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Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes

2015; Elsevier BV; Volume: 14; Issue: 11 Linguagem: Inglês

10.1074/mcp.m115.053983

ISSN

1535-9484

Autores

Dayoung Park, Kristin A. Brune, Anupam Mitra, Alina I. Marusina, Emanual Maverakis, Carlito B. Lebrilla,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. Proliferative stem cells located in the base of intestinal crypts form specialized differentiated cell types as they migrate up the villi. A continuous cell turnover occurs every four to eight days as newly differentiated cells eventually replace older cells at the tip of the villus. Self-renewing intestinal epithelial cells (IECs)1 are highly susceptible to malignant growths, which arise from imbalances in cellular proliferation, differentiation, and apoptosis. If the number of developing cells outbalances the number of mature cells undergoing apoptosis, an abnormal growth of tissue can form which, in some cases, may lead to malignant tumors. Thus, a greater understanding of the molecular details of IEC differentiation may lead to novel insight into the pathophysiology of a variety of GI diseases, including cancer. 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Comparative N-glycan profiling of colorectal cancer cell lines reveals unique bisecting GlcNAc and alpha-2,3-linked sialic acid determinants are associated with membrane proteins of the more metastatic/aggressive cell lines.J. Proteome Res. 2014; 13: 277-288Crossref PubMed Scopus (85) Google Scholar, 17.Balog C.I. Stavenhagen K. Fung W.L. Koeleman C.A. McDonnell L.A. Verhoeven A. Mesker W.E. Tollenaar R.A. Deelder A.M. Wuhrer M. N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.Mol. Cell. Proteomics. 2012; 11: 571-585Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although the mechanisms that control these specific changes are not well understood, it is evident that glycosylation is critical for these cellular transformations. Determining the surface glycosylation changes of IECs in relation to their differentiation may lead to a better understanding of this malignancy. Among the various intestinal epithelial cell types, absorptive enterocytes are the most abundant (18.Crosnier C. Stamataki D. Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control.Nat. Rev. Genet. 2006; 7: 349-359Crossref PubMed Scopus (568) Google Scholar). Differentiation of enterocytes results in the formation of a brush border on their apical surface, a structure that controls membrane-associated hydrolases and nutrient transport. The human cell line Caco-2 has been widely accepted and used as an in vitro model for absorptive intestinal epithelial cells since its establishment in 1974 (19.Fogh J. Wright W.C. Loveless J.D. Absence of HeLa cell contamination in 169 cell lines derived from human tumors.J. Natl. Cancer Inst. 1977; 58: 209-214Crossref PubMed Scopus (570) Google Scholar, 20.Rousset M. 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Briefly, upon differentiation, increased activity was observed for GlcNAc transferase II and V, which are involved in N-glycosylation (27.Brockhausen I. Romero P.A. Herscovics A. Glycosyltransferase changes upon differentiation of CaCo-2 human colonic adenocarcinoma cells.Cancer Res. 1991; 51: 3136-3142PubMed Google Scholar), and for β-3-galactosyltransferase, α-2-fucosyltransferase, sialyltransferase, and β-6-GlcNAc transferase, which are relevant to O-glycan biosynthesis (28.Amano J. Kobayashi K. Oshima M. Comparative study of glycosyltransferase activities in Caco-2 cells before and after enterocytic differentiation using lectin-affinity high-performance liquid chromatography.Arch. Biochem. Biophys. 2001; 395: 191-198Crossref PubMed Scopus (9) Google Scholar). Additionally, differentiation-dependent changes in mRNA expression were observed for α-2,6-sialyltransferase (29.Dall'Olio F. Malagolini N. Guerrini S. Lau J.T. Serafini-Cessi F. Differentiation -dependent expression of human beta-galactoside alpha 2,6-sialyltransferase mRNA in colon carcinoma CaCo-2 cells.Glycoconj. J. 1996; 13: 115-121Crossref PubMed Scopus (35) Google Scholar). A more global glycan analysis was performed by lectin array profiling of the surfaces of Caco-2 cells, demonstrating that lectins which recognize branched fucose and α-2,6-sialic acid were effective at Caco-2 cell binding (30.Zheng T. Peelen D. Smith L.M. Lectin arrays for profiling cell surface carbohydrate expression.J. Am. Chem. Soc. 2005; 127: 9982-9983Crossref PubMed Scopus (175) Google Scholar, 31.Tao S.C. Li Y. Zhou J. Qian J. Schnaar R.L. Zhang Y. Goldstein I.J. Zhu H. Schneck J.P. Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers.Glycobiology. 2008; 18: 761-769Crossref PubMed Scopus (151) Google Scholar). 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Isomer-specific chromatographic profiling yields highly sensitive and specific potential N-glycan biomarkers for epithelial ovarian cancer.J. Chromatogr. A. 2013; 1279: 58-67Crossref PubMed Scopus (76) Google Scholar). Using membrane enrichment methods compatible with mass spectrometry (46.An H.J. Gip P. Kim J. Wu S. Park K.W. McVaugh C.T. Schaffer D.V. Bertozzi C.R. Lebrilla C.B. Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells.Mol. Cell. Proteomics. 2012; 11M111.010660Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we have targeted our analysis to the cell membrane compartment to identify the specific glycan features that accompany Caco-2 cell differentiation. Additionally, the corresponding membrane-localized proteins, from which glycans were released, were identified. Finally, select glycosylation-related mRNA expression levels were quantified during differentiation. Monitoring changes in specific structures is important to identify disease-associated deviations from normal processes of intestinal cell growth and differentiation, which will allow for therapeutic interventions to be initiated earlier. Caco-2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and grown in Eagle's Minimum Essential Medium (EMEM) supplemented with nonessential amino acids, 2 mm l-glutamine, 10% (v/v) fetal bovine serum (Life Technologies, Grand Island, NY), 1 mm sodium pyruvate, 1.5 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were subcultured at 80% confluency and maintained at 37 °C in a humidified incubator with 5% CO2. On days 5, 7, 14, 21, and 24, cells were collected in biological triplicates by scraping. For consistency, ∼2 × 106 cells were used per replicate for analysis. Cells were grown on 13 mm round coverslips and fixed in 2% paraformaldehyde and 2.5% glutaraldehyde (pH 7.2) in 0.1 m phosphate buffer at 4 °C on days 5, 7, 10, 14, 18, 21, and 24. Samples were dehydrated in a critical point dryer (Tousimis, Rockville, MD) and coated with gold using a sputter coater (Ted Pella Inc., Redding, CA). Images were taken on a FEI XL30 electron microscope (FEI, Hillsboro, OR). Alkaline phosphatase (ALP) activity was measured in cell homogenates using a colorimetric assay as per the manufacturer's instructions (Abcam, Cambridge, MA). Cells lysed at different growth times were incubated with 50 μm p-nitrophenyl phosphate (p-NPP) for 60 min. When the substrate is dephosphorylated, p-nitrophenyl is produced, which turns the solution to a visible yellow color. Absorbance readings were taken at 405 nm with an electronic microplate reader (Fisher Scientific, Pittsburgh, PA). Enzyme activities were determined with a standard curve generated using intestinal ALP enzyme (Abcam). ALP activity was measured in mU, where a Unit is defined as the amount of enzyme to hydrolyze 1 μmol of p-NPP per minute at 37 °C. Details of the isolation of the cell membrane fraction have been described previously (47.An H.J. Gip P. Kim J. Wu S. Park K.W. McVaugh C.T. Schaffer D.V. Bertozzi C.R. Lebrilla C.B. Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells.Mol. Cell. Proteomics. 2012; 11M111.010660Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In brief, after harvesting cell pellets were resuspended in homogenization buffer containing 0.25 m sucrose, 20 mm HEPES-KOH (pH 7.4), and 1:100 protease inhibitor mixture (EMD Milipore, Billerica, MA). Cells were lysed on ice using a probe sonicator (Qsonica, Newtown, CT) and lysates were pelleted by centrifugation at 2000 × g for 10 min to remove the nuclear fraction and unlysed cells followed by a series of ultracentrifugation steps at 200,000 × g for 45 min to remove other nonmembrane subcellular fractions. Membrane pellets were suspended with 100 μl of 100 mm NH4HCO3 in 5 mm dithiothreitol and heated for 10 s at 100 °C to thermally denature the proteins. To release the glycans, 2 μl of peptide N-glycosidase F (New England Biolabs, MA) was added to the samples, which were then incubated at 60 °C in a microwave reactor (CEM Corporation, Matthews, NC) for 10 min at 20 watts. After addition of 400 μl of ice-cold ethanol, samples were frozen for 1 h at −80 °C to precipitate deglycosylated proteins and centrifuged for 20 min at 15,000 rpm. The supernatant containing N-glycans was collected and dried. Released N-glycans were purified by solid-phased extraction using porous graphitized carbon packed cartridges (Grace, Deerfield, IL). Cartridges were first equilibrated with alternating washes of nanopure water and a solution of 80% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid in water. Samples were loaded onto the cartridge and washed with nanopure water at a flow rate of 1 ml/min to remove salts and buffer. N-Glycans were eluted with a solution of 40% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid in water and dried. Glycan samples were reconstituted in nanopure water and analyzed using an Agilent nano-LC/ESI QTOF MS system (Agilent, Santa Clara, CA). Samples are introduced to the MS with a microfluidic chip, which consists of enrichment and analytical columns packed with porous graphitized carbon and a nanoelectrospray tip. The programmed 32-min run applies a binary gradient to separate and elute glycans at a flow rate of 0.4 μl/min: (A) 3% (v/v) acetonitrile and 0.1% (v/v) formic acid in water and (B) 90% (v/v) acetonitrile in 1% (v/v) formic acid in water. MS spectra were acquired at 1.5 s per spectrum over a mass range of m/z 500–2000 in positive ionization mode. Mass inaccuracies were corrected with reference masses m/z 622.029, 922.010, 1221.991, and 1521.971. Collision-induced dissociation (CID) was performed with nitrogen gas using a series of collision energies (Vcollision) dependent on the m/z values of the N-glycans, based on the equation: where the slope and offset were set at (1.8/100 Da) V and −2.4 V, respectively. N-Glycan compositions were identified with an in-house retrosynthetic library according to accurate mass (48.Kronewitter S.R. An H.J. de Leoz M.L. Lebrilla C.B. Miyamoto S. Leiserowitz G.S. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.Proteomics. 2009; 9: 2986-2994Crossref PubMed Scopus (105) Google Scholar). This library was constructed based on knowledge of the mammalian N-glycan biosynthetic pathway. Quantitative reproducibility and tandem MS confirmation of library searches were previously validated, enabling rapid and accurate assignment of glycan compositions (36.Hua S. An H.J. Ozcan S. Ro G.S. Soares S. DeVere-White R. Lebrilla C.B. Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers.Analyst. 2011; 136: 3663-3671Crossref PubMed Scopus (125) Google Scholar, 45.Hua S. Williams C.C. Dimapasoc L.M. Ro G.S. Ozcan S. Miyamoto S. Lebrilla C.B. An H.J. Leiserowitz G.S. Isomer-specific chromatographic profiling yields highly sensitive and specific potential N-glycan biomarkers for epithelial ovarian cancer.J. Chromatogr. A. 2013; 1279: 58-67Crossref PubMed Scopus (76) Google Scholar, 48.Kronewitter S.R. An H.J. de Leoz M.L. Lebrilla C.B. Miyamoto S. Leiserowitz G.S. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.Proteomics. 2009; 9: 2986-2994Crossref PubMed Scopus (105) Google Scholar, 49.Ruhaak L.R. Miyamoto S. Kelly K. Lebrilla C.B. N-Glycan profiling of dried blood spots.Anal. Chem. 2012; 84: 396-402Crossref PubMed Scopus (61) Google Scholar). Signals above a signal-to-noise ratio of 5.0 were filtered and deconvoluted using MassHunter Qualitative Analysis B.03.01 software (Agilent). Deconvoluted masses were compared with theoretical masses using a mass tolerance of 20 ppm and a false discovery rate of 0.6%. Relative abundances were determined by integrating peak areas for observed glycan masses and normalizing to the summed peak areas of all glycans detected. Statistical evaluation of glycan abundances were performed using a two-tailed, unpaired Student's t test. Extracted membrane proteins were resolubilized in 8 m urea. After reduction of proteins with dithiothreitol and alkylation with iodoacetamide, samples were diluted to 1 m urea and incubated with trypsin at 37 °C overnight. The resulting peptides were enriched using C18 packed pipette tips (Agilent). Columns were washed and conditioned with acetonitrile followed by 0.1% (v/v) trifluoroacetic acid in water. Samples were introduced to the column, washed with 0.1% (v/v) trifluoroacetic acid in water, and eluted with a solution of 80% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid in water. Peptides were concentrated in vacuo prior to mass spectrometric analysis. Membrane peptides were reconstituted in 2% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid in water and separated using a reverse-phase Michrom Magic C18AQ column (200 μm, 150 mm) coupled with a Q Exactive Plus mass spectrometer through a Proxeon nano-spray source (Thermo Scientific, Fremont, CA). A 90-min binary gradient was applied at a flow rate of 2 μl/min with (A) 0.1% (v/v) formic acid in water and (B) 100% acetonitrile: 0–75 min, 5–35% (B); 75–82 min, 35–80% (B); 82–84 min, 80% (B); 84–85 min, 80–5% (B). Per acquisition, the instrument was run in a data-dependent mode as follows: spray voltage, 2.2 kV; ion transfer capillary temperature, 200 °C; full scan mass range, m/z 350–1600; MS automatic gain control, 1 × 106; MS maximum injection time, 30 ms; MS/MS automatic gain control, 5 × 104; MS/MS maximum injection time, 50 ms; precursor resolution, 70,000; product ion resolution, 17,500; precursor ion isolation width of m/z 1.6; normalized collision energy, 27. Raw data were exported using xCalibur, version 2.0 (Thermo Scientific). Proteins were identified from the tandem mass spectra using X! Tandem (2015.04.01) against the Ensembl v76 human protein database (86137 entries) (50.Craig R. Beavis R.C. TANDEM: matching proteins with tandem mass spectra.Bioinformatics. 2004; 20: 1466-1467Crossref PubMed Scopus (1989) Google Scholar) with the following parameters: mass tolerances of 10 ppm for the precursor and 20 ppm for fragment ions; peptide probability >0.95 using PeptideProphet (51.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3897) Google Scholar); carbamidomethylation of cysteine as a fixed modification; oxidation of methionine and tryptophan and deamidation of asparagine and glutamine as variable modifications; two missed cleavage sites; at least two unique peptides. Protein identifications were filtered at a 1% false discovery rate and were accepted if E-values were less than or equal to 0.02. Additional protein information was retrieved through functional annotation analysis using the PANTHER database (52.Thomas P.D. Kejariwal A. Campbell M.J. Mi H. Diemer K. Guo N. Ladunga I. Ulitsky-Lazareva B. Muruganujan A. Rabkin S. Vandergriff J.A. Doremieux O. PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification.Nucleic Acids Res. 2003; 31: 334-341Crossref PubMed Scopus (509) Google Scholar). N-glycosylation sites were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc). Undifferentiated, partially differentiated, and fully differentiated Caco-2 cells (2 × 106 cells per preparation) were scraped on day 5, 14, and 21 respectively and washed twice with PBS (1×), followed by resuspension in RNAlater (Life Technologies). Briefly, total RNA were extracted using RNeasy plus mini kit (Qiagen, Valencia, CA) and the quantity and quality of RNA were determined by using Qubit Fluorometer (Life Technologies) and TapeStation 2200 (Agilent) following manufacturer's protocol. Total RNA were reverse transcribed to cDNA using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA) following manufacturer's instructions. The predesigned

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