Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells
2011; Elsevier BV; Volume: 11; Issue: 4 Linguagem: Inglês
10.1074/mcp.m111.010660
ISSN1535-9484
AutoresHyun Joo An, Phung Gip, Jae‐Han Kim, Shuai Wu, Kun Wook Park, Cheryl T. McVaugh, David V. Schaffer, Carolyn R. Bertozzi, Carlito B. Lebrilla,
Tópico(s)Infant Nutrition and Health
ResumoMost cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. Glycosylation is the process by which oligosaccharides, termed glycans, are appended onto membrane and secreted proteins and lipids. It is the most common and complex form of post-translational modification, with ∼50% of all eukaryotic proteins glycosylated (1Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim. Biophys. Acta. 1999; 1473: 4-8Crossref PubMed Scopus (1492) Google Scholar, 2Lebrilla C.B. Mahal L.K. Post-translation modifications.Curr. Opin. Chem. 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Human embryonic stem cells (hESCs) 1The abbreviations used are:hESChuman embryonic stem cellsSPEsolid phase extractionHexhexoseHexNAcN-acetylhexosamineNeuAcsialic acidFucfucoseManmannoseGCCgraphitized carbon cartridgeICRion cyclotron resonanceIRMPDinfrared multiphoton dissociationCon ACanavaliaensiformisGNAGalanthusnivalisMan9GlcNAc2Man9Man8GlcNAc2Man8Man7GlcNAc2Man7JACArtocarpusintegrifolia. 1The abbreviations used are:hESChuman embryonic stem cellsSPEsolid phase extractionHexhexoseHexNAcN-acetylhexosamineNeuAcsialic acidFucfucoseManmannoseGCCgraphitized carbon cartridgeICRion cyclotron resonanceIRMPDinfrared multiphoton dissociationCon ACanavaliaensiformisGNAGalanthusnivalisMan9GlcNAc2Man9Man8GlcNAc2Man8Man7GlcNAc2Man7JACArtocarpusintegrifolia. are of particular biomedical interest because they hold enormous potential for regenerative medicine and drug discovery. As a model system, they can also contribute to the understanding of human development and potentially help to guide cancer research because hESCs and cancer cells share similar characteristics (17Lovell-Badge R. The future for stem cell research.Nature. 2001; 414: 88-91Crossref PubMed Scopus (105) Google Scholar, 18Nagano K. Yoshida Y. Isobe T. Cell surface biomarkers of embryonic stem cells.Proteomics. 2008; 8: 4025-4035Crossref PubMed Scopus (60) Google Scholar, 19McNeish J. Embryonic stem cells in drug discovery.Nat. Rev. Drug Discov. 2004; 3: 70-80Crossref PubMed Scopus (160) Google Scholar). Therefore, structural elucidation of the components present on hESC membranes may provide a basis for understanding their role in hESC maintenance and differentiation. Recent studies suggest that glycans on the plasma membrane of hESCs change during differentiation; these changes can have profound effects on cellular function (20Lanctot P.M. Gage F.H. Varki A.P. The glycans of stem cells.Curr. Opin. Chem. Biol. 2007; 11: 373-380Crossref PubMed Scopus (133) Google Scholar) and could be harnessed to meet the need to identify cell surface markers for isolating and purifying specific cell populations for therapeutic application. Indeed, one of the earliest pluripotent stem cell markers is SSEA-1 (stage-specific embryonic antigen-1), a glycan, otherwise known as Lexis X antigen, expressed on mouse embryonic stem cells (21Solter D. Knowles B.B. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).Proc .Natl. Acad. Sci. U.S.A. 1978; 75: 5565-5569Crossref PubMed Scopus (1127) Google Scholar, 22Gooi H.C. Feizi T. Kapadia A. Knowles B.B. Solter D. Evans M.J. Stage-specific embryonic antigen involves α-1-]3 fucosylated type-2 blood-group chains.Nature. 1981; 292: 156-158Crossref PubMed Scopus (494) Google Scholar). Two other antigenic epitopes discovered were SSEA-3 and SSEA-4 (23Kannagi R. Cochran N.A. Ishigami F. Hakomori S. Andrews P.W. Knowles B.B. Solter D. Stage-specific embryonic antigens (SSEA-3 and SSEA-4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.EMBO J. 1983; 2: 2355-2361Crossref PubMed Scopus (438) Google Scholar), which are both glycolipids that have become the most common cell surface markers used to characterize hESCs (24Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12274) Google Scholar). human embryonic stem cells solid phase extraction hexose N-acetylhexosamine sialic acid fucose mannose graphitized carbon cartridge ion cyclotron resonance infrared multiphoton dissociation Canavaliaensiformis Galanthusnivalis GlcNAc2Man9 GlcNAc2Man8 GlcNAc2Man7 Artocarpusintegrifolia. human embryonic stem cells solid phase extraction hexose N-acetylhexosamine sialic acid fucose mannose graphitized carbon cartridge ion cyclotron resonance infrared multiphoton dissociation Canavaliaensiformis Galanthusnivalis GlcNAc2Man9 GlcNAc2Man8 GlcNAc2Man7 Artocarpusintegrifolia. Cell surface glycosylation may play an important role in development and may provide important new sources of markers for differentiation. Studies regarding the glycosylation of stem cell surfaces are limited. Wearne et al. (12Wearne K.A. Winter H.C. O'Shea K. Goldstein I.J. Use of lectins for probing differentiated human embryonic stem cells for carbohydrates.Glycobiology. 2006; 16: 981-990Crossref PubMed Scopus (58) Google Scholar) recently reported the use of fluorescence-labeled lectins to identify a number of specific structural motifs including mannose residues, α2,3- and α2,6-linked N-acetylneuraminic acid, α1,6-linked l-fucosyl, and β-d-galactosyl groups. In addition, they also found a number of common antigens including T, Tn, and sialyl-Tn. A more structurally intensive study of whole stem cell glycosylation was reported by Satomaa et al. (25Satomaa T. Heiskanen A. Mikkola M. Olsson C. Blomqvist M. Tiittanen M. Jaatinen T. Aitio O. Olonen A. Helin J. Hiltunen J. Natunen J. Tuuri T. Otonkoski T. Saarinen J. Laine J. The N-glycome of human embryonic stem cells.BMC Cell Biol. 2009; 10: 42Crossref PubMed Scopus (82) Google Scholar). The cell surface studies were limited to lectins, which cannot be used quantitatively. Structural methods including nuclear magnetic resonance, mass spectrometry, and glycosidase digestion were used on whole cells where they showed that the N-glycan profile was rich in high mannose glycans as well as complex type structures that are terminated by both α2,3- and α2,6-sialylated oligosaccharides and fucosyl structures. Here we characterize the N-glycan profile of human embryonic stem cell membrane glycans using a method that enables specific detection and quantitation and acquisition of structural information. Interestingly, hESCs have high levels of high mannose glycans on the cell surface, which is largely unprecedented in mammalian cells. Moreover, the hESCs were particularly rich in Man8 and Man9 structures, including Man9 with terminal glycoside still intact. This unusual glycomic signature might have functional implications, as well as practical utility in the characterization and isolation of hESCs. The National Institutes of Health-approved hESC lines, H1 and HSF-6, were maintained under feeder-free conditions using a chemically defined medium, X-VIVO 10 (Cambrex, Walkersville, MD), supplemented with human recombinant growth factors fibroblast growth factor and transforming growth factor-β1 (80 and 0.5 ng/ml, respectively; R & D Systems (10La Belle J.T. Gerlach J.Q. Svarovsky S. Joshi L. Label-free impedimetric detection of glycan-lectin interactions.Anal. Chem. 2007; 79: 6959-6964Crossref PubMed Scopus (112) Google Scholar). The cells are propagated on hESC-qualified Matrigel-coated plates (BD Biosciences). Medium was exchanged daily after the first 48 h in culture, and the cells were passaged every 5–7 days using collagenase IV (200 units/ml; Invitrogen) and mechanically removed. For glycan analysis, the cells were collected after collagenase IV treatment, centrifuged, washed with PBS (Invitrogen) pelleted, and frozen on dry ice. Approximately 50 million cells were counted and collected at different passage numbers to obtain biological triplicates. Karyotype analysis was routinely performed and indicated that all samples were diploid and had no chromosomal abnormalities. The cells were routinely stained with pluripotent markers Oct4 and SSEA-4. IMR-90 human fibroblast cells (University of California Berkeley Tissue Culture Facility) were grown in Dulbecco's modified Eagle's high glucose medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). The cells were passaged every 3 days using trypsin 0.25% and EDTA solution (Invitrogen). For glycan analysis, IMR-90s were collected using 0.5 mm EDTA, centrifuged, and washed with PBS, pelleted, and frozen on dry ice. MCF10A human breast epithelial cells (ATCC, CRL-10317) were grown in Dulbecco's modified Eagle's medium with high glucose (Invitrogen; 31053) supplemented with 10% fetal bovine serum (PAA Laboratories), penicillin/streptomycin (10 units/ml and 10 μg/ml; Invitrogen), 2.5 μg/ml fungizone (Invitrogen), 20 ng/ml epidermal growth factor (Biovision), 0.5 μg/ml hydrocortisone (VWR), 100 ng/ml cholera toxin (VWR), and 10 μg/ml recombinant human insulin (Sigma). MCF10As were passaged weekly by trypsinization. For glycan analysis, the cells were detached using a cell scraper, washed with PBS, pelleted, and frozen on dry ice. Approximately 50 million cells were counted and collected from both cell lines at different passages to obtain biological triplicates. Membrane extraction was performed using ultracentrifugation. The pellets were thawed on ice with the addition of a homogenization buffer consisting of 0.25 m sucrose, 20 mm Hepes-KOH, pH 7.4, and protease inhibitor mixture (1:100; Calbiochem/EMD Chemicals). The cells were sonicated on ice, and cell lysates were centrifuged at 1,000 × g for 10 min to remove the nuclear fraction and debris. The supernatant was collected, and additional homogenization buffer was added for ultracentrifugation at 200,000 × g for 45 min at 4 °C to remove the cytoplasmic fraction. The pellets were resuspended in 0.2 m Na2CO3 (pH 11) to break up the microsomes. The samples were spun twice more at 200,000 × g for 45 min to wash the samples of the cytoplasmic fraction. The supernatant was removed, and the membrane fractions were frozen at −20 °C. All of the fractions (nuclear, cytoplasmic, and membranes) were analyzed by SDS-PAGE followed by Western blot using known organelle-specific markers for the nucleus (nuclear pore complex proteins; Covance), endoplasmic reticulum (Bip/GRP78; BD Biosciences), cytosol (α-tubulin; Sigma), and the plasma membrane (CD49b; BD Biosciences). Primary antibodies were probed with a horseradish peroxidase conjugated anti-mouse secondary antibody (IgG). Before Western blot analysis, membrane pellets were resuspended in 4% SDS buffer, and protein concentration was determined by the BCA assay (Pierce). The samples (4 μg) were separated by SDS/PAGE (4–12%; Bio-Rad). For the analysis of N-glycans, 100 μl of 100 mm ammonium bicarbonate (NH4HCO3), 5 mm DTT (Promega) was added to the samples and heated to 100 °C for 2 min to denature the protein. After cooling at room temperature, 2 μl of peptide N-glycosidase F (New England Biolabs) was added to the mixture (pH 7.5) and incubated at 37 °C for 12 h in a water bath. 800 μl of chilled ethanol was added, and the mixture was frozen at −80 °C for 1 h and then centrifuged to separate glycans from deglycosylated proteins. The supernatant was completely dried down to remove the ethanol prior to solid phase extraction (SPE) using a graphitized carbon cartridge (GCC; Alltech). For O-glycan analysis, alkaline borohydride solution (500 μl, mixture of 1.0 m sodium borohydride and 0.1 m sodium hydroxide) was added to the membrane fraction. The mixture was incubated at 42 °C for 12 h in a water bath. The addition of 1.0 m hydrochloric acid solution was slowly added in ice bath to stop the reaction and destroy excess sodium borohydride. Released N- and O-glycans were purified and enriched by SPE-GCC. Prior to use, graphitized carbon cartridge (150 mg of bed weight, 4 ml of cartridge volume) was washed with nanopure water followed by 80% ACN in 0.05% TFA (v/v) and again with nanopure water. Glycan solutions were applied to the GCC cartridge and subsequently washed with several cartridge volumes of nanopure water at a flow rate of 1 ml/min to remove salts. Glycans were eluted stepwise with 10% ACN in H2O (v/v), 20% ACN in H2O (v/v), and 40% ACN in 0.05% TFA in H2O (v/v). Each fraction was collected and concentrated in vacuo prior to mass spectrometry analysis. Fractions were reconstituted in nanopure water prior to MS analysis. Mass spectra were recorded on a Fourier transform ion cyclotron resonance (ICR) mass spectrometer with an external source HiResMALDI (IonSpec Corporation) equipped with a 7.0 Tesla magnet. The HiResMALDI was equipped with a pulsed Nd:YAG laser (355 nm). 2,5-Dihydroxy-benzoic acid was used as a matrix (5 mg/100 ml in 50% ACN:H2O) for both positive and negative modes. A saturated solution of NaCl in 50% ACN in H2O was used as a cation dopant to increase signal sensitivity. The glycan solution (0.7 μl) was applied to the MALDI probe followed by matrix solution (0.7 μl). The sample was dried under vacuum prior to mass spectrometric analysis. A desired ion was readily selected in the analyzer with the use of an arbitrary wave form generator and a frequency synthesizer. A continuous wave Parallax CO2 laser with 20-W maximum power and 10.6-μm wavelength was installed at the rear of the magnet and was used to provide the photons for IRMPD. The laser beam diameter is 6 mm as specified by the manufacturer. The laser beam was expanded to 12 mm by means of a 2× beam expander (Synrad) to ensure complete irradiation of the ion cloud through the course of the experiment. The laser was aligned and directed to the center of the ICR cell through a BaF2 window (Bicron Corporation). Photon irradiation time was optimized to produce the greatest number and abundance of fragment ions. The laser was operated at an output of ∼13 W. GCC fractions were analyzed using a microfluidic HPLC-ChIP-TOF MS (Agilent, CA). The microfluidic HPLC-Ch consists of an enrichment column, an LC separation column packed with porous graphitized carbon, and a nanoelectrospray tip. Separation was performed by a binary gradient A: 3% acetonitrile in 0.1% formic acid solution and B: 90% acetonitrile in 0.1% formic acid solution. The column was initially equilibrated and eluted with the flow rate at 0.3 μl/min for nanopump and 4 μl/min for capillary pump. The 65-min gradient was programmed as follows: 2.5–20 min, 0–16% B; 20–30 min, 16–44% B; 30–35 min, B increased to 100%, then continued 100% B to 45 min, finally 0% B for 20 min to equilibrate the ChIP column before next sample injection. Each possible composition of N-glycans was identified with the in-house program GlycoX (26An H.J. Tillinghast J.S. Woodruff D.L. Rocke D.M. Lebrilla C.B. A new computer program (GlycoX) to determine simultaneously the glycosylation sites and oligosaccharide heterogeneity of glycoproteins.J. Proteome Res. 2006; 5: 2800-2808Crossref PubMed Scopus (54) Google Scholar) and the N-glycan library (27Kronewitter 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) according to the mass tolerance with additional retention times and abundance information noted at the same time. The membrane proteins were dried and solubilized with 60 μl of 8 m urea. The samples were then reduced with DTT and alkylated with iodoacetamide. After dilution in 180 μl of water, an overnight digestion with trypsin was performed. The peptides were then concentrated and desalted using C18 peptide trap (MichromeBioResources, Inc. Auburn, CA) before LC separation and online MS/MS. A nanoLC-2Dsystem (Eksigent, Dublin, CA) coupled with an LTQ ion trap mass spectrometer (Thermo Finnigan) was used with a homemade fritless reverse phase microcapillary column (75 μm × 180 mm; packed with Magic C18AQ, 3 μm 100 Å: Michrom Bio Resources) and vented column configuration. Digested samples were transferred from the autosampler to the online trap column (0.15 mm × 20 mm; packed with Magic C18AQ, 3 μm, 100 Å) and desalted. The peptides were eluted from the trap and separated on the capillary column using a reverse phase gradient at a flow rate of 300 nl/min and directly electrosprayed into the mass spectrometer. A cycle of one MS survey scan followed by 10 MS/MS scans was repeatedly acquired over the LC gradient. Dynamic exclusion for 1-min duration was utilized. Buffers were 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). A 107-min gradient (2–40% B for 95 min, followed by 40–80% B for 12 min) was used. Protein identification based on LC-MS/MS was performed using X! Tandem with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 1.8 Da. Iodoacetamide derivatization of cysteine was specified as a fixed modification. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified peptides. hESCs were fixed with 2% paraformaldehyde and rinsed three times in PBS. The cells were blocked with staining buffer (2% fetal bovine serum in PBS) and then stained with pluripotent marker SSEA-4 (2.5 μg/500 μl/well; Millipore) for 30 min at room temperature. The wells were rinsed in PBS before adding FITC-conjugated lectins (20 μg/ml; EY Labs) and Alexa Fluor 594-conjugated goat anti-mouse secondary antibody to SSEA-4 (1:400; Invitrogen). The wells were rinsed in PBS and stained with a solution of 1× Hoechst 23187 (Sigma) as a nuclear stain and analyzed using an Olympus IX71 fluorescent microscope. Control wells were stained with mouse IgG3 isotype (Invitrogen), and the lectins were incubated with their respective inhibitory sugar. hESCs were collected after incubation with collagenase IV (200 units/ml; Invitrogen) and mechanically removed. The colonies were dissociated into single-cell suspensions in 0.5 mm EDTA, then filtered through a 40-micron cell strainer, and counted. The cells were blocked with staining buffer (2% fetal bovine serum in PBS) and then stained with pluripotent marker SSEA-4 (2.5 μg/500 μl/500,000 cells; Millipore) for 30 min on ice. The cells were washed and stained with APC-conjugated goat anti-mouse secondary antibody to SSEA-4 and 5, 10, 20, or 40 μg/ml of the following FITC-conjugated lectins: Canavaliaensiformis or Galanthusnivalis (EY Labs). To validate binding specificity, hESCs were also stained with lectins preincubated with sugar haptens: methy-α-mannoside and yeast mannan, respectively (Sigma). After 30 min on ice, the cells were washed and resuspended in staining buffer with propidium iodide to distinguish dead cells from live cells. Flow cytometry (BD FACs Calibur from BD Biosciences) was performed, and the data were analyzed using FlowJo software (TreeStarInc). At least three independent assays were carried out for each lectin. The final quantitation represents live hESCs that were double-labeled with SSEA-4 and FITC-conjugated lectins. hESCs were also stained with mouse IgG3 isotype (Invitrogen), as a control for SSEA-4 labeling. The experimental strategy, including: (i) the purification of cell membrane fractions from whole cell lysates by ultracentrifugation, (ii) release and enrichment of surface glycans by SPE using a graphitized carbon, (iii) glycan profiling by high performance mass spectrometry, and (iv) isomer separation and quantitation by nanoLC, is outlined in supplemental Fig. S1. These methods were developed and streamlined to profile cell membrane glycans effectively and selectively by mass spectrometry. As further described below, the quantitation and localization of glycans on the hESCs membrane were also validated by flow cytometry and immunocytochemistry. Selective isolation of membrane fractions from whole cells with a compatible buffer that allows MS detection of glycans was a crucial component of the methodology development. Although plasma membrane purification would have been a more rigorous approach, the sensitivity of the method at this point is still insufficient for the analysis of glycans from less than 50 million cells. Ultracentrifugation was employed as the technique to isolate membrane fractions from cells. However, although mass spectrometry can be a powerful method for analyzing biomolecules because of its intrinsic speed and sensitivity, coupling the two techniques has proven challenging in part because of such inc
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