Mass Spectrometry-Based Identification Of The Tumor Antigen UN1 as the Transmembrane CD43 Sialoglycoprotein
2011; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.1074/mcp.m111.007898
ISSN1535-9484
AutoresAnnamaria de Laurentiis, Marco Gaspari, Camillo Palmieri, Cristina Falcone, Enrico Iaccino, Giuseppe Fiume, Ornella Massa, Mariorosario Masullo, Franca Maria Tuccillo, Laura Roveda, U. Prati, Olga Fierro, Immacolata Cozzolino, Giancarlo Troncone, Pierfrancesco Tassone, Giuseppe Scala, Ileana Quinto,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation.In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis. The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis. Glycoproteins play a major role in cell signaling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure variability and binding specificity to lectin ligands (1Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Abstract Full Text Full Text PDF PubMed Scopus (1987) Google Scholar). Mucin-type glycoproteins are characterized by a high content of O-linked carbohydrate chains (O-glycans), and are secreted or expressed on the membrane of hematopoietic and epithelial cells. O-glycan biosynthesis initiates in the Golgi apparatus with the attachment of N-acetyl-galactosamine (GalNAc) to serine or threonine residues by an UDP-N-acetyl-d galactosamine: polipeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) generating the Tn antigen structure of O-glycans. Subsequent elongation of the O-linked glycan branch is catalyzed by tissue-specific glycosyltransferases through the addition of other carbohydrates, such as galactose, fucose, and sialic acid, which results in the synthesis of a complex array of O-glycan structures that differ for the nature and length of O-linked carbohydrate chains (2Wopereis S. Lefeber D.J. Morava E. Wevers R.A. Mechanisms in protein O-glycan biosynthesis and clinical and molecular aspects of protein O-glycan biosynthesis defects: a review.Clin. Chem. 2006; 52: 574-600Crossref PubMed Scopus (124) Google Scholar). In addition, the oligosaccharides can be modified by sialylation, fucosylation, sulfatation, methylation, or acetylation. Truncation of O-glycan structures as well as abnormal expression of specific O-glycans occur in cancer cells, suggesting that aberrant glycosylation may contribute to cancer progression by modifying cell signaling, adhesion, and antigenicity (3Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10231-10233Crossref PubMed Scopus (786) Google Scholar, 4Brockhausen I. Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions.EMBO Rep. 2006; 7: 599-604Crossref PubMed Scopus (409) Google Scholar). The UN1 monoclonal antibody (mAb) 1The abbreviations used are:mAbmonoclonal antibodyGalNAcN-acetyl-galactosamineGalNAc-transferaseUDP-N-acetyl-D galactosamine: polipeptide N-acetylgalactosaminyltransferaseMAL IIMaackia Amurensis lectinPNAPeanut AgglutininDEAEdiethylaminoethylSPEreversed-phase solid phase extractionSCXstrong-cation exchangenanoLCnanoscale liquid chromatographyMS/MStandem mass spectrometry., which was initially selected for high reactivity with human immature thymocytes (CD3dim) (5Tassone P. Bond H. Bonelli P. Tuccillo F. Valerio G. Petrella A. Lamberti A. Cecco L. Turco M.C. Cerra M. et al.UN1, a murine monoclonal antibody recognizing a novel human thymic antigen.Tissue Antigens. 1994; 44: 73-82Crossref PubMed Scopus (12) Google Scholar), recognized the UN1 antigen as a highly sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa and binding specificity to the lectins Maackia Amurensis (MAL II) and Peanut Agglutinin (PNA), which are binders of the sialic acid in (α2–3) linkage and the disaccharide Galβ1,3GalNAc, respectively (6Cecco L. Bond H.M. Bonelli P. Tuccillo F. Cerra M. Tassone P. Sorice R. Lamberti A. Morrone G. Venuta S. Purification and characterization of a human sialoglycoprotein antigen expressed in immature thymocytes and fetal tissues.Tissue Antigens. 1998; 51: 528-535Crossref PubMed Scopus (8) Google Scholar). The UN1 antigen was immunodetected on the membrane of human thymocytes, a subpopulation of peripheral blood CD4+ T-lymphocytes and leukemic T-cell lines, such as HPB-ALL, H9, and MOLT-4 (5Tassone P. Bond H. Bonelli P. Tuccillo F. Valerio G. Petrella A. Lamberti A. Cecco L. Turco M.C. Cerra M. et al.UN1, a murine monoclonal antibody recognizing a novel human thymic antigen.Tissue Antigens. 1994; 44: 73-82Crossref PubMed Scopus (12) Google Scholar, 6Cecco L. Bond H.M. Bonelli P. Tuccillo F. Cerra M. Tassone P. Sorice R. Lamberti A. Morrone G. Venuta S. Purification and characterization of a human sialoglycoprotein antigen expressed in immature thymocytes and fetal tissues.Tissue Antigens. 1998; 51: 528-535Crossref PubMed Scopus (8) Google Scholar). In addition, UN1 was expressed at early stages of development in fetal tissues, including thymus, spleen, adrenal cortex, bronchial epithelium, and skin, and was down-regulated in ontogeny (7Tassone P. Tuccillo F. Bonelli P. D'Armiento F.P. Bond H.M. Palmieri C. Tagliaferri P. Venuta S. Fetal ontogeny and tumor expression of the early thymic antigen UN1.Int. J. Oncol. 2002; 20: 707-711PubMed Google Scholar). UN1 was also expressed in a variety of solid tumors, including breast, colon, gastric, and squamous cell lung carcinomas, whereas undetected in normal tissues and benign lesions (7Tassone P. Tuccillo F. Bonelli P. D'Armiento F.P. Bond H.M. Palmieri C. Tagliaferri P. Venuta S. Fetal ontogeny and tumor expression of the early thymic antigen UN1.Int. J. Oncol. 2002; 20: 707-711PubMed Google Scholar). In particular, a direct correlation was observed between the expression level of UN1 and the stage of malignancy in breast tissue (8Tassone P. Bonelli P. Tuccillo F. Bond H.M. D'Armiento F.P. Galea E. Palmieri C. Tagliaferri P. Natali P.G. Venuta S. Differential expression of UN1, early thymocyte-associated sialoglycoprotein, in breast normal tissue, benign disease and carcinomas.Anticancer Res. 2002; 22: 2333-2340PubMed Google Scholar). In fact, UN1 was not expressed in normal cells and nonproliferative lesions, whereas it was poorly expressed in fibroadenoma, moderately expressed in atypical hyperplasia, and highly expressed in proliferative lesions of in situ breast carcinoma (stage 0 of disease) and infiltrating breast carcinoma (stages I–III) with the highest expression level in metastatic lesions (stage IV) (8Tassone P. Bonelli P. Tuccillo F. Bond H.M. D'Armiento F.P. Galea E. Palmieri C. Tagliaferri P. Natali P.G. Venuta S. Differential expression of UN1, early thymocyte-associated sialoglycoprotein, in breast normal tissue, benign disease and carcinomas.Anticancer Res. 2002; 22: 2333-2340PubMed Google Scholar). The expression pattern in primary cells suggested that UN1 behaved as an oncofetal antigen with a potential value for cancer immunophenotyping and clinical applications; however, the role of UN1 in tumorigenesis was not further addressed because of the lack of knowledge of its primary structure. monoclonal antibody N-acetyl-galactosamine UDP-N-acetyl-D galactosamine: polipeptide N-acetylgalactosaminyltransferase Maackia Amurensis lectin Peanut Agglutinin diethylaminoethyl reversed-phase solid phase extraction strong-cation exchange nanoscale liquid chromatography tandem mass spectrometry. In this study, we demonstrate that UN1 is the transmembrane CD43 glycoprotein. The primary structure of UN1 was determined by mass spectrometry through the identification of two tryptic peptides that matched the intracytoplasmic domain of CD43. The CD43 identity of UN1 antigen was confirmed by immunological cross-reactivity of the two proteins and strict dependence of UN1 detection on CD43 gene expression. We also show that the single monosaccharide GalNAc-O-linked to the CD43 peptide core is a component of the UN1 epitope, and that CD43 molecules harboring the UN1 epitope are peculiarly expressed in breast, colon, and sigmoid colon carcinomas. The evidence that UN1-type glycosylated CD43 molecules are expressed in cancer tissues supports the possibility of using the UN1 mAb for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis. The plasmid pCMV-CD43 expressing the human CD43 was purchased from Origene Company (Rockville, MD). The CD43 nucleotide sequence was amplified by PCR from pCMV-CD43 using the forward primer CCCAAGCTTATGGCCACGCTTCTCCTTCTCCTT and reverse primer GGGGTACCGAAGGGGCAGCCCCGTCTCC; the PCR product was digested with HindIII and KpnI and ligated to HindIII-KpnI-digested p3xFLAG-CMV-14 (Sigma-Aldrich) to generate p3xFLAG-CMV-CD43 expressing 3xFLAG fused to C terminus of CD43. The UN1 mAb (IgG1, k) was produced by the hybridoma technology and selected for high reactivity with human immature thymocytes (5Tassone P. Bond H. Bonelli P. Tuccillo F. Valerio G. Petrella A. Lamberti A. Cecco L. Turco M.C. Cerra M. et al.UN1, a murine monoclonal antibody recognizing a novel human thymic antigen.Tissue Antigens. 1994; 44: 73-82Crossref PubMed Scopus (12) Google Scholar); immunoglobulins were purified from hybridoma supernatant by affinity chromatography on protein G-Sepharose (GE Healthcare, Uppsala, Sweden). CD43 (C-20) against a C terminus peptide of human CD43, CD43 (MEM-59) against a neuraminidase-sensitive epitope of CD43, donkey anti-goat IgG-HRP and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-FLAG M2 and anti-γ-Tubulin were from Sigma-Aldrich; sheep anti-mouse IgG horseradish peroxidase conjugated was from GE Healthcare. HPB-ALL and CEM, human leukemic T-cell lines expressing the UN1 antigen (5Tassone P. Bond H. Bonelli P. Tuccillo F. Valerio G. Petrella A. Lamberti A. Cecco L. Turco M.C. Cerra M. et al.UN1, a murine monoclonal antibody recognizing a novel human thymic antigen.Tissue Antigens. 1994; 44: 73-82Crossref PubMed Scopus (12) Google Scholar), were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum/phosphate-buffered saline (PBS), 2 mm l-glutamine, 100U/ml penicillin, 100 μg/ml streptomycin, at 5% CO2 and 37 °C. Media were purchased from GIBCO, Invitrogen, Carlsbad, CA. HPB-ALL cells (5 × 106) were transfected with p3xFLAG-CMV-CD43 (40 μg) by electroporation (0.2 kV, 950 μF) using a Bio-Rad Gene Pulser apparatus (Bio-Rad, Hercules, CA), as previously reported (9Mallardo M. Dragonetti E. Baldassarre F. Ambrosino C. Scala G. Quinto I. An NF-kappaB site in the 5′-untranslated leader region of the human immunodeficiency virus type 1 enhances the viral expression in response to NF-kappaB-activating stimuli.J. Biol. Chem. 1996; 271: 20820-20827Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). For flow cytometry, HPB-ALL cells (5 × 106) were electroporated with CD43 siRNA or control siRNA (500 pmol) (Dharmacon, Lafayette, CO), and 48 h later the transfected cells (1 × 106) were 1 h-incubated with the UN1 mAb (1 μg) or CD43 (MEM-59) antibody (1 μg) in 100 μl of PBS at 4 °C, washed in PBS, and incubated with Alexa Fluor 488 rabbit anti-mouse IgG (Molecular Probes, Eugene, Oregon) for 30 min at 4 °C. Following extensive washing in PBS, cells were analyzed by flow cytometry using FacScan (Becton Dickinson, Franklin Lakes, NJ). Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare) by electroblotting in 150 mm glycine-20 mm Tris at 20 mA for 16 h. Following pre-incubation in PBS plus 5% blocking reagent (Bio-Rad), membranes were incubated with primary antibody in PBS plus 5% blocking reagent for 2 h at room temperature, followed by incubation with secondary antibody in PBS plus 5% blocking reagent for 1 h at room temperature. Strips were washed, treated with ECL Western blotting kit (GE Healthcare), and exposed to x-ray film. The UN1 antigen was purified from HPB-ALL membrane extracts by sequential steps, which included anion exchange chromatography, immunoprecipitation, mimotope-based displacement from immunocomplex, and lectin affinity binding, as previously described (10de Laurentiis A. Caterino M. Orrù S. Ruoppolo M. Tuccillo F. Masullo M. Quinto I. Scala G. Pucci P. Palmieri C. Tassone P. Salvatore F. Venuta S. Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.Int. J. Biol. Macromol. 2006; 39: 122-126Crossref PubMed Scopus (6) Google Scholar) (supplemental Fig. S1). Briefly, HPB-ALL cells (2.4 × 1010) were suspended in 230 ml of Buffer R containing 20 mm Tris/HCl, pH 7.8, supplemented with Protease Inhibitor Mixture (Roche, Mannheim, Germany), and sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany). Cell lysate was 10 min-centrifuged at 800 × g to remove nuclei and intact cells. Supernatant was further centrifuged for 2 h at 100,000 × g at 4 °C. The pellet (membrane fraction) was lysed in buffer R containing 1% Triton X-100 for 16 h on ice, and centrifuged for 60 min at 15,000 × g to recover the supernatant. Membrane proteins were separated by anion-exchange chromatography on a diethylaminoethyl (DEAE)-Sepharose Fast Flow (Sigma-Aldrich) column connected to the AKTA FPLC System (GE Healthcare). The column (2.6 cm × 28 cm) was equilibrated with 20 mm Tris/HCl, pH 7.8, containing 0.1% Triton X-100 (buffer A) at a flow rate of 2 ml/min. Membrane proteins (1 g) were applied to the column, washed with buffer A, and bound proteins were eluted with 500 mm NaCl in buffer A; the elution profile was monitored by absorbance at 280 nm. Collected fractions (24 ml) were analyzed for the presence of the UN1 antigen by Western blotting using the UN1 mAb. For UN1 quantization, films were analyzed by scanning densitometry using NIH Image Software (http://rsbweb.nih.gov/nih-image/); specific signal was evaluated as number of pixels/μg of protein. UN1-positive fractions were pooled and dialyzed against PBS buffer containing 0.1% Triton X-100. Dialyzed sample was adjusted to 0.5% Triton X-100 final concentration and preincubated with normal mouse IgG (474 μg) coupled to 4.5 ml of a 50% (v/v) slurry of Protein G-Sepharose (GE Healthcare) on a rotating agitator for 16 h at 4 °C. Following centrifugation at 800 × g, the pellet was recovered as a negative control for mass spectrometry, whereas supernatant (about 240 mg of proteins) was incubated with UN1 mAb (474 μg) bound to 4.5 ml of a 50% (v/v) slurry of Protein G-Sepharose for 16 h on a rotating agitator at 4 °C. The immunocomplex was collected by centrifugation at 800 × g for 5 min at 4 °C, and the pellet was washed and resuspended in 15 ml of PBS buffer containing 0.5% Triton X-100. By screening a random peptide library displayed on filamentous fd phages with UN1 mAb, we previously identified the G-23 peptide (SFAATPHTCKLLDECVPLWPAEG) as a mimotope of the UN1 antigen (10de Laurentiis A. Caterino M. Orrù S. Ruoppolo M. Tuccillo F. Masullo M. Quinto I. Scala G. Pucci P. Palmieri C. Tassone P. Salvatore F. Venuta S. Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.Int. J. Biol. Macromol. 2006; 39: 122-126Crossref PubMed Scopus (6) Google Scholar). The UN1 antigen was displaced from the binding to the UN1 mAb by incubation with G23 peptide at a peptide/UN1 mAb molar ratio of 1 × 103 for 16 h at 4 °C; the displaced UN1 antigen was recovered in supernatant following centrifugation at 800 × g for 5 min at 4 °C, as previously described (10de Laurentiis A. Caterino M. Orrù S. Ruoppolo M. Tuccillo F. Masullo M. Quinto I. Scala G. Pucci P. Palmieri C. Tassone P. Salvatore F. Venuta S. Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.Int. J. Biol. Macromol. 2006; 39: 122-126Crossref PubMed Scopus (6) Google Scholar). The UN1 antigen was separated from contaminant G-23 peptide by 16 h-incubation with biotinylated MAL II (5 μg/ml; Vector Laboratories, Burlingame, CA), for which sialic acid (α2–3) is a ligand, followed by 2 h-incubation with Streptavidin MagneSphere Paramagnetic Particles (Promega, Madison, WI) on a rotating agitator at 4 °C. The UN1 antigen/MAL II complex was collected with a magnetic separator and, following extensive washing in PBS buffer containing 0.5% Triton X-100, the lectin binding to the UN1 antigen was competed with 250 mm sialic acid in 3.6 ml of PBS containing 0.1% Triton X-100, which released the purified UN1 sample for mass spectrometry. The membrane purified UN1 antigen was trichloroacetic acid-precipitated and resuspended in 50 μl of 200 mm Tris-HCl buffer, pH 8.0, containing 0.1% Triton X-100. UN1-positive DEAE fractions immunoprecipitated with IgG were used as control sample of mass spectrometry. Protein samples were 1 h-reduced with 10 mm dithiothreitol (DTT) at 37 °C followed by 1 h-incubation with 30 mm iodoacetamide at 37 °C for cysteine alkylation. Iodoacetamide was neutralized by 20 min incubation with DTT (15 mm final concentration) and calcium chloride was added to 1 mm final concentration. Protein samples were digested with sequencing-grade modified trypsin (3.2 ng/μl) (Sigma-Aldrich) overnight at 37 °C, as previously reported (11Gaspari M. Abbonante V. Cuda G. Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples.J. Sep. Sci. 2007; 30: 2210-2216Crossref PubMed Scopus (18) Google Scholar). To avoid nonionic detergent Triton X-100 contamination, a two-step purification method was applied based on reversed-phase solid phase extraction (SPE) followed by strong-cation exchange (SCX) chromatography (11Gaspari M. Abbonante V. Cuda G. Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples.J. Sep. Sci. 2007; 30: 2210-2216Crossref PubMed Scopus (18) Google Scholar). Briefly, tryptic peptides were purified by reversed-phase SPE with Oasis HLB cartridges (10 mg packing bed, Waters, Milford, MA). SPE column was conditioned with 500 μl of H2O/methanol 1/1 (v/v); the column was equilibrated with 500 μl of H2O/methanol/trifluoroacetic acid 97.9/2/0.1 (v/v/v) (Wash A). The peptide solution (62 μl) was diluted to a final volume of 500 μl in Wash A, and loaded onto the SPE cartridge. Following two consecutive 400 μl washings with Wash A and H2O/methanol/formic acid mixture 97.9/2/0.1 (v/v/v), respectively, peptides were eluted off the SPE cartridge with 250 μl of H2O/methanol/formic acid 19.9/80/0.1 (v/v/v). The eluted peptides were evaporated to dryness in a vacuum centrifuge and stored at 4 °C until use. Peptides were dissolved in 30 μl of H2O/methanol/formic acid mixture 84/15/1 (v/v/v) (Wash SCX) and then applied to SCX Zip TipsTM (Millipore, Billerica, MA), previously equilibrated with Wash SCX. Following extensive washing with Wash SCX, the detergent-free peptide mixture was eluted in 2 μl of H2O/methanol/ammonium hydroxide 80/15/5 (v/v/v) and diluted with 28 μl of H2O/acetonitrile/trifluoroacetic acid 97.95/2/0.05 (v/v/v) (loading pump solvent). Nanoscale liquid chromatography (NanoLC) was performed on Ultimate nanoLC system (Dionex, Sunnyvale, CA), using a valveless setup, as previously described (11Gaspari M. Abbonante V. Cuda G. Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples.J. Sep. Sci. 2007; 30: 2210-2216Crossref PubMed Scopus (18) Google Scholar). Briefly, a peptide sample (10 μl) was loaded at the rate of 12 μl/min onto Integra FritTM trapping column (100 μm bore ID; packing bed length 0.5 cm) (New Objective, Cambridge, MA) in-house packed with 5 μm C18 particles (Dr. Maisch GmbH, Entringen, Germany). Following 4-min washing with loading pump solvent, the trapping column was switched on-line to a Pico FritTM column (50 μm bore ID; packing bed length 10 cm) (New Objective) in-house packed with 3 μm C18 particles. Peptides were separated at 100 nL/min with a binary gradient consisting of H2O/acetonitrile/formic acid/trifluoroacetic acid 97.9/2/0.09/0.01 (v/v/v/v) (mobile phase A), and H2O/acetonitrile/formic acid/trifluoroacetic acid 29.9/70/0.09/0.01 (v/v/v/v) (mobile phase B). Gradient was from 0 to 40% mobile phase B in 25 min. Following 5 min at 95% mobile phase B, the column was re-equilibrated at 0% mobile phase B for 15 min before the following injection. MS detection was performed on a QSTAR XL hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA) operating in positive ion mode, with nanoelectrospray potential at 1300 V, curtain gas at 15 units, collision-activated dissociation gas at 3 units. Information-dependent acquisition was performed by selecting the two most abundant peaks for MS/MS analysis following a full TOF-MS scan from 400 to 1200 m/z lasting 2 s. MS/MS analysis was performed in enhanced mode (2 s/scan). Threshold value for peak selection for MS/MS was 10 counts. MS/MS spectra were converted in Mascot Generic Format by the Analyst software (Applied Biosystems, version 1.1). A script running on Analyst was used to determine peptide charge state and to perform centroiding and de-isotoping on MS/MS data. Data were analyzed on the local Mascot search engine (www.matrixscience.com), version 1.9, against the International Protein Index Human Database (version 3.38; 70856 entries; accessed on January 2008) using the following parameters: MS tolerance 20 ppm; MS/MS tolerance 0.2 Da; fixed modifications carbamidomethyl cysteine; variable modifications methionine oxidized; enzyme trypsin; maximum missed cleavages 1. HPB-ALL membrane extracts (300 μg) were precipitated by adding nine sample volumes of ice-cold acetone for 2 h at - 20 °C. Following centrifugation at 15,000 × g for 30 min, pellets were resuspended in 125 μL of rehydration buffer containing 8 m urea, 2% Triton X-100, 70 mm DTT, 0.8% (v/v) IPG buffer, pH 3–10 nonlinear (GE Healthcare) by mixing (800 rpm, 30 min, 30 °C) in thermomixer (Eppendorf, Hamburg, Germany), and loaded onto 7-cm Immobiline DryStrip gels (pH 3–10 nonlinear) (GE Healthcare). Isoelectric focusing was conducted on an IPGphor (GE Healthcare) with a current limit of 50 μA per strip at 20 °C. Following 16 h-rehydration, the isoelectric focusing was performed by applying 300 V for 30 min, a gradient to 1000 V for 30 min, a gradient to 5000 V for 90 min, and finally 5000 V for 90 min. Following isoelectric focusing, strips were equilibrated in SDS equilibration buffer [50 mm Tris-HCl, pH 8.8, 6 m urea, 30% glycerol, 2% SDS, and 0.002% (w/v) bromphenol blue] containing 2% DTT for 15 min at room temperature followed by further incubation in fresh SDS equilibration buffer containing 2.5% (w/v) iodoacetamide for 15 min at room temperature. The equilibrated strips were then applied to 8% SDS-PAGE for 2-DE gel separation, transferred to nitrocellulose membranes, and immunoblotted with UN1 or anti-CD43 antibodies. HPB-ALL membrane extracts were sequentially digested with glycosidases (CalBiochem brand Deglycosylation Kit, EMD Biosciences, San Diego, CA), according to the manufacturer's instructions. Briefly, protein extract (100 μg) was incubated in 100 μl with α2–3,6,8,9-neuraminidase (2.5 mU) for 16 h at 37 °C followed by sequential digestion with β1,4-galactosidase (1.5 mU), β-N-acetylglucosaminidase (23 mU), β1,3-galactosidase (2000 mU) or endo-α-N-acetylgalactosaminidase (0.6 mU) for 24 h at 37 °C. Aliquots of digested samples were analyzed by Western blotting using CD43 (C-20) and UN1 antibodies. The Consortium for Functional Glycomics (www.functionalglycomics.org) performed the glycan array screening, as previously described (12Blixt O. Head S. Mondala T. Scanlan C. Huflejt M.E. Alvarez R. Bryan M.C. Fazio F. Calarese D. Stevens J. Razi N. Stevens D.J. Skehel J.J. van Die I. Burton D.R. Wilson I.A. Cummings R. Bovin N. Wong C.H. Paulson J.C. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 17033-17038Crossref PubMed Scopus (950) Google Scholar). The printed array version 3.2 consisted of 406 glycans in six replicates (http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh12.shtml). Further details are given in supplemental Fig. S2. Tissues were obtained from patients of the Oncology Surgery, Cancer Center of Excellence, Fondazione Tommaso Campanella, Catanzaro, upon the written consent of the patients and the approval of the Ethics Committee Board. Surgical specimens were derived from cancer tissues of sigmoid colon, colon, and breast; noncancerous tissues were resected 5–10 cm away from the cancer tissues of the same patient. Samples were suspended in OCT compound (Sakura Finetek, Torrance, CA) and frozen at −80 °C. Serial 4-μm sections were obtained from the block using a Leica CM 1950 cryostat (Leica Mycrosystems, Wetzlar, Germany). For optical microscopy, cryostat sections were stained with hematoxylin and eosin (H&E) and analyzed for cell morphology. For confocal microscopy, sections were 1-h incubated with the UN1 mAb (1 μg/ml) or normal mouse IgG (1:400) (Santa Cruz) diluted in PBS on ice in a humidified chamber, washed in PBS, and incubated with rabbit anti-mouse-Alexa Fluor488 (1:400) for 30 min in the dark. Following washing in PBS, sections were fixed and permeabilized using Cytofix/CytoPerm kit (BD Biosciences Pharmingen, San Diego, CA) for 20 min on ice, and then washed in washing buffer (PBS containing 1% BSA, 0.2% Triton X-100). Then, sections were 1-h incubated with CD43 (C-20) antibody (1:200) or normal goat IgG (1:400) (Santa Cruz) in a humidified chamber on ice; following washing, sections were incubated with donkey anti-goat-Alexa Fluor568 (1:400) (Molecular Probes) for 30 min in the dark. Nuclei were stained with DAPI (Sigma-Aldrich) diluted in PBS (2 μg/ml). Following washing with PBS, coverslips were mounted on glass slides by using ProLong Antifade Kit (Molecular Probes). Pictures were captured with a Leica TCS SP2 confocal microscope (Leica Mycrosystems) with a HC PL FLUOTAR 40× Apo PLA oil immersion objective (NA 1.4) in glycerol and acquired with Leica Confocal Software Version 2.61. Z stacks of images were collected using a step increment of 0.2 μm between planes. UN1 was visualized by excitation with an argon laser at 488 nm and photomultiplier tube voltage of 420 mV. CD43 was detected using a krypton laser at 543 nm and photomultiplier tube voltage of 650 mV. Nuclei were detected using an argon laser at 405 nm and photomultiplier tube voltage of 450 mV. Single optical sections using 4× averaging were acquired by sequential scanning to collect the images in three channels. Scan format was 1024 × 1024 pixels. Images were processed with Adobe Photoshop CS. Purification of the UN1 antigen from membranes of HPB-ALL cells was performed in sequential biochemical steps, as previously described (10de Laurentiis A. Caterino M. Orrù S. Ruoppolo M. Tuccillo F. Masullo M. Quinto I. Scala G. Pucci P. Palmieri C. Tassone P. Salvatore F. Venuta S. Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.Int. J. Biol. Macromol. 2006; 39: 122-126Crossref PubMed Scopus (6) Google Scholar) (supplemental Fig. S1). In the first step, the membrane extract was fractionated by anion-exchange chromatography followed by immunoprecipitation of pooled UN1-positive fractions with the UN1 mAb. Then, the UN1 antigen was displaced from the immunocomplex using the G-23 peptide, a UN1 mimotope (10de Laurentiis A. Caterino M. Orrù S. Ruopp
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