydrated with 100 µL of 100% acetonitrile for 20 min and then dried in a SpeedVac concentrator (Thermo Savant, USA) for 30 min. Then 2 µL of 5 µg/mL trypsin in 25 mM ammonium bicarbonate was added to each well. The 96-well plates were then sealed with Parafilm, re-swelled at 4 °C for 45 min, and incubated at 37 °C for 10 h. After tryptic digestion, 8 µL of the extraction solution consisting of 0.25% TFA and 5 mM n-octylβ-D-glucopyranoside was added to each well and shaked at 37 °C for 1 h to extract the peptides.

Membrane Skeleton Proteomics

MALDI-TOF and MALDI-TOF-TOF MS. MALDI samples were prepared according to a thin layer method as described before.21,22 In brief, saturated R-cyano-4-hydroxycinnamic acid (CHCA) in a solution containing 97% acetone and 0.003% TFA was dragged with an Eppendorf tip across the surface of AnchorChipTM 600/384 to form a thin layer of crystalline CHCA. Four microliters of each tryptic digested sample solution was deposited onto a spot. After incubating for 3 min, the remaining liquid was removed with tips, and then each spot was washed with 6 µL of a solution containing 10 mM ammonium citrate and 0.3% TFA. One microliter of 0.1 pmol/µL standard peptide mixtures which were used for external mass calibration were also performed as above. Mass spectra were recorded on an Ultraflex MALDI-TOF-TOF mass spectrometer (Bruker Daltonik) under the control of FlexControlTM 2.2 software (Bruker Daltonik GmbH). MALDI-TOF spectra were recorded in the positive ion reflector mode in a mass range from 800 to 4000 Da and the ion acceleration voltage was 25 kV. After being processed by the FlexAnalysis 2.2 (Bruker Daltonik GmbH) and Biotool 2.2 (Bruker Daltonik GmbH) software provided by the manufacturer, the MALDI-TOF spectra were automatically searched against Swiss-Prot or IPI database (IPI.HUMAN.v3.07) using the Mascot software (Matrix Science, London, UK). The main parameters were set as follows: S/N g 3.0; Fixed modification: Carbamidomethyl (C); Variable modification: Oxidation (M); Maximum number of missing cleavages 1; MS tolerance ( 100 ppm. All accepted results had a Mascot score of at least 64 for Swiss-Prot accessions and 69 for IPI accessions (p < 0.005), whereas the scores greater than 54 or 59 were normally significant (p < 0.05), respectively. After analysis of the TOF results, the skeleton proteins were subjected to TOFTOF analysis in “LIFT” mode. Some strongest peaks of each TOF spectra were selected as precursor ions which were accelerated in TOF1 at a voltage of 8 KV and fragmented by lifting the voltage to 19 KV. The MS/MS spectra were also processed by FlexAnalysis 2.2, Biotool 2.2, and MASCOT. The main parameters of Mascot were the same as TOF search, except that the MS tolerance was ( 0.7 Da.

Results and Discussion Morphological Evaluation of the Isolated Plasma Membranes by Electron Microscopy. The quality of the plasma membrane fraction was morphologically evaluated by electron microscopy (Figure 1). Some nuclei, mitochondria, membrane sheets, and vesicles were clearly observed in the liver homogenate (Figure 1A). However, the predominant structures in the plasma membrane fraction were membrane sheets, and an occasional mitochondrion could also be seen (Figure 1B). This indicates that the plasma membranes are remarkably enriched after subcellular fractionation. Figure 1C shows a higher magnification image of plasma membranes. Verification of Enrichment of Plasma Membranes by Western Blotting Analysis. The quality of the plasma membranes was also examined by Western blotting. We compared the liver homogenate with the plasma membranes using antibodies against organelle-specific protein markers (Figure 2). To estimate the enrichment degree of plasma membranes, we detected the plasma membrane-specific protein flotillin-1 and finally found that the protein was enriched about 25-fold in plasma membrane fraction than in liver homogenate (Figure 2A). As is well-known, the plasma membranes prepared by sucrose density gradient centrifugation are typically heavily contaminated by mitochondria and endoplasmic reticulum

research articles (ER) due to the overlap of their density. In the present work, Western blotting analysis showed that both mitochondria and ER were about 2-fold less in plasma membrane fraction than in liver homogenate (Figure 2B, 2C). Furthermore, we also evaluated the contamination of Golgi and found it was effectively decreased in plasma membrane fraction (about 5-fold less than in liver homogenate) (Figure 2D). These results indicate that our plasma membranes are significantly enriched and the major contaminants were mitochondria and ER, although they were also decreased to a certain extent. Evaluation of the Membrane Skeleton Fraction by Western Blotting Analysis. By Western blotting analysis, we found that transmembrane proteins (Na+/K+ ATPase) that were not associated with membrane skeletons were almost extracted into the supernatant by TEB (Figure 3A). At the same time, membrane skeleton proteins (for example, beta actin) and proteins in lipid raft (for example, flotillin-1) were greatly enriched in the detergent-resistant pellet (Figure 3B and 3C). The resistance of the membrane skeletons to Triton X-100 was conferred by the interactions between cytoskeletal proteins. Consequently, these interconnected proteins co-pelleted as Triton-insoluble assembly. Meanwhile, lipid rafts could be also resistant against disruption by Triton X-100, as shown in Figure 3C. Protein Extraction and Separation. Protein extraction is very important to get good 2-DE results; therefore, we have to determine the optimal solubilization procedure for our membrane skeleton fraction. The solubilization buffer is usually composed of 7 M urea, 2 M thiourea, 65 mM DTT, and a kind of zwitterionic or nonionic detergent. It is absolutely essential to optimize the detergent. In the present work, we evaluated the solubilization power of different detergents using three high-abundance membrane skeleton proteins (alpha-fodrin, alpha-actinin, and nonmuscle myosin heavy chain IIa (MYH9)). Two zwitterionic detergents (CHAPS and ASB-14) and one nonionic detergent (Triton X-100) were compared. As shown in Figure 4, 2% ASB-14 allowed the best recovery of these skeleton proteins. This was in agreement with previous observations for the solubilization of membrane proteins.23,24 Surprisingly, CHAPS, which was the most commonly used detergent, had the worst solubilization power for skeleton proteins. This was probably caused by the high molecular weight and low solubility of these proteins. Finally, 2% ASB-14 was selected in this work. Proteins were separated within a pH 3-10 IPG strip and a 10% SDS polyacrylamide gel. The 2-D gel analysis of the sample was repeated three times, and the match rate of protein spots was higher than 90%. Figure 5 shows a representative gel. After staining with “blue silver”, approximately 250 spots per gel were detected, wherein only about 30 spots with high abundance while most of the other spots were rather weak. Compared with a previous work25 about proteomic study of human liver plasma membranes, the number of spots detected on the 2-D gels was drastically reduced after TEB extraction, especially the high-abundance spots. This indicates that the protein complexity in our membrane skeleton fraction is obviously reduced. Protein Identification. Protein spots were mainly analyzed by PMF using MALDI-TOF MS after tryptic in gel digestion. The MS spectra were automatically searched against Swiss-Prot or IPI database using the Mascot search engine. Due to its excellent annotation, Swiss-Prot was mainly considered, and IPI was used to verify and complement the protein identifications by the former database. Proteins with Mascot scores g64 Journal of Proteome Research • Vol. 6, No. 9, 2007 3511

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Figure 2. Western blotting analysis of the plasma membranes prepared by subcellular fractionation. Thirty-six micrograms of the liver homogenate and plasma membrane proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. The blots were probed with antibodies against organellespecific proteins: (A) anti-Flotillin-1 for plasma membranes (PM); (B) anti-Oxphos complex I 39 kDa subunit (Oxphos C1 39S) for mitochondria; (C) anti-KDEL for ER; (D) anti-Golgi 97 for Golgi. LH represents liver homogenate; PM represents plasma membranes; MC represents mitochondria.

Figure 3. Western blotting analysis of detergent extraction. The plasma membrane preparation was extracted by Triton extraction buffer (TEB). Na+/K+ ATPase, a plasma membrane marker protein with multiple transmembrane domains, was almost extracted into the supernatant (A). At the same time, beta actin, the major membrane skeleton protein, was greatly enriched in the detergentresistant pellet (B). Meanwhile, flotillin-1, a marker protein of lipid raft, was also enriched in the pellet (C).

Figure 1. Electron micrographs of human liver homogenate and the isolated plasma membranes. (A) Liver homogenate. Nuclei, mitochondria, membrane sheets, and vesicles could be clearly observed. Bar, 2 µm × 5000. (B) Plasma membranes. The predominant structures in this fraction were membrane sheets, and an occasional mitochondrion could also be seen. Bar, 2 µm × 5000. (C) Plasma membranes with higher magnification. Bar, 500 nm × 20 000. 3512

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or g69 (P < 0.005) for Swiss-Prot or IPI database respectively were considered as high-confidence proteins. As a result, 104 high-confidence proteins were identified, corresponding to 220 spots. We labeled the 104 proteins on the 2-D gel and summarized in Supporting Information Figure S1. The detailed information of these proteins was listed in Supporting Information Table S1. Thirty-eight of the 104 proteins were cytoskeletal proteins according to Swiss-Prot annotation or literature, corresponding to 94 spots. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. The detailed information of the 38 cytoskeletal proteins was listed in Table 1, and their distribution on the 2-D gel was shown in Figure 5. Obviously, most of the high-abundance proteins on the gel were skeleton proteins, which suggested that our membrane skeleton fraction was significantly pure. All of the cytoskeletal proteins were further confirmed by MALDI-TOFTOF MS/MS analysis of selected peptides in “LIFT” mode. Consequently, 33 of the 38 proteins had at least one peptide identified. The sequences of these peptides were summarized in Supporting Information Table S2. An example of PMF spectrum was shown in Figure 6A. This protein was identified

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Figure 4. Optimization of detergents used for the solubilization of human liver membrane skeleton proteins. Proteins (300 µg) were separated on pH 3-10 IPG strips, followed by 10% SDS polyacrylamide gels. Alpha-fodrin, alpha-actinin, and nonmuscle myosin heavy chain IIa (MYH9) are indicated by arrows. The proteins were solubilized and focused in a solution containing 7 M urea, 2 M thiourea, 65 mM DTT, 0.5% ampholytes, and (A) 4% CHAPS; (B) 2% ASB-14; (C) 2% Triton X-100.

all of which were associated with actin or cytokeratin. These proteins were classified into several groups as shown in Table 2. The interconnected proteins actin and fodrin were two of the most abundant proteins, which suggests that the liver cells may contain an actin- and fodrin-based meshworks just as the previous observations with neutrophils.15,26 Actin filaments are often nucleated at the plasma membrane. Consequently, the highest density of actin filaments in most cells is in the cortex, just beneath the plasma membrane. The presence of an actincontaining filamentous network at the rat hepatocytes cortex has been verified by immunofluorescent light microscopy.27 Fodrin is an actin-binding protein which was first isolated from the particulate fraction of brain.28 The protein was localized by immunofluorescence in the cortical cytoplasm of cultured fibroblasts, neurons, and intestinal epithelial cells. Bennett et al.7 named the protein brain spectrin and reported its presence in liver plasma membrane. The interconnected actin filaments and fodrin may form a meshwork adjacent to the plasma membrane, determining the shape of the cell surface.

Figure 5. Distribution of the cytoskeletal proteins on the 2-D gel. The first dimension is a pH 3-10 linear IPG strip, and the second dimension is a 10% SDS polyacrylamide gel. The proteins were solubilized and focused in a solution containing 7 M urea, 2 M thiourea, 2% ASB-14, 65 mM DTT, and 0.5% ampholytes. 300 µg proteins were loaded on the 2-D gel. All of the 38 skeleton proteins (see Table 1) identified from the human liver membrane skeleton fraction were annotated on the gel.

as beta-actin by PMF, and the sequence tags yielded from the MS/MS spectra of the two peptide ions (m/z 1515.779; Figure 6B and m/z 1790.920; Figure 6C) strongly supported this result. The combined database search using PMF and Lift MS/MS data resulted in a MOWSE score of 293, which was much higher than the PMF score (MOWSE score 129). Apparently, the MS/ MS data made the protein identification more confident. Classification of Membrane Skeleton Proteins and their Biological Roles. The most striking result of the present work was the identification of so many cytoskeletal proteins, almost

A big group of the membrane skeleton proteins identified in the present work were adhesion proteins, including those involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes, and tight junctions. These proteins play important roles in determining the shape of the cell and in signal transduction pathways. Adherens junctions connect bundles of actin filaments from cell to cell. We identified some proteins that formed adherens junctions, including actin, R-actinin, R-catenin, β-catenin, plakoglobin (also called γ-catenin), p120 catenin (also called catenin δ-1), and vinculin (see Table 2). R-Actinin cross-links actin filaments, and it is phosphorylated on its actin-binding domain by the focal adhesion kinase.29 β-Catenin and plakoglobin interact directly with cell adhesion molecules cadherins,14,30,31 and they constitute a bridge between cadherins and alpha-catenin. A similar protein p120 catenin, which was originally identified as a substrate of several receptor tyrosine kinases, is bound to a small proportion of E-cadherin.32,33 Vinculin is a component of adhesion plaques and adherens junctions in nonmuscle cells. It exhibits sequence homologies to R-actinin and binds directly to actin filament34 and R-actinin.35 Journal of Proteome Research • Vol. 6, No. 9, 2007 3513

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Table 1. Cytoskeletal Proteins Identified from the Membrane Skeleton Fraction of Human Liver 2D No.a

accession no.b

protein name

scorec

PepCountd

MW

pI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

PLEC1_HUMAN DESP_HUMAN SPTA2_HUMAN FLNB_HUMAN SPTB2_HUMAN MYH14_HUMAN MYH9_HUMAN IPI00307829 CING_HUMAN VINC_HUMAN DSG2_HUMAN CO6A1_HUMAN CTND1_HUMAN ACTN4_HUMAN ACTN1_HUMAN DSC2_HUMAN CTN1_HUMAN CTNB1_HUMAN GELS_HUMAN PLAK_HUMAN LAMA_HUMAN LAM1_HUMAN COR1C_HUMAN K2C8_HUMAN TBA1_HUMAN TBB2_HUMAN K1C18_HUMAN ARP3_HUMAN ARP2_HUMAN ACTG_HUMAN ACTB_HUMAN ARPC2_HUMAN CAZA1_HUMAN CAZA2_HUMAN CAPZB_HUMAN MLRM_HUMAN HSPB1_HUMAN SRBS1_HUMAN

Plectin 1 Desmoplakin Alpha-fodrin Beta-filamin Beta-fodrin Nonmuscle myosin heavy chain IIc Nonmuscle myosin heavy chain IIa Cingulin-like 1 Cingulin Vinculin Desmoglein-2 precursor Collagen alpha 1(VI) chain precursor Catenin delta-1 (p120 catenin) Non-muscle alpha-actinin 4 Non-muscle alpha-actinin-1 Desmocollin-2 precursor Alpha-1 catenin Beta-catenin Gelsolin precursor Junction plakoglobin Lamin A/C Lamin B1 Coronin-1C Type II cytoskeletal 8 Tubulin alpha-1 chain Tubulin beta-2 chain Type I cytoskeletal 18 Actin-related protein 3 Actin-related protein 2 Gamma-actin Beta-actin ARP2/3 complex 34 kDa subunit F-actin capping protein alpha-1 subunit F-actin capping protein alpha-2 subunit F-actin capping protein beta subunit Nonsarcomeric myosin RLC Heat shock 27 kDa protein(HSP 27) CAP/ponsin; SH3P12

378 438 535 74 357 112 178 207 204 144 73 186 221 206 389 360 143 336 84 596 162 218 132 389 511 537 411 182 346 318 293 194 381 360 217 130 189 220

3 2 4 0 3 1 2 0 0 1 1 3 3 1 4 4 1 4 0 5 1 1 1 3 5 5 4 1 3 3 2 1 3 3 2 1 0 2

531733 331774 274539 278195 274609 228002 226532 149079 136386 123799 122385 108529 108170 104854 103058 99962 100071 85497 85698 81630 74139 66408 53249 53704 49924 49671 48058 47371 44761 41793 41737 34333 32923 32949 31350 19794 22783 142455

5.73 6.44 5.22 5.49 5.41 5.76 5.5 5.51 5.46 5.51 5.15 5.29 5.86 5.27 5.25 5.91 5.95 5.53 5.9 5.95 6.57 5.11 6.65 5.52 4.95 4.78 5.34 5.61 6.3 5.31 5.29 6.84 5.45 5.58 5.36 4.67 5.98 6.4

a Protein numbers on the 2-D gel (see Figure 5). b Swiss-Prot or IPI accession numbers. c MOWSE scores obtained by the combined search (PMF and LIFT data) using Mascot engine. For those proteins without LIFT data, the scores represent MOWSE scores by PMF search. d Numbers of peptides identified by MALDI-TOF-TOF MS/MS using the Ultraflex instrument in “LIFT” mode.

Focal adhesions connect cells to the extracellular matrix through integrins that link intracellularly to actin filaments. Among those proteins identified in this work, actin, R-actinin, vinculin, filamin B and collagen R1 (VI) chain precursor belong to focal adhesions.(see Table 2) Integrin can be linked to actin filaments via R-actinin, filamin B, and an unidentified protein talin.36 Vinculin may cross-link actin and talin, whereas filamin B stabilizes actin webs and links actin filaments to various transmembrane proteins.37 Collagen VI is a ubiquitous component of extracellular matrix. Desmosomes connect intermediate filaments from cell to cell. The particular type of intermediate filaments depends on the cell type, and they are keratin filaments in most epithelial cells. We identified most of the proteins involved in desmosomes, including cytokeratin 8, cytokeratin 18, plakoglobin, demoplakin, desmoglein-2, and desmocollin-2 (see Table 2). Cytokeratin 8 belongs to type II keratin, and it associates to cytokeratin 18 which belongs to type I keratin. Plakoglobin, which is a component of adherens junctions as stated above, is also present in desmosomes. It associates with two desmosomal cadherins desmoglein38 and desmocollin.39 Desmoplakins are the most abundant proteins in the innermost portion of the desmosomal plaque and connect intermediate filaments to desmosomes.40 Hemidesmosomes anchor a variety of epithelial cells to extracellular matrix through integrins that link intracellularly 3514

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to intermediate filaments. We identified some proteins in hemidesmosomes, including cytokeratin 8, cytokeratin 18, and plectin 1. Plectin is a versatile cytoskeletal linker. It attaches intermediate filaments bundles to hemidesmosomes by direct interaction with integrin beta4 subunit,41 as well as links intermediate filaments to microtubules, actin filament bundles, and filaments of the motor protein myosin II.42 Tight junctions are belt-like regions of contact between epithelial cells, and serve as a selective permeability barrier across these cells. Among the proteins identified in the present work, actin, cingulin, and cingulin-like 1 were molecules of tight junctions (see Table 2). Cingulin is an acidic, heat-stable protein, with a highly elongated shape, and it is localized at the surfaces of tight junctions by immunofluorescence and immunoelectron microscopy analysis.43 Actin filaments have a similar distribution to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton of the tight junctions.44 Cingulin-like 1 is a novel protein involved in anchoring tight junctions to actin-based cytoskeletons. Using immunoelectron microscopy, it was found to be localized to the undercoat of tight junctions in liver.11 The adhesion proteins mentioned above play important roles not only in determining the cell shape and tissue integrity, but also in some signal transduction pathways.14 The signals transduced by these adhesion proteins may essentially influence the cell development and be involved in some diseases.

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Figure 6. Mass spectra of beta-actin. (A) Peptide mass fingerprint after tryptic digestion. (B) Lift MS/MS spectrum of ion 1515.779. (C) Lift MS/MS spectrum of ion 1790.920. In the two MS/MS spectra, the y ions and the corresponding peptide sequence are shown.

Therefore, the study of the signals mediated by the adhesion proteins is promising, and there may be some great breakthrough in the near future. Another important group of the membrane skeleton proteins identified in this work were those proteins that regulate F-actin dynamics (see Table 2). Actin filaments are dynamic in cells, and the kinetic of their assembly and disassembly can be either slowed or accelerated by accessory proteins. Among the proteins identified in the present work, actin-related protein 2, actin-related protein 3, and ARP2/3 complex 34kDa subunit are components of ARP2/3 complex, which nucleates actin filaments at the plasma membrane.45 Alpha-1, alpha-2, and beta subunits of F-actin capping protein (CapZ) were also identified. They can cap most of the actin filaments at their plus end, which thus slows the filaments growth rate by making the plus end inactive.46,47 Gelsolin is an actin-severing protein which can prevent actin capping, promote actin filaments nucleation, and sever filaments already formed.48,49 Another F-actin dynamics regulation protein identified in the work was heat shock 27 kDa protein (HSP 27), which acts as an inhibitor of actin polymerization.50 Another group of skeleton proteins were motor proteins. The actin-based motor proteins belong to myosin superfamily. In the present work, we identified nonmuscle myosin heavy chain IIa, IIc, and myosin regulatory light chain, all of which are associated with contractile activity in nonmuscle cells.

We also identified some other cytoskeletal proteins, including Coronin-1C, CAP/ponsin, tubulin, and lamin. Coronin-1C is an actin-binding protein which is mainly found at the submembraneous area. F-actin interaction and membrane association of Coronin-1C are mediated by its carboxyl terminus.51 CAP/ponsin contains a conserved region homologous to the active peptide sorbin, as well as three SH3 domains. It colocalizes with actin filaments and involves in the formation of actin stress fibers and focal adhesions.52 This protein is part of the SHIP2 complex53 that binds filamin and regulates submembraneous actin.54 CAP/ponsin is also a protein kinase A anchoring protein, putting it at the cross-roads for regulating cytoskeletal structure in several ways.55 Tubulin is a major constituent of microtubules. It may be introduced into the membrane skeleton fraction by plectin, which links intermediate filaments to microtubules. Lamin is a component of nuclear lamina, which may be introduced by its interaction with cytokeratins.56 This protein has also been identified in some previous proteomic study of Triton-extracted plasma membranes.17,18 Besides those cytoskeletal proteins, we also identified 66 other proteins, including some plasma membrane marker proteins, such as flotillin-1, flotillin-2, 5′-nucleotidase, and so on. Most of these proteins were corresponding to weak spots on 2-D gels. However, there were also four high-abundance mitochondrial proteins, including carbamoyl-phosphate synJournal of Proteome Research • Vol. 6, No. 9, 2007 3515

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Table 2. Classification of the Cytoskeletal Proteins Identified from the Membrane Skeleton Fraction of Human Liver Classification

Fodrin-based meshworks

Adherens junction

Protein Name

Beta-actin Gamma-actin Alpha-fodrin Beta-fodrin Beta-actin Gamma-actin Non-muscle alpha-actinin-1 Non-muscle alpha-actinin 4 Alpha-1 catenin Beta-catenin Junction plakoglobin Catenin delta-1 (p120 catenin)

Vinculin

Focal adhesion

Desmosomes

Hemidesmosome

Tight junction

F-actin dynamics

Motor proteins

Other cytoskeletal proteins

Beta-actin Gamma-actin Non-muscle alpha-actinin-1 Non-muscle alpha-actinin 4 Vinculin Beta-filamin Collagen alpha 1(VI) chain precursor Type II cytoskeletal 8 Type I cytoskeletal 18 Junction plakoglobin Desmoplakin Desmoglein-2 precursor Desmocollin-2 precursor Type II cytoskeletal 8 Type I cytoskeletal 18 Plectin 1 Collagen alpha 1(VI) chain precursor Beta-actin Gamma-actin Cingulin Cingulin-like 1 Actin-related protein 2 Actin-related protein 3 ARP2/3 complex 34 kDa subunit F-actin capping protein alpha-1 subunit F-actin capping protein alpha-2 subunit F-actin capping protein beta subunit Gelsolin precursor Heat shock 27 kDa protein(HSP 27) Nonmuscle myosin heavy chain IIa Nonmuscle myosin heavy chain IIc Nonsarcomeric myosin RLC Coronin-1C Tubulin alpha-1 chain Tubulin beta-2 chain Lamin A/C Lamin B1 CAP/ponsin; SH3P12

Description

Beta- and Gamma- actin are found together in almost all nonmuscle cells. The actin filaments in the layer underlying the plasma membrane called the cell cortex Fodrin is an F-actin binding protein, and may form a two-dimensional meshwork adjacent to the membrane See above F-actin cross-linking proteins which are thought to anchor actin to a variety of intracellular structures Found at cell-cell boundaries and probably at cell-matrix boundaries Involved in the regulation of cell adhesion and in the Wnt signaling pathway It is gamma-catenin and presents in adherens junctions and desmosomes Efficient tyrosine kinase substrate in ligand-induced receptor signaling through the EGF, PDGF, CSF-1, and ERBB2 receptors. It associates with E-cadherin/Catenin complex A component of adhesion plaques and adherence junctions in nonmuscle cells See above See above See above Stabilizes three-dimensional actin webs and connects them to cell membrane Biological process: cell adhesion Anchor to the plasma membrane at desmosomes and hemidesmosomes. Keratin 18 associates with keratin 8. See above Involved in the anchoring of intermediate filaments to the desmosomes Involved in the interaction of plaque proteins and intermediate filaments mediating cellscell adhesion See above Interlinks intermediate filaments with microtubules and microfilaments and anchors intermediate filaments to desmosomes or hemidesmosomes See above See above Plays a role in the formation and regulation of tight junctions It was found to be localized to the undercoat of tight junctions in the liver Components of ARP2/3 complex which catalyzes the nucleation of actin filaments Slows the rate of both filament growth and filament depolymerization

Promotes actin filaments nucleation as well as severs filaments already formed Actin polymerization modulator Associates with contractile activity in nonmuscle cell Regulates nonmuscle cell contractile activity Actin-associated protein Major constituent of microtubules. It may be introduced into the membrane skeleton fraction by plectin Components of the nuclear lamina. Interact with cytokeratins Colocalizes with actin filaments and involves in the formation of actin stress fibers and focal adhesions

thase I (CPS1), pyruvate carboxylase, elongation factor Tu and agmatinase, wherein CPS1 was especially abundant. This indicates that mitochondria are the major contaminants of our membrane skeleton fraction. In a previous work, 2-DE was used to separate proteins from cytoskeletal compartments of rat hepatocytes, and immunoblotting with over 20 different antibodies was used to characterize the distribution profiles of cytoskeletal proteins, resulting 3516

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in the identification of 8 cytoskeletal proteins in 2-D gels.4 Considering immunoblotting is a low-throughput and timeconsuming method to identify proteins, more cytoskeletal proteins might be identified from rat hepatocytes with a highthroughput identification method. In a recent research, SDSPAGE was applied to separate the proteins from a detergentresistant membrane skeleton of neutrophil, and 11 cytoskeletal proteins were identified by MALDI-TOF MS.15 Although mass

Membrane Skeleton Proteomics

spectrometry is a high-throughput identification method, the resolution of SDS-PAGE is not high enough to separate every protein. In our present work, proteins were separated by highresolving 2-D gels and identified by high-throughput mass spectrometry, resulting in a comprehensive proteome map of membrane skeletons from human liver. 2-D gels can resolve most of the cytoskeletal proteins and provide molecular weight and pI information. However, they exclude most of integral membrane proteins. In this work, two integral membrane skeleton proteins integrin and cadherin were not identified from the 2-D gels. To solve this problem, the coupling of liquid chromatography with tandem mass spectrometry may be a choice.

Conclusions We prepared plasma membranes from human normal liver by subcellular fractionation. Electron microscopy and Western blotting analysis showed that plasma membrane was greatly enriched. Then the plasma membranes were extracted by a solution containing Triton X-100 to produce a membrane skeleton fraction. Western blotting was used to verify the enrichment of skeleton proteins. After getting an ideal membrane skeleton fraction, we separated its protein components by 2-D gels. We optimized the detergent used for the solubilization of skeleton proteins and finally selected 2% ASB-14. After analyzing the protein spots by MALDI-TOF and MALDITOF-TOF MS, 104 nonredundant proteins were identified, 38 of which were cytoskeletal proteins. These membrane skeleton proteins were classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes, and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. Taken together, our results revealed that the liver cells had actin- and cytokeratin-based membrane skeletons. This work is supposed to provide a comprehensive proteome map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons which are now largely uncharacterized.

Acknowledgment. We thank Prof. Songping Liang for the help in plasma membrane preparation. Human normal liver tissues were provided by Chinese HLPP. This work was supported by grants from National Basic Research Program of China (No. 2006CB910103, 2001CB510207) and National High Technology Research and Development Program of China (No. 2004BA711A18). Supporting Information Available: Distribution of the 104 proteins on the 2-D gel (Figure S1). Detailed information of the 104 proteins identified from the 2-D gels (Table S1). Sequenced peptides of the cytoskeletal proteins (Table S2). This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Therien, H. M.; Gruda, J.; Carrier, F. Interaction of filamentous actin with isolated liver plasma membranes. Eur. J. Cell Biol. 1984, 35, 112-121. (2) Tsukita, S. Isolation of cell-to-cell adherens junctions from rat liver. J. Cell Biol. 1989, 108, 31-41. (3) Baffet, G.; Loyer, P.; Glaise, D.; Corlu, A.; Etienne, P. L.; GuguenGuillouzo, C. Distinct effects of cell-cell communication and corticosteroids on the synthesis and distribution of cytokeratins in cultured rat hepatocytes. J. Cell Sci. 1991, 99, 609-615.

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