Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery*
2015; Elsevier BV; Volume: 14; Issue: 6 Linguagem: Inglês
10.1074/mcp.m114.046862
ISSN1535-9484
AutoresDiana Campos, Daniela Freitas, Joana Gomes, Ana Magalhães, Catharina Steentoft, Catarina Gomes, Malene Bech Vester-Christensen, José Alexandre Ferreira, Luís Pedro Afonso, Lúcio Lara Santos, João Pinto-de-Sousa, Ulla Mandel, Henrik Clausen, Sergey Y. Vakhrushev, Celso A. Reis,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoCirculating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer. Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer. Most broad proteomic studies for discovery of cancer biomarkers in serum have been designed to interrogate the proteome and not taking into account that cancer cells often produce aberrant glycoforms (1Surinova S. Schiess R. Huttenhain R. Cerciello F. Wollscheid B. Aebersold R. On the development of plasma protein biomarkers.J. Proteome Res. 2011; 10: 5-16Crossref PubMed Scopus (248) Google Scholar). Many cancer biomarkers currently used in the clinic are based on circulating O-glycoproteins that are detected in established serological assays (CA125, CA15–3, CEA, and CA19.9) (2Reis C.A. Osorio H. Silva L. Gomes C. David L. Alterations in glycosylation as biomarkers for cancer detection.J. Clin. Pathol. 2010; 63: 322-329Crossref PubMed Scopus (350) Google Scholar). In addition to being overexpressed in cancer, these proteins also carry aberrant glycans, which open for the opportunity to selectively detect aberrant glycoforms. An inherent problem with most cancer biomarker assays is that they often have poor specificity because the detected glycoprotein is found in elevated levels in nonmalignant conditions (2Reis C.A. Osorio H. Silva L. Gomes C. David L. Alterations in glycosylation as biomarkers for cancer detection.J. Clin. Pathol. 2010; 63: 322-329Crossref PubMed Scopus (350) Google Scholar, 3Drake P.M. Cho W. Li B. Prakobphol A. Johansen E. Anderson N.L. Regnier F.E. Gibson B.W. Fisher S.J. Sweetening the pot: adding glycosylation to the biomarker discovery equation.Clin. Chem. 2010; 56: 223-236Crossref PubMed Scopus (259) Google Scholar). We recently found that the specificity of the widely used CA125 biomarker assay can be increased by selectively detecting aberrant O-glycoforms of the MUC16 mucin probed in the CA125 assay (4Chen K. Gentry-Maharaj A. Burnell M. Steentoft C. Marcos-Silva L. Mandel U. Jacobs I. Dawnay A. Menon U. Blixt O. Microarray glycoprofiling of CA125 improves differential diagnosis of ovarian cancer.J. Proteome Res. 2013; 12: 1408-1418Crossref PubMed Scopus (79) Google Scholar). Thus, the truncated O-glycan STn (NeuAcα2–6GalNAcα1-O-Ser/Thr) 1The abbreviations used are:STnNeuAcα2–6GalNAcα1-O-Ser/ThrTnGalNAcα1-O-Ser/ThrSTNeuAcα2–3Galβ1–3[NeuAcα2–6]±GalNAcα1-O-Ser/ThrSCSimpleCellTCLtotal cell lysatesVVAVicia villosa agglutinins. 1The abbreviations used are:STnNeuAcα2–6GalNAcα1-O-Ser/ThrTnGalNAcα1-O-Ser/ThrSTNeuAcα2–3Galβ1–3[NeuAcα2–6]±GalNAcα1-O-Ser/ThrSCSimpleCellTCLtotal cell lysatesVVAVicia villosa agglutinins. (Fig. 1) was particularly suited for discrimination of MUC16 circulating in cancer patients in contrast to MUC16 circulating in benign conditions (4Chen K. Gentry-Maharaj A. Burnell M. Steentoft C. Marcos-Silva L. Mandel U. Jacobs I. Dawnay A. Menon U. Blixt O. Microarray glycoprofiling of CA125 improves differential diagnosis of ovarian cancer.J. Proteome Res. 2013; 12: 1408-1418Crossref PubMed Scopus (79) Google Scholar). NeuAcα2–6GalNAcα1-O-Ser/Thr GalNAcα1-O-Ser/Thr NeuAcα2–3Galβ1–3[NeuAcα2–6]±GalNAcα1-O-Ser/Thr SimpleCell total cell lysates Vicia villosa agglutinins. NeuAcα2–6GalNAcα1-O-Ser/Thr GalNAcα1-O-Ser/Thr NeuAcα2–3Galβ1–3[NeuAcα2–6]±GalNAcα1-O-Ser/Thr SimpleCell total cell lysates Vicia villosa agglutinins. One of the most characteristic phenotypes of cancer cells is the expression of truncated O-glycans, and the structures T (Galβ1–3GalNAcα1-O-Ser/Thr), STn, and Tn (GalNAcα1-O-Ser/Thr) (Fig. 1) are considered pancarcinoma antigens (2Reis C.A. Osorio H. Silva L. Gomes C. David L. Alterations in glycosylation as biomarkers for cancer detection.J. Clin. Pathol. 2010; 63: 322-329Crossref PubMed Scopus (350) Google Scholar, 5Springer G.F. T and Tn, general carcinoma autoantigens.Science. 1984; 224: 1198-1206Crossref PubMed Scopus (941) Google Scholar). These truncated O-glycans are essentially not produced in normal and benign cells, which suggests that circulating O-glycoproteins in normal and benign conditions should have more mature O-glycans, whereas O-glycoproteins shed from cancer cells are expected to display truncated glycan structures. Cancer cells produce, secrete, and shed many different O-glycoproteins with truncated O-glycans, and provided these glycoproteins reach the circulation they may be detectable in serum. However, it is also known that nonsialylated glycoproteins are cleared from circulation through innate immune lectin receptors (6Wahrenbrock M.G. Varki A. Multiple hepatic receptors cooperate to eliminate secretory mucins aberrantly entering the bloodstream: are circulating cancer mucins the "tip of the iceberg?".Cancer Res. 2006; 66: 2433-2441Crossref PubMed Scopus (51) Google Scholar). In fact, we were previously unable to detect circulating T and Tn glycoforms of MUC1 and MUC16, while the sialylated ST (NeuAcα2–3Galβ1–3[NeuAcα2–6]±GalNAcα1-O-Ser/Thr) and STn glycoforms were readily detectable (4Chen K. Gentry-Maharaj A. Burnell M. Steentoft C. Marcos-Silva L. Mandel U. Jacobs I. Dawnay A. Menon U. Blixt O. Microarray glycoprofiling of CA125 improves differential diagnosis of ovarian cancer.J. Proteome Res. 2013; 12: 1408-1418Crossref PubMed Scopus (79) Google Scholar, 7Wandall H.H. Blixt O. Tarp M.A. Pedersen J.W. Bennett E.P. Mandel U. Ragupathi G. Livingston P.O. Hollingsworth M.A. Taylor-Papadimitriou J. Burchell J. Clausen H. Cancer biomarkers defined by autoantibody signatures to aberrant O-glycopeptide epitopes.Cancer Res. 2010; 70: 1306-1313Crossref PubMed Scopus (203) Google Scholar). Furthermore, two classical serological biomarker assays, CA19–9 (8Magnani J.L. Nilsson B. Brockhaus M. Zopf D. Steplewski Z. Koprowski H. Ginsburg V. 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Oncol. 2013; 7: 719-731Crossref PubMed Scopus (62) Google Scholar) carcinomas, whereas expression of STn on normal tissues is highly restricted (11Kjeldsen T. Clausen H. Hirohashi S. Ogawa T. Iijima H. Hakomori S. Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2–6 alpha-N-acetylgalactosaminyl (sialosyl-Tn) epitope.Cancer Res. 1988; 48: 2214-2220PubMed Google Scholar, 18Cao Y. Stosiek P. Springer G.F. Karsten U. Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study.Histochem. Cell Biol. 1996; 106: 197-207Crossref PubMed Scopus (149) Google Scholar). In addition, STn expression is associated with carcinoma aggressiveness and poor prognosis (15Leivonen M. Nordling S. Lundin J. von Boguslawski K. Haglund C. STn and prognosis in breast cancer.Oncology. 2001; 61: 299-305Crossref PubMed Scopus (58) Google Scholar, 19Miles D.W. Happerfield L.C. Smith P. Gillibrand R. 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One mechanism is the altered expression of the sialyltransferase ST6GalNAc-I, which is believed to be the main STn synthase (21Marcos N.T. Bennett E.P. Gomes J. Magalhaes A. Gomes C. David L. Dar I. Jeanneau C. DeFrees S. Krustrup D. Vogel L.K. Kure E.H. Burchell J. Taylor-Papadimitriou J. Clausen H. Mandel U. Reis C.A. ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues.Front. Biosci. 2011; 3: 1443-1455PubMed Google Scholar, 22Marcos N.T. Pinho S. Grandela C. Cruz A. Samyn-Petit B. Harduin-Lepers A. Almeida R. Silva F. Morais V. Costa J. Kihlberg J. Clausen H. Reis C.A. Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen.Cancer Res. 2004; 64: 7050-7057Crossref PubMed Scopus (175) Google Scholar) (Fig. 1), and in fact overexpression of this enzyme in cell lines appears to override the normal O-glycan elongation machinery and result in expression of STn (22Marcos N.T. Pinho S. Grandela C. Cruz A. Samyn-Petit B. Harduin-Lepers A. Almeida R. Silva F. Morais V. Costa J. Kihlberg J. Clausen H. Reis C.A. Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen.Cancer Res. 2004; 64: 7050-7057Crossref PubMed Scopus (175) Google Scholar, 23Sewell R. Backstrom M. Dalziel M. Gschmeissner S. Karlsson H. Noll T. Gatgens J. Clausen H. Hansson G.C. Burchell J. Taylor-Papadimitriou J. The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer.J. Biol. Chem. 2006; 281: 3586-3594Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Another mechanism may be reduced core1 elongation that leads to accumulation of Tn, which serves as substrate for ST6GalNAc-I (22Marcos N.T. Pinho S. Grandela C. Cruz A. Samyn-Petit B. Harduin-Lepers A. Almeida R. Silva F. Morais V. Costa J. Kihlberg J. Clausen H. Reis C.A. Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen.Cancer Res. 2004; 64: 7050-7057Crossref PubMed Scopus (175) Google Scholar). The core1 synthase C1GALT1 is dependent on a private chaperone Cosmc, and several studies have reported that somatic mutations in COSMC gene (24Ju T. Lanneau G.S. Gautam T. Wang Y. Xia B. Stowell S.R. Willard M.T. Wang W. Xia J.Y. Zuna R.E. Laszik Z. Benbrook D.M. Hanigan M.H. Cummings R.D. Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc.Cancer Res. 2008; 68: 1636-1646Crossref PubMed Scopus (216) Google Scholar), or hypermethylation of COSMC gene in cancer (25Radhakrishnan P. Dabelsteen S. Madsen F.B. Francavilla C. Kopp K.L. Steentoft C. Vakhrushev S.Y. Olsen J.V. Hansen L. Bennett E.P. Woetmann A. Yin G. Chen L. Song H. Bak M. Hlady R.A. Peters S.L. Opavsky R. Thode C. Qvortrup K. Schjoldager K.T. Clausen H. Hollingsworth M.A. Wandall H.H. Immature truncated O-glycophenotype of cancer directly induces oncogenic features.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E4066-4075Crossref PubMed Scopus (205) Google Scholar) lead to increased expression of Tn and STn. We have further shown that knockout (KO) of COSMC in a number of human cancer cell lines produce cells that express different levels of Tn and STn truncated O-glycans ranging from exclusive Tn to exclusive STn (26Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (264) Google Scholar). A third potential mechanism offered recently may be related to cancer-associated relocation of the polypeptide GalNAc-transferases (GalNAc-Ts) that initiate O-glycosylation (Fig. 1) from Golgi to ER, which appear to induce expression of the Tn truncated O-glycans, although expression of STn has not been explored yet (27Gill D.J. Tham K.M. Chia J. Wang S.C. Steentoft C. Clausen H. Bard-Chapeau E.A. Bard F.A. Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E3152-3161Crossref PubMed Scopus (141) Google Scholar). In the present study, we applied a glycoproteomics strategy to explore potential biomarker O-glycoproteins with the STn glycoform in gastric cancer. We first characterized the O-glycoproteome and including the secretome of two gastric cancer cell lines, AGS (intestinal type gastric carcinoma) and MKN45 (diffuse type gastric carcinoma), using our SimpleCell (SC) discovery platform where we identified a total of 499 O-glycoproteins (1236 O-glycosites). This strategy involves genetic engineering of cell lines to produce homogenous truncated O-glycans (Tn and/or STn) by KO of COSMC, followed by Vicia Villosa lectin (VVA) enrichment of Tn glycoproteins and/or glycopeptides for sensitive identification of O-glycoproteins and O-glycosites by mass spectrometry (26Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (264) Google Scholar, 28Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar) (Fig. 1). We applied the same glycoproteomics workflow to a wild type (wt) gastric cancer cell line, KATO III (diffuse type gastric carcinoma), which naturally expresses Tn and STn O-glycans in a mixture with more complex structures, and identified a significantly smaller O-glycoproteome (total of 47 O-glycoproteins) compared with SimpleCells (total of 499 O-glycoproteins). We next modified the strategy to enrich for STn O-glycoproteins in pools of serum from cancer patients and normal controls using pretreatment with neuraminidase to remove sialic acid and expose Tn for VVA capture. This approach enabled us to isolate and identify 37 O-glycoproteins (49 O-glycosites) in gastric cancer serum. Finally, we confirmed that two of the identified serum O-glycoproteins (CD44 and GalNAc-T5) were expressed in gastric cancer tumors by immunohistology, and further used proximity ligation assay (PLA) to show that STn glycoforms of CD44 was expressed in cancer tissue. This study clearly shows that cancer patients have a variety of circulating O-glycoproteins with the STn glycoform, and supports the hypothesis that these glycoproteins originate from the cancer tissue. The identified secreted and circulating aberrant O-glycoproteins serve as a discovery set for biomarkers of gastric cancer. We targeted the COSMC gene in two human gastric cell lines, AGS and MKN45, using zinc finger nuclease (ZFN) precise gene editing as previously described (26Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (264) Google Scholar, 29Schjoldager K.T. Vakhrushev S.Y. Kong Y. Steentoft C. Nudelman A.S. Pedersen N.B. Wandall H.H. Mandel U. Bennett E.P. Levery S.B. Clausen H. Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 9893-9898Crossref PubMed Scopus (95) Google Scholar, 30Steentoft C. Bennett E.P. Schjoldager K.T. Vakhrushev S.Y. Wandall H.H. Clausen H. Precision genome editing: a small revolution for glycobiology.Glycobiology. 2014; 24: 663-680Crossref PubMed Scopus (47) Google Scholar). Briefly, cells were transfected with 4 μg of compoZr® C1GalT1C1 DNA using an AmaxaTM NucleofectorTM according to the manufacture's cell lines specific protocols (Lonza, Basel, Switzerland). Cells were screened on acetone fixed slides by immunocytochemistry using monoclonal antibodies (MAbs) to the Tn (5F4) and STn (3F1) O-glycans (See Supplemental Table S1 for MAbs used in study). The cells were then cloned by limited dilution and the final clones were further characterized by staining with monoclonal antibodies to either the C1GalT1 enzyme (5B6) or the T antigen (3C9) O-glycan structure with and without pretreatment with neuraminidase (Sigma). Slides were examined using a Zeiss Optical Microscope. Finally, we analyzed selected clones by PCR followed by sequencing to define the induced mutations in COSMC. Serum samples from gastric carcinoma patients were collected from The Portuguese Institute of Oncology (IPO-Porto, Portugal), and control samples from individuals undergoing hernia surgery from the Hospital de São João in Porto, Portugal. All samples were collected with informed consent and use of samples was approved by the local Ethical committees (CHSJ and IPO). A total of 29 individual samples (∼0.69 ml each, from a sample taken at time of surgery) from both intestinal and unclassified subtype, according to Lauren's classification (31Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. an attempt at a histo-clinical classification.Acta Pathol. Microbiol. Scand. 1965; 64: 31-49Crossref PubMed Scopus (5066) Google Scholar), with different disease stages, were used for the gastric carcinoma serum pool. Individuals were selected for blood group B and O to avoid potential cross-reactivity of VVA lectin with blood group A, although glycan array data from the Consortium for Functional Glycomics show no such cross-reactivity (http://www.functionalglycomics.org). Although some O-glycoproteins may potentially be lost in coagulation, most comparative proteomic studies of serum and plasma have found similar glycoprotein composition (24Ju T. Lanneau G.S. Gautam T. Wang Y. Xia B. Stowell S.R. Willard M.T. Wang W. Xia J.Y. Zuna R.E. Laszik Z. Benbrook D.M. Hanigan M.H. Cummings R.D. Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc.Cancer Res. 2008; 68: 1636-1646Crossref PubMed Scopus (216) Google Scholar, 32Adamczyk B. Struwe W.B. Ercan A. Nigrovic P.A. Rudd P.M. Characterization of fibrinogen glycosylation and its importance for serum/plasma N-glycome analysis.J. Proteome Res. 2013; 12: 444-454Crossref PubMed Scopus (44) Google Scholar). The age of carcinoma patients varied from 49 to 81 with an average value of 65 years old, and included 17 males and 12 females. Age of control individuals varied from 21 to 83 years old with an average of 69 and included 20 males and 5 females. Total cell lysates (TCL) and culture medium (SEC) were processed as previously described (28Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar, 33Yang Z. Halim A. Narimatsu Y. Joshi H.J. Steentoft C. Schjoldager K.T. Schulz M.A. Sealover N.R. Kayser K.J. Bennett E.P. Levery S.B. Vakhrushev S.Y. Clausen H. The GalNAc-type O-glycoproteome of CHO cells characterized by the SimpleCell strategy.Mol. Cell. Proteomics. 2014; 13: 3224-3235Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In brief, spent culture medium (∼80 ml harvested from two 175 ml T-flasks seeded at 5 × 105 cells and cultured for 3 days) was cleared, dialyzed, and subjected to neuraminidase treatment (10 U Clostridium perfringens neuraminidase Type VI (Sigma)) before loaded on a 0.3 ml Vicia villosa agglutinin (VVA) agarose (Vector laboratories, Burlingame, CA) column. The column was washed with 10 column volumes (CV) of 0.4 m Glucose in LAC A buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 m Urea, 1 mm CaCl2, MgCl2, MnCl2, and ZnCl2) followed by 1 ml 50 mm AmBic. The glycoproteins were then eluted by 4 × 500 μl 0.05% RapiGest with heating to 90 °C for 10 min as described previously (28Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar). Fractions were pooled and directly reduced with 5 mm DTT for 40 min at 60 °C, alkylated with 10 mm iodoacetamide in dark for 45 min, and digested with trypsin (25 μg). Cell pellets were obtained by removing media, washing with phosphate buffer solution (PBS) and, followed by scraping of the cells in PBS, lysed in RapiGest and sonicated. The cleared lysate was reduced, alkylated with iodoacetamide, and then digested overnight with trypsin. For AGS SC we also used chymotrypsin and Glu-C in addition to trypsin. Thus, for this cell line three data sets for TCL and three for SEC were generated. Serum pools (20 ml) were diluted in PBS to 100 ml and neuraminidase treated (10 U), and loaded twice on a 0.3 ml VVA agarose column. The glycoproteins were eluted and reduced and alkylated as described above, and digested with chymotrypsin (25 μg) overnight at 37 °C. All digests were finally subjected to lectin weak affinity chromatography (LWAC) on VVA agarose as previously described (26Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (264) Google Scholar, 28Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar). Briefly, the digestion was quenched with 1% trifluoroacetic acid (TFA), and the resulting peptides were desalted with C18 stage tips and dried in a SpeedVac. The digest was then neuraminidase treated (New England Biolabs, Ipswich, MA), diluted in 2 ml LAC A buffer and injected onto a pre-equilibrated 2.6 m long VVA agarose (Vector Laboratories, Burlingame, CA) column, similar to the system described previously (28Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar, 34Chalkley R.J. Thalhammer A. Schoepfer R. Burlingame A.L. Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8894-8899Crossref PubMed Scopus (19
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