Liver Membrane Proteome Glycosylation Changes in Mice Bearing an Extra-hepatic Tumor
2010; Elsevier BV; Volume: 10; Issue: 9 Linguagem: Inglês
10.1074/mcp.m900538-mcp200
ISSN1535-9484
AutoresAlbert Lee, Joel M. Chick, Daniel Kolarich, Paul A. Haynes, Graham Robertson, Maria Tsoli, Lucy Jankova, Stephen Clarke, Nicolle H. Packer, Mark S. Baker,
Tópico(s)Ubiquitin and proteasome pathways
ResumoCancer is well known to be associated with alterations in membrane protein glycosylation (Bird, N. C., Mangnall, D., and Majeed, A. W. (2006) Biology of colorectal liver metastases: A review. J. Surg. Oncol. 94, 68–80; Dimitroff, C. J., Pera, P., Dall'Olio, F., Matta, K. L., Chandrasekaran, E. V., Lau, J. T., and Bernacki, R. J. (1999) Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem. Biophys. Res. Commun. 256, 631–636; and Arcinas, A., Yen, T. Y., Kebebew, E., and Macher, B. A. (2009) Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns. J. Proteome Res. 8, 3958–3968). Equally, it has been well established that tumor-associated inflammation through the release of pro-inflammatory cytokines is a common cause of reduced hepatic drug metabolism and increased toxicity in advanced cancer patients being treated with cytotoxic chemotherapies. However, little is known about the impact of bearing a tumor (and downstream effects like inflammation) on liver membrane protein glycosylation.In this study, proteomic and glycomic analyses were used in combination to determine whether liver membrane protein glycosylation was affected in mice bearing the Engelbreth-Holm Swarm sarcoma. Peptide IPG-IEF and label-free quantitation determined that many enzymes involved in the protein glycosylation pathway specifically; mannosidases (Man1a-I, Man1b-I and Man2a-I), mannoside N-acetylglucosaminyltransferases (Mgat-I and Mgat-II), galactosyltransferases (B3GalT-VII, B4GalT-I, B4GalT-III, C1GalT-I, C1GalT-II, and GalNT-I), and sialyltransferases (ST3Gal-I, ST6Gal-I, and ST6GalNAc-VI) were up-regulated in all livers of tumor-bearing mice (n = 3) compared with nontumor bearing controls (n = 3). In addition, many cell surface lectins: Sialoadhesin-1 (Siglec-1), C-type lectin family 4f (Kupffer cell receptor), and Galactose-binding lectin 9 (Galectin-9) were determined to be up-regulated in the liver of tumor-bearing compared with control mice. Global glycan analysis identified seven N-glycans and two O-glycans that had changed on the liver membrane proteins derived from tumor-bearing mice. Interestingly, α (2,3) sialic acid was found to be up-regulated on the liver membrane of tumor-bearing mice, which reflected the increased expression of its associated sialyltransferase and lectin receptor (siglec-1). The overall increased sialylation on the liver membrane of Engelbreth-Holm Swarm bearing mice correlates with the increased expression of their associated glycosyltransferases and suggests that glycosylation of proteins in the liver plays a role in tumor-induced liver inflammation Cancer is well known to be associated with alterations in membrane protein glycosylation (Bird, N. C., Mangnall, D., and Majeed, A. W. (2006) Biology of colorectal liver metastases: A review. J. Surg. Oncol. 94, 68–80; Dimitroff, C. J., Pera, P., Dall'Olio, F., Matta, K. L., Chandrasekaran, E. V., Lau, J. T., and Bernacki, R. J. (1999) Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem. Biophys. Res. Commun. 256, 631–636; and Arcinas, A., Yen, T. Y., Kebebew, E., and Macher, B. A. (2009) Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns. J. Proteome Res. 8, 3958–3968). Equally, it has been well established that tumor-associated inflammation through the release of pro-inflammatory cytokines is a common cause of reduced hepatic drug metabolism and increased toxicity in advanced cancer patients being treated with cytotoxic chemotherapies. However, little is known about the impact of bearing a tumor (and downstream effects like inflammation) on liver membrane protein glycosylation. In this study, proteomic and glycomic analyses were used in combination to determine whether liver membrane protein glycosylation was affected in mice bearing the Engelbreth-Holm Swarm sarcoma. Peptide IPG-IEF and label-free quantitation determined that many enzymes involved in the protein glycosylation pathway specifically; mannosidases (Man1a-I, Man1b-I and Man2a-I), mannoside N-acetylglucosaminyltransferases (Mgat-I and Mgat-II), galactosyltransferases (B3GalT-VII, B4GalT-I, B4GalT-III, C1GalT-I, C1GalT-II, and GalNT-I), and sialyltransferases (ST3Gal-I, ST6Gal-I, and ST6GalNAc-VI) were up-regulated in all livers of tumor-bearing mice (n = 3) compared with nontumor bearing controls (n = 3). In addition, many cell surface lectins: Sialoadhesin-1 (Siglec-1), C-type lectin family 4f (Kupffer cell receptor), and Galactose-binding lectin 9 (Galectin-9) were determined to be up-regulated in the liver of tumor-bearing compared with control mice. Global glycan analysis identified seven N-glycans and two O-glycans that had changed on the liver membrane proteins derived from tumor-bearing mice. Interestingly, α (2,3) sialic acid was found to be up-regulated on the liver membrane of tumor-bearing mice, which reflected the increased expression of its associated sialyltransferase and lectin receptor (siglec-1). The overall increased sialylation on the liver membrane of Engelbreth-Holm Swarm bearing mice correlates with the increased expression of their associated glycosyltransferases and suggests that glycosylation of proteins in the liver plays a role in tumor-induced liver inflammation Despite membrane proteins constituting ∼70% of all human protein based drug targets, studying membrane proteins is often hindered by their low abundance, large size, and relatively high hydrophobicity. Isolation and separation of membrane proteins by two-dimensional gel electrophoresis has been particularly problematic because of their poor solubility in various isoelectric focusing buffers. Alternative proteomic approaches such as shotgun proteomics have recently become the preferred method for identifying membrane proteins using high resolution separation of proteolytic peptides by liquid chromatography (4Washburn M.P. Wolters D. Yates 3rd, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4077) Google Scholar), capillary isoelectric focusing (5Wang W. Guo T. Rudnick P.A. Song T. Li J. Zhuang Z. Zheng W. Devoe D.L. Lee C.S. Balgley B.M. Membrane proteome analysis of microdissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS.Anal. Chem. 2007; 79: 1002-1009Crossref PubMed Scopus (58) Google Scholar) or peptide immobilized pH gradient isoelectric focusing (peptide IPG-IEF) 1The abbreviations used are:Peptide IPG-IEFpeptide immobilized pH gradient-isoelectric focusingEHSEngelbreth Holm SwarmNSAFnormalised spectral abundance factorMgat-IMannoside N-acetylglucosaminyltransferase 1Mgat-IIMannoside N-acetylglucosaminyltransferase 2Mgat-VMannoside N-acetylglucosaminyltransferase 5B3GalT-VIIBeta-1,3-galactosyltransferase 7C1GalT-IBeta1,3-galactosyltransferase 1NeuAcN-acetyl neuraminic acidNeuGcN-glycolyl neuraminic acid. (6Chick J.M. Haynes P.A. Molloy M.P. Bjellqvist B. Baker M.S. Len A.C. Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing.J. Proteome Res. 2008; 7: 1036-1045Crossref PubMed Scopus (51) Google Scholar) in combination with reverse phase chromatography and tandem mass spectrometry. peptide immobilized pH gradient-isoelectric focusing Engelbreth Holm Swarm normalised spectral abundance factor Mannoside N-acetylglucosaminyltransferase 1 Mannoside N-acetylglucosaminyltransferase 2 Mannoside N-acetylglucosaminyltransferase 5 Beta-1,3-galactosyltransferase 7 Beta1,3-galactosyltransferase 1 N-acetyl neuraminic acid N-glycolyl neuraminic acid. We have recently demonstrated the use of broad and narrow range peptide IPG-IEF as an alternative method for shotgun proteomics of rat liver membrane proteins (6Chick J.M. Haynes P.A. Molloy M.P. Bjellqvist B. Baker M.S. Len A.C. Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing.J. Proteome Res. 2008; 7: 1036-1045Crossref PubMed Scopus (51) Google Scholar). Peptide IPG-IEF has several advantages as a high-resolution shotgun proteomics technique most notably because of its high sample loading capacity. It has previously been demonstrated to provide more confident and reproducible protein identifications compared with 1D SDS-PAGE (7Krijgsveld J. Gauci S. Dormeyer W. Heck A.J. In-gel isoelectric focusing of peptides as a tool for improved protein identification.J. Proteome Res. 2006; 5: 1721-1730Crossref PubMed Scopus (94) Google Scholar) and/or strong cation exchange (8Slebos R.J. Brock J.W. Winters N.F. Stuart S.R. Martinez M.A. Li M. Chambers M.C. Zimmerman L.J. Ham A.J. Tabb D.L. Liebler D.C. Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2008; 7: 5286-5294Crossref PubMed Scopus (79) Google Scholar) by using the theoretical pI of peptides as an additional filtering criteria to reduce false discovery rates (6Chick J.M. 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Glycans can act as markers to characterize cell types and states and they are also involved in numerous biological functions and processes such as modulating structure and function of proteins, fertilization, cell division, anticoagulation, and inflammatory responses (13Rudd P.M. Elliott T. Cresswell P. Wilson I.A. Dwek R.A. Glycosylation and the immune system.Science. 2001; 291: 2370-2376Crossref PubMed Scopus (1378) Google Scholar, 14Dwek R.A. Glycobiology: Toward Understanding the Function of Sugars.Chem. Rev. 1996; 96: 683-720Crossref PubMed Scopus (2840) Google Scholar, 15Varki A. et al.Essentials in Glycobiology. 1999; Google Scholar). Glycans are composed of monosaccharides that are covalently linked by glycosidic bonds, either in α or β conformations. 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Glycosyltransferases are a group of enzymes that catalyze the addition of monosaccharides to core proteins. Most glycoproteins are either secreted into the extracellular environment or attached to the plasma membrane of the cancer cell; therefore changes to glycosyltransferases can ultimately influence the overall adhesive properties (2Dimitroff C.J. Pera P. Dall'Olio F. Matta K.L. Chandrasekaran E.V. Lau J.T. Bernacki R.J. Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells.Biochem. Biophys. Res. Commun. 1999; 256: 631-636Crossref PubMed Scopus (38) Google Scholar). Reduced levels of sialyltransferases and increased expression of fucosyltransferases, which are known to occur during cancer carcinogenesis and metastasis, extensively modify carbohydrate moieties on the cell surface (25Petretti T. Kemmner W. Schulze B. Schlag P.M. 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A carbohydrate domain common to both sialyl Le(a) and sialyl Le(X) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1.J. Biol. Chem. 1991; 266: 14869-14872Abstract Full Text PDF PubMed Google Scholar). The degree of sialylation of these antigens can affect the polar orientation and adhesion of tumor cells during cancer metastasis (28Berg E.L. Robinson M.K. Mansson O. Butcher E.C. Magnani J.L. A carbohydrate domain common to both sialyl Le(a) and sialyl Le(X) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1.J. Biol. Chem. 1991; 266: 14869-14872Abstract Full Text PDF PubMed Google Scholar). The clinical relevance of inflammation both in contributing to the development of cancer and in determining prognosis is well established (29Kacevska M. Robertson G.R. Clarke S.J. Liddle C. Inflammation and CYP3A4-mediated drug metabolism in advanced cancer: impact and implications for chemotherapeutic drug dosing.Expert Opin. Drug Metab. 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Inflammation is a symptom of a disease or infection that regulates the expression of specific proteins that mediate innate and adaptive immune responses to promote healing of the affected site. Previous studies using lectin and immunoblot analyses have shown that turpentine oil-induced inflammation in mice leads to changes in the sialylation patterns of serum glycoproteins (31Yasukawa Z. Sato C. Kitajima K. Inflammation-dependent changes in alpha2,3-, alpha2,6-, and alpha2,8-sialic acid glycotopes on serum glycoproteins in mice.Glycobiology. 2005; 15: 827-837Crossref PubMed Scopus (66) Google Scholar). Reverse transcription- polymerase chain reaction (RT-PCR) analysis of the expression profiles of mRNA for the known sialyltransferases involved in the synthesis of these sugars identified increased levels of β-galactoside α (2,3)-sialyltransferases (ST3Gal-I and ST3Gal-III), β-N-acetylgalactosaminide α (2,6)-sialyltransferase (ST6GalNAc-VI), and β-galactoside α (2,6)-sialyltransferase (ST6Gal-I) (31). These sialyltransferases are mostly involved in the addition of α (2,3) and α (2,6) sialic acids onto N- and O-glycans. Most plasma proteins are derived from the liver and significant increases in serum acute phase glycoproteins and their sialylation have generally been observed with most rodent experimental inflammation models (31Yasukawa Z. Sato C. Kitajima K. Inflammation-dependent changes in alpha2,3-, alpha2,6-, and alpha2,8-sialic acid glycotopes on serum glycoproteins in mice.Glycobiology. 2005; 15: 827-837Crossref PubMed Scopus (66) Google Scholar, 32Lin S.Y. Chen Y.Y. Fan Y.Y. 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The impact of the EHS tumor and its associated inflammatory response on the liver membrane proteins has recently been reported (30Robertson G.R. Liddle C. Clarke S.J. Inflammation and altered drug clearance in cancer: transcriptional repression of a human CYP3A4 transgene in tumor-bearing mice.Clin. Pharmacol. Ther. 2008; 83: 894-897Crossref PubMed Scopus (43) Google Scholar, 36Charles K.A. Rivory L.P. Brown S.L. Liddle C. Clarke S.J. Robertson G.R. Transcriptional repression of hepatic cytochrome P450 3A4 gene in the presence of cancer.Clin. Cancer Res. 2006; 12: 7492-7497Crossref PubMed Scopus (63) Google Scholar), however little is known about whether tumor-associated liver inflammation can influence membrane protein glycosylation. In the present study, peptide IPG-IEF and label-free quantitation were used to identify and determine the relative abundance of glycosyltranferases and cell surface lectins on the liver membrane of mice bearing a distal benign tumor. Triton X-114 phase partitioning (37Lee A. Kolarich D. Haynes P.A. Jensen P.H. Baker M.S. Packer N.H. Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture.J. Proteome Res. 2009; 8: 770-781Crossref PubMed Scopus (61) Google Scholar) was employed to further enrich membrane proteins for global glycan profiling to determine whether the changes in glycosyltransferases identified by peptide IPG-IEF reflected the structures of the glycans presented on the membrane. The combination of proteomics and glycomics approaches could help determine potentially important biological changes resulting from the EHS tumor on liver cellular membrane. Tris, sodium chloride, sodium hydroxide, sodium carbonate, EDTA, Bradford reagent, HEPES, phosphate-buffered saline tablets, ammonium bicarbonate (NH4HCO3), ammonium acetate (CH3COONH4), formic acid, Triton X-114, dithiothreitol, iodoacetamide, LC grade acetonitrile (ACN) and sodium borohydride (NaBH4) were obtained from Sigma. NuPAGE 10% Bis-Tris precast gradient gels and 3-(N-morpholino) propane sulfonic acid running buffer were purchased from Invitrogen (San Diego, CA). IPG strips were a generous gift from GE Healthcare (Uppsala, Sweden). Sequencing-grade trypsin was obtained from Promega (Madison, WI). N-Glycosidase F (PNGase F, recombinant cloned from Flavobacterium meningosepticum and expressed in Escherichia coli) lyophilized in 10 mm sodium phosphate buffer, pH 7.2 was acquired from Roche Diagnostics (Basel, Switzerland). Immobolin-P Polyvinylidene Fluoride (PVDF) was obtained from Millipore (Billerica, MA). AG50W-X8 cation-exchange resin was obtained from Bio-Rad (Hercules CA) and C18 tips were obtained from Eppendorf (Hamburg, Germany). Eight to 10 week old transgenic FvB male mice (n = 3) were aseptically inoculated using a 14-gauge needle with 0.3 ml suspension of EHS sarcoma cells (containing 106 cells) in 0.9% (w/v) phosphate-buffered saline into the quadriceps of the right hind leg. Control mice (n = 3) were injected with 0.3 ml of 0.9% (w/v) phosphate-buffered saline. Tumor mass reached ∼3 g or 10% (w/w) total body weight over 18 to 21 days. Tumor and control mice were euthanized and livers were harvested. To address concerns of biological reproducibility, all proteomic and glycomic experiments were conducted on three biological replicates of individual control and tumor-bearing mice. All animal work was approved by the Concord Repatriation General Hospital local area health service Animal Ethics Committee. Mouse livers (n = 3, ∼1.5 g) were perfused with 0.9% (w/v) phosphate buffered saline and homogenized in 10 mm HEPES buffer (pH 7.8) supplemented with 2 mm NaCl, 10 mm NaOH, 500 mm EDTA, and protease inhibitors (3 mg of antipain-dihydrochloride, 0.5 mg of aprotinin, 0.5 mg of bestatin, 1 mg of chymostatin, 3 mg of E-64, 10 mg of EDTA-Na2, 0.5 mg of leupeptin, 20 mg of Pefabloc SC, 0.5 mg of pepstatin, 3 mg of phosphoramidon) one tablet per 100 ml of buffer (Roche Diagnostics, Switzerland) using an Omni TH homogenizer (Omni International, Inc., Kennesaw, GA). The homogenized tissue was then centrifuged at 13,000 × g for 15 min at room temperature to remove unbroken cells and cell debris. Total cell membrane proteins were isolated from the supernatant using a modified sodium carbonate stripping method (38Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1382) Google Scholar). Briefly, the supernatant was diluted to a final volume of 20 ml in 0.1 m sodium carbonate (pH 11), then incubated for 1 h rotating at 4 °C. The carbonate-treated membranes were sedimented by ultracentrifugation at 120,000 × g for 1 h at 4 °C. The supernatant was then removed and the membrane pellet was washed twice with ammonium bicarbonate (10 mm NH4HCO3, pH 7.8), then 1 ml of 0.5 m triethylammonium bicarbonate, 0.05% SDS was added and pellet was transferred to a 20 ml glass scintillation vial and pulse-sonicated using a Branson 450 Sonifier (Branson, Danbury, CT) using 2-s bursts for 15 intervals on ice, and stored at −80 °C if not used immediately. Approximately 100 μl (equivalent to ∼0.5–1 mg) of the mouse liver membrane pellet was homogenized in 4 volumes Tris-buffered saline buffer containing 1% (v/v) Triton X-114 and chilled on ice with intermittent vortexing for 20 min (37Lee A. Kolarich D. Haynes P.A. Jensen P.H. Baker M.S. Packer N.H. Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture.J. Proteome Res. 2009; 8: 770-781Crossref PubMed Scopus (61) Google Scholar). The samples were heated to 37 °C for 20 min and phase partitioned followed by centrifugation at 300 g for 5 min. The upper aqueous phase was removed and stored. The detergent phase was further diluted with 4 volumes of Tris-buffered saline buffer containing 1% (v/v) Triton X-114 and phase partitioning repeated. The proteins in the combined aqueous phases and detergent phase were each precipitated with 9 volumes of ice-cold acetone and incubated overnight at −20 °C. Acetone-precipitated proteins were resuspended in 20 μl 8 m urea. The sonicated membrane sample was reduced with 10 mm dithiothreitol for 1 h at 37 °C and subsequently alkylated with 55 mm iodoacetamide in the dark at room
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