Targeted Identification of Metastasis-associated Cell-surface Sialoglycoproteins in Prostate Cancer
2011; Elsevier BV; Volume: 10; Issue: 6 Linguagem: Inglês
10.1074/mcp.m110.007294
ISSN1535-9484
AutoresLifang Yang, Julius O. Nyalwidhe, Siqi Guo, Richard R. Drake, O. John Semmes,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoCovalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells. Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells. Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications with more than 50% of eukaryotic proteins thought to be glycosylated (1Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim. Biophys. 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Larsen et al. took advantage of the high affinity of titanium dioxide microcolumns toward SA residues to isolate SA-containing peptides from serum under highly acidic conditions (25Larsen M.R. Jensen S.S. Jakobsen L.A. Heegaard N.H. Exploring the sialiome using titanium dioxide chromatography and mass spectrometry.Mol. Cell Proteomics. 2007; 6: 1778-1787Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Two similar approaches, involving hydrazide and boronic acid chemistry, capitalize on the cis-diols present in monosaccharides. Zhang et al. described the use of hydrazide chemistry for purification by directly coupling of glycoproteins to a solid support (26Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1266) Google Scholar, 27Zhang H. Yi E.C. Li X.J. Mallick P. Kelly-Spratt K.S. 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In this study, we describe a glycoproteomic identification strategy for the selective detection, isolation and identification of cell-surface sialoglycoproteins from cultured cell lines. The method utilizes the sialic acid biosynthetic pathway for the incorporation of monosaccharide bearing bioorthogonal functional handles (tetraacetylated N-azidoacetyl-d- mannosamine) into cellular sialic acid (29Laughlin S.T. Agard N.J. Baskin J.M. Carrico I.S. Chang P.V. Ganguli A.S. Hangauer M.J. Lo A. Prescher J.A. Bertozzi C.R. Metabolic labeling of glycans with azido sugars for visualization and glycoproteomics.Methods Enzymol. 2006; 415: 230-250Crossref PubMed Scopus (107) Google Scholar, 30Laughlin S.T. Bertozzi C.R. Imaging the glycome.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12-17Crossref PubMed Scopus (254) Google Scholar, 31Prescher J.A. Bertozzi C.R. Chemistry in living systems.Nat. Chem. Biol. 2005; 1: 13-21Crossref PubMed Scopus (1093) Google Scholar). These reagents have previously been used to label and visualize cell surface expression of glycoproteins via microscopy. To illustrate the potential of using this cell labeling procedure in biomarker discovery, we combined it with an MS-based proteomics approach as applied to a syngeneic metastatic prostate cancer cell line model. CompleteTM protease inhibitors were purchased from Roche Applied Sciences (Indianapolis, IN), sequencing grade trypsin was from Promega (Madison, WI), and Immobilon-FL PDVF membrane was from Millipore (Billerica, MA). Protein-free blocking buffer and high capacity streptavidin agarose resin was from Thermo Scientific (Rockford, IL). Dulbecco's modified Eagle media (DMEM) and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). Antibiotics-antimycotic, Click-iTTM ManNAz metabolic glycoprotein reagent, Click-iT Biotin Protein Analysis Detection Kit, propidium iodide, fluorescein conjugated streptavidin (streptavidin-FITC), 4–12% NuPAGE® Bis-Tris gels, and lithium dodecyl sulfate buffer were from Invitrogen (Carlsbad, CA). 2× Laemmli buffer, 7.5% Criterion® Tris-HCl Gel, and Bio-Safe Coomassie blue were from Bio-Rad (Hercules, CA). The streptavidin-IR 800 and species-specific secondary antibodies conjugated to IR 800 or IR700 were from Li-COR Biosciences (Lincoln, PA). PC3-N2 and PC3-ML2, two sublines of PC3 prostate cancer cells, were kindly provided by Dr. Mark Stearns (Drexel University). The PC3 cell line was originally derived from a skeletal metastasis in a patient with primary prostate adenocarcinoma. The N2 and ML2 cell lines have been developed on the basis of their invasiveness in vitro and metastatic potential in vivo. Both cells were tumorigenic when injected subcutaneously in Severe combined immunodeficiency mice. However, N2 cells were unable to migrate through a Matrigel-coated membrane in vitro as well as induce metastases in severe combined immunodeficiency mice, whereas ML2 cells were highly invasive in vitro and induced skeletal metastases in more than 80% of mice (32Wang M. Stearns M.E. Isolation and characterization of PC-3 human prostatic tumor sublines which preferentially metastasize to select organs in S.C.I.D. mice.Differentiation. 1991; 48: 115-125Crossref PubMed Scopus (111) Google Scholar). N2 and ML2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics at 37 °C with 5% CO2. For metabolic labeling, growth medium was replaced at 70% cell confluence with complete DMEM medium containing the indicated concentration of an azido-modified sugar, tetraacetylated N-azidoacetyl-d- mannosamine (AC4ManNAz), or a control sugar, N-acetyl-d-mannosamine (ManNAc), and cells were incubated for 1–3 days. Cells then were dissociated from the plastic surface by nonenzyme dissociation buffer. After metabolic labeling, N2 and ML2 cells were harvested, washed with 0.1% FBS/phosphate-buffered saline (PBS), and resuspended (106 cells) in 100 μl click reaction solution with the indicated amount of each component. The reaction was incubated at room temperature for 30 min, and then cells were washed three times with 0.1% FBS/PBS. Cells were subsequently stained with streptavidin-FITC (1 μg/sample in 100 μl 2% FBS/PBS) for 30 min at 4 °C, and washed with 2% FBS/PBS three times. Prior to flow cytometric analysis, cells were incubated with 1 μg/ml propidium iodide in 500 μl 2% FBS/PBS at 4 °C for 20 min. Data was acquired by FACScalibur (BD Biosciences, San Jose, CA) and analyzed by Flowjo software (Tree Star Inc., Ashland, OR). N2 and ML2 cells were seeded onto glass coverslips in 6-well plates containing 10% FBS/DMEM. Growth medium was supplemented with 40 μm AC4ManNAz or ManNAc for 3 days. Cells were washed with ice-cold PBS, fixed with 2% paraformaldehyde, and then subjected to click reaction solution (25 μl biotin-alkyne, 2.5 μl CuSO4, 2.5 μl and 5 μl for component D and E, 65 μl PBS). Subsequently, the fixed and labeled cells were washed with PBS and stained with streptavidin-FITC and the nuclei were counterstained with propidium iodide. The coverslips were inverted onto glass slides and mounted with VectorShield medium (Vector Labs, Burlingame, CA), and sealed with nail polish. Fluorescent images were examined and captured with a Zeiss confocal microscope. N2 and ML2 cells were seeded in 15-cm dishes and treated with the optimized labeling conditions for AC4ManNAz and ManNAc (20 μm for 1 day) in growth medium. The cells were then harvested with nonenzymatic dissociation buffer, 5 × 107 cells were collected and washed with 0.1% FBS/PBS. Harvested cells were subjected to click reaction solution as described above. After chemical conjugation, cell pellets were washed with ice-cold PBS twice to remove unreacted reagents and then lysed in lysis buffer I (1% Nonidet P-40, 150 mm NaCl, protease inhibitor, 100 mm sodium phosphate, pH7.5) using a Dounce homogenizer. The total cell lysate was further cleared by methanol-chloroform precipitation and resolved in lysis buffer II (1% SDS, protease inhibitor, 50 mm Tris-HCl, pH 8.0). Protein concentrations were measured using the BCA protein assay (Pierce). To detect biotin-labeled sialoglycoproteins in cell extracts, 20 μg of labeled protein lysate was resolved by SDS-PAGE. Electrophoresed proteins were transferred onto PVDF membrane, blocked with Odyssey Blocking Buffer (Rockland Immunochemicals, Gilbertsville, PA), probed with streptavidin-IR 800, and visualized and quantified using an Odyssey infrared imaging system. Streptavidin beads were pretreated with protein-free blocking buffer overnight at 4 °C and washed five times with PBS. Cell lysate (2 mg) was incubated with 100 μl beads in 0.3% Nonidet P-40/PBS overnight at 4 °C with a rotating shaker. The captured glycoproteins were washed intensively with modified RIPA buffer (150 mm NaCl, 2% SDS, 1% Nonidet P-40, 1% Na Deoxycholate, 50 mm Tris-HCl, pH 7.5). Bound material was eluted by boiling for 10 min in 100 μl 2× Laemmli sample buffer. For assessment of capture efficiency, 10 μl of the eluent, along with the input and flow-through fractions were separated by SDS-PAGE and probed with streptavidin-IR 800 by Western blot. Captured glycoproteins (45 μl) were separated on a 1.0 mm 7.5% Tris-HCl polyacrylamide gels. Gels were stained with Coomassie blue and imaged on a Typhoon 9410 (GE Healthcare, Piscataway, NJ). Twenty-seven equally spaced gel pieces were excised from each lane, spanning the full height of the gel (30–300 kDa). Individual gel pieces were destained with 25 mm NH4HCO3 in 50% acetonitrile (ACN), reduced with 20 mm dithiothreitol in 25 mm NH4HCO3 for 45 min at 56 °C, and alkylated with 55 mm iodoacetamide in 25 mm NH4HCO3 in the dark for 1 h at room temperature. After washing, the gel pieces were dehydrated with ACN and dried using a speed vac. Trypsin (12.5 ng/μl in 25 mm NH4HCO3, pH 8.0) was added to each gel piece, and the gel pieces were allowed to swell on ice for 1 h. Excess trypsin was removed, replaced with 25 mm NH4HCO3, 10% ACN, pH 8.0, and the mixture was incubated overnight at 37 °C. The digest was collected and peptides were extracted 2X with 50% ACN/0.5% formic acid. The combined extracts were then dried using a SpeedVac for subsequent LC-MS/MS analysis. For N2 and ML2 cell lysate experiments; both cell lines were cultured in complete growth medium to 80% confluence. Cells were then washed with PBS and the pellet resuspended in 160 μl dissolution buffer containing 100 mm NH4HCO3 and TFE (1:1 v/v). The samples were sonicated 3× for 20 s and incubated at 60 °C for 1 h. The lysates were centrifuged to remove cell debris and unbroken cells and the supernatant was collected. The protein concentration of the samples was determined by BCA assay and normalized for each sample and before reduction and alkylation with TCEP and iodoacetamide respectively. The concentration of TFE was reduced to 5% by the addition of 1.4 ml 100 mm NH4HCO3. Trypsin was added at a ratio of 1:50 protease to protein and the digestion proceeded at 37 °C for 18 h with constant mixing. After digestion the sample was dried down in a SpeedVac, desalted and lyophilized before liquid chromatography-tandem MS (LC-MS/MS) analysis. Digests were resuspended in 20 μl Buffer A (5% ACN, 0.1% formic acid, 0.005% heptafluorobutyric acid) and 15 μl loaded onto a 12-cm × 0.075 mm fused silica capillary column packed with 5 μm diameter C-18 beads (The Nest Group, Southborough, MA) using a programmed automatic injection. Peptides were eluted over 80 min, by applying a 0–80% linear gradient of Buffer B (95% acetonitrile, 0.1% formic acid, 0.005% HFBA) at a flow rate of 200 μl/min with a precolumn flow splitter resulting in a final flow rate of ∼300 nl/min directly into the source. In some cases, the gradient was extended to 150 min to acquire more MS/MS spectra. An LTQ™ Linear Ion Trap (ThermoFinnigan, San Jose, CA) was run in an automated collection mode with an instrument method composed of a single segment and five data-dependent scan events with a full MS scan followed by four MS/MS scans of the highest intensity ions. Normalized collision energy was set at 35, activation Q was 0.250 with minimum full scan signal intensity at 1 × 105 with no minimum MS2 intensity specified. Dynamic exclusion was turned on utilizing a three-minute repeat count of 2 with the mass width set at 1.0 m/z. Peak lists were generated using Xcaliber (version 2.1). Sequence analysis was performed with MASCOT (version 2.2.03) using the SwissProt 57.1 database with a human taxonomy filter enabled that contained 20,405 entires. The database searches were performed with fixed modification as carbamidomethyl (Cys) and variable modifications as oxidation (Met) and deamidation (Asn, Gln). Enzyme specificity was selected to trypsin with 1 missed cleavage. The mass tolerance was set at 1 for precursor ions and 0.8 for fragment ions. Threshold score for acceptance of individual spectra was set at 0.05. All the MS/MS spectra were manually inspected to verify the validity of the database search results. False discovery rates were estimated to be 0.25% on the protein level by searching a decoy version of the SwissProt protein database. The relative abundance of peptides was estimated by spectral counting. Enymatic deglycosylation of samples was performed prior to analysis by western. For PNGase F digestions, cell lysates (20 μg) were denatured and reduced for 10 min at 100 °C in 0.5% SDS and 1% 2-mercaptoethanol. The samples were then adjusted to 1% Nonidet P 40 and 50 mm sodium phosphate, pH 7.5, and incubated with PNGase F (1,000 NEB units) overnight at 37 °C. The experiments using neuraminidase reactions were adjusted to 50 mm sodium acetate, pH 6.0 prior to incubation overnight at 37 °C with 50 NEB units neuraminidase per 20 μg of protein. Deglycosylation digestion of Fetuin was used to optimize conditions (data not shown). Whole-cell lysates were collected in M-PER lysis buffer containing 1x protease inhibitor mixture. The protein concentration was determined by the BCA protein assay. Cell lysates, streptavidin pull-down fractions and deglycosylated protein samples were separated by electrophoresis through 4–12% or 7.5% SDS-PAGE and then transferred to Immobilon-FL PDVF membranes. Membranes were blocked in LiCor blocking buffer (Rockland Immunochemicals, Gilbertsville, PA) diluted with PBS (1:1), then incubated with anti-CDCP1 polyclonal (#4115, 1:1000, Cell Signaling Technology, Danvers, MA), or anti-integrin β1 mouse monoclonal (#610467, 0.1 μg/ml, BD PharMingen, San Diego, CA) primary antibodies overnight at 4 °C. After 5× washes in PBS, membranes were incubated with species-specific (goat anti-rabbit, 1:15,000; goat anti-mouse, 1:15,000) IRDye700- or IRDye800-conjugated secondary antibodies for 1 h at room temperature, and visualized with a LiCor Odyssey infrared imager (LiCor, Lincoln, NE). Consistent protein loading was determined by reprobing membranes stripped in Restore Western blot stripping buffer with anti-actin antibody (#612656, 0.1 μg/ml, BD PharMingen, San Diego, CA) or anti-GAPDH antibody (#25778, 1:2000, Santa Cruz Biotechnology, Santa Cruz, CA). Initial basic information on the structure of the identified proteins was obtained through literature reports. The ProteinID Finder (Proteome Solutions) program was employed to extract such available information from the UniProt database. Because of the limited annotation of protein glycosylation for most human proteins and the shortage of subcellular location information for hypothetical proteins and functionally uncharacterized proteins, we also subjected each identified protein to prediction algorithms based on protein sequence analysis. For protein glycosylation we used NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/) to predict the possible presence of the consensus NXS/T glycosylation motif. For subcellular location, all identified proteins were analyzed with two transmembrane prediction algorithms SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) indicating hydrophobic protein sequence regions. To generate the lists of cell-surface glycoproteins uniquely expressed in N2 or ML2 cells, identified proteins were required to meet the following 3 criteria: (a) identified from one cell line; (b) unique peptide ≥ 1, and NXS/T motif ≥ 1 or UniProt indicated N-linked or O-linked glycosylation; and (c) transmembrane region ≥ 1, or UniProt membrane subcellular location. The list of identified proteins that were unique to each experimental group was subjected to the commercially available curator database software Ingenuity Pathways Analysis (IPA) to determine their molecular function and interacting networks. Our strategy for interrogation of cell surface sialoglycoproteins using selective chemical tagging followed by high-affinity enrichment and GeLC-MS/MS analysis is summarized in Fig. 1. The method consists of several steps: (1) metabolic labeling of N2 and ML2 cells with the azide-containing mannose analog, peracetylated azido-mannose (AC4ManNAz); (2) chemoselective conjugation of azide sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry in live cells; (3) affinity enrichment of the labeled cell-surface sialylated proteins by streptavidin capture; and (4) separation by one-dimensional gel electrophoresis and identification by LC-MS/MS. When compared with other published approaches, the theoretical advantage of our approach is the targeted selectivity for sialyl glycosylated proteins on the cell surface. A critical parameter in the proposed strategy is the efficient labeling and surface expression of the
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