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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2010 Elsevier Inc.
- ISSN
- 1535-9476
- eISSN
- 1535-9484
- DOI
- 10.1074/mcp.m900559-mcp200
- Publisher site
- See Article on Publisher Site
Abstract
Research Threshold in Stage-specific Embryonic Glycotypes Uncovered by a Full Portrait of Dynamic N-Glycan Expression during Cell Differentiation* Maho Amano‡, Misa Yamaguchi‡, Yasuhiro Takegawa‡, Tadashi Yamashita‡, Michiyo Terashima§, Jun-ichi Furukawa‡, Yoshiaki Miura¶, Yasuro Shinohara‡, Norimasa Iwasaki§, Akio Minami§, and Shin-Ichiro Nishimura‡¶ has contributed to basic understanding of ESC biology (1). Although various glycoforms appear to participate inde- pendently in multiple molecular interactions in cellular Functional genomics and proteomics during cellular differen- adhesion that contribute to embryogenesis and organo- tiation have also been intensively studied during recent years genesis, a full portrait of the glycome diversity and the (2–4). However, it was suggested that there is no significant effect of the structural variations of cellular glycoforms on change of the protein expression profile in the differenti- individual cell stages in proliferation and differentiation ated cells in comparison with that of undifferentiated (progen- remain unclear. Here we describe a novel concept for the itor) cells. It is generally recognized that work on ESCs has characterization of dynamic glycoform alteration during proceeded with a general lack of standards for adequate cell differentiation by means of “glycoblotting-based cel- quantification of ESCs (5). It is likely that this has led to lular glycomics,” the only method allowing for rapid and inconsistencies and difficulties in reproducing work from in- quantitative glycan analysis. We demonstrated that pro- cesses of dynamic cellular differentiation of mouse em- dependent laboratories because this field has relied upon a bryonic carcinoma cells, P19CL6 and P19C6, and mouse variety of antibodies against a few cell surface antigens, which embryonic stem cells into cardiomyocytes or neural cells has been useful but not adequate. Therefore, it should be can be monitored and characterized quantitatively by pro- noted that there has been no reliable set of molecular markers filing entire N-glycan structures of total cell glycoproteins. that can easily establish the quality and differentiation status Whole N-glycans enriched and identified by the glycob- of a cell line. lotting method (67 glycans for P19CL6, 75 glycans for Numerous important roles of mammalian glycans are now P19C6, and 72 glycans for embryonic stem cells) were evident, and the variation in the cellular glycome is a molec- profiled and bar-coded quantitatively with respect to the ular basis for modulating dynamic cellular mechanisms such ratio of subgroups composed of characteristic glyco- forms, namely glycotypes. Molecular & Cellular Pro- as cell-cell adhesion, cell activation, and malignant alterations teomics 9:523–537, 2010. (6–9). Structural variations of the glycome in cell surface glycoproteins and/or glycosphingolipids appear to produce some useful biomarkers such as stage-specific embryonic The global characterization of the genome, transcriptome, antigens and ligands for endogenous lectins during cell pro- epigenome, and proteome of embryonic stem cells (ESCs) liferation and differentiation (10–13). Loss of some key N- glycans or expression of unusual N-glycans disrupts normal From the ‡Laboratory of Advanced Chemical Biology, Graduate cell-cell adhesion in early mammalian embryos that is asso- School of Life Science, and Frontier Research Center for Post- ciated with fertilization (14–17). In addition, it seems likely that Genome Science and Technology, Hokkaido University, N21 W11, cellular responsiveness to growth or arrest is greatly depen- Kita-ku, Sapporo 001-0021, Japan, §Department of Orthopedic Surgery, Hokkaido University School of Medicine, N15 W7, Kita- dent on total N-glycan number and the degree of branching of ku, Sapporo 060-8638, Japan, and ¶Ezose Sciences, Inc., cell surface glycoproteins (12). Judging from the fact that Pine Brook, New Jersey 07058 N-glycans found in most proteins are an overwhelming ma- Received, November 18, 2009, and in revised form, December 14, jority of the glycome compared with other subgroups such as O-glycans classified as mucin glycoproteins or glycosphingo- Published, MCP Papers in Press, December 14, 2009, DOI 10.1074/mcp.M900559-MCP200 lipids, our attention should be focused on the expression The abbreviations used are: ESC, embryonic stem cell; iPS, in- profiles of cellular N-glycans. duced pluripotent stem; FBS, fetal bovine serum; RA, retinoic acid; We hypothesized that drastic changes in the ratio of some HRP, horseradish peroxidase; NeuGc, N-glycolylneuraminic acid; key subtypes of the N-glycans with mutual core structure HM, high mannose type; MF, monofucosylated type; DF, difucosy- lated type; O, others; BS, bisect type. against total N-glycan expression level may be crucial to This is an open access article under the CC BY license. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 9.3 523 This paper is available on line at http://www.mcponline.org Glycoblotting-based Cellular Glycomics of ESCs an intermediate of differentiation, by treatment with 0.05% trypsin, switch cell stages/types during cell differentiation in which EDTA (Invitrogen) and were replated onto a poly-L-lysine-coated tis- generated N-glycan subtypes must exchange their major 4 2 sue culture dish (6 cm inner diameter) at a density of 5 10 cells/cm partner molecules in differentiated cell adhesion, namely cell in Dulbecco’s modified Eagle’s medium/F-12 medium (Invitrogen) surface carbohydrate-binding proteins or complementary gly- containing N-2 supplement and fibronectin. The cells were then cans in sugar-sugar interaction (18). In other words, there allowed to adhere and were cultured for 5 days. Cardiac muscle differentiation of P19CL6 cells was induced by DMSO under ad- might be a significant “threshold” or “critical point” in the herent conditions as described previously (27). Briefly, 3.7 10 expression level of the key N-glycan subtypes against cell cells were plated onto a tissue culture dish (6 cm inner diameter) surface area to initiate this dynamic cellular differentiation. For (Corning) and cultured in a standard medium containing 1% DMSO example, it is well documented that Lewis X trisaccharide for 16 days. As a control, undifferentiated cells were cultured in antigen (SSEA-1), a prominent member of the Lewis blood standard medium for 16 days. The media were changed every 2 group antigen family, is one of the most important subtypes days. All experiments were performed in triplicate and repeated twice independently (n 6). Mouse ESCs were differentiated under that have key functional roles during developmental pro- conditions reported previously (30, 31). cesses or cancer progression (10, 19). Glycosphingolipids Basic Protocol of Glycoblotting-based Quantitative Cellular N-Gly- SSEA-3 and SSEA-4 are also among the most commonly comics—A preliminary trial for the cellular N-glycomics was reported utilized markers to characterize ESCs, and they could also be previously in human prostate cancer cells (PC-3) and normal human classified as a sort of subtype composed of globoseries core prostate epithelial cells on the basis of the protocol designed for TM human serum N-glycomics using the BlotGlyco ABC bead, a pro- oligosaccharide structures (13). Sialic acid-containing oligo- totype bead prepared by conjugating N-(2-aminobenzoyl)cysteine saccharides are also supposed to be involved in some essen- hydrazide and thiopropyl-Sepharose 6B (26). However, we had to tial subtypes having various functions as potential markers in re-examine and establish a comprehensive protocol feasible for cell differentiation or malignant alteration (20). Therefore, it is mammalian cellular glycomics using BlotGlyco H, a commercially not surprising that real time monitoring of entire N-glycan available synthetic polymer bead (Sumitomo Bakelite Co., Ltd., To- expression levels in the course of proliferation and differenti- kyo, Japan) (see Fig. 1), because our recent studies on the functional glycomics using BlotGlyco H bead have demonstrated improved ation, a full portrait of the cellular glycoforms, may allow for performance of this new platform in terms of quantification, repro- the identification of target cells and assessment of the quality ducibility, and application (25). of individual stem cells and differentiated cells. The advent of Release of total N-glycans was carried out directly using whole cell such a versatile and comprehensive protocol for quantitative lysates as follows. After inducing differentiation, P19C6 and ESCs cellular glycomics is now urgently needed because it should were cultured by using a poly-L-lysine-coated tissue culture dish (6 cm inner diameter) for the appropriate days indicated above, then greatly contribute to the quality control of human ESCs and a were scraped in PBS containing 10 mM EDTA, and washed with PBS. variety of human induced pluripotent stem (iPS) cells in terms Following suspension in PBS, cells were lysed by incubation with 1% of the warranty of safety and reproducibility of required stem Triton X-100 for 1 h on ice. The lysates were centrifuged at 15,000 cell engineering (21–23). rpm for 10 min at 4 °C, and the obtained supernatant was added to Until now, technical limitations have restricted acquisition cold acetone (1:4) to precipitate proteinaceous materials. The precip- of the total glycan structures of mammalian cells and evalu- itates were collected by centrifugation at 12,000 rpm for 15 min at 4 °C followed by serial washing with acetonitrile. The resulting pre- ation of the cell type-specific glycoforms (24). We report cipitates were dissolved in 50 lof80mM ammonium bicarbonate herein that the glycoblotting method (24), a PCR-like technol- containing 0.2% of 1-propanesulfonic acid, 2-hydroxyl-3-myrista- ogy developed for rapid and large scale enrichment analysis mido and incubated at 60 °C for 10 min. The solubilized proteina- of human serum glycans (25, 26), can be used for rapid and ceous materials were reduced by 10 mM DTT at 60 °C for 30 min quantitative cellular glycomics to monitor dynamic glycoform followed by alkylation with 20 mM iodoacetamide by incubation in the dark at room temperature for 30 min. The mixture was then treated alteration during differentiation of mouse embryonic carci- with 400 units of trypsin (Sigma-Aldrich) at 37 °C overnight followed noma cells (P19CL6 and P19C6 cells) and mouse ESCs. by heat inactivation of the enzyme at 90 °C for 10 min. After cooling to room temperature, N-glycans of glycopeptides were released from MATERIALS AND METHODS trypsin-digested samples by incubation with 2 units of peptide-N- glycosidase F (Roche Applied Science) at 37 °C overnight. Then the Cell Culture and Differentiation—P19C6 and P19CL6 cells were sample mixture was dried by SpeedVac and stored at 20 °C until subcloned from pluripotent mouse embryonal carcinoma, and both use. Approximately 1–5 10 cells (200 g of total protein) corre- were obtained from RIKEN Cell Bank (Ibaragi, Japan) (27, 28). P19C6 sponding to cell confluence on the culture dish (6 cm inner diameter) and P19CL6 were maintained with Dulbecco’s modified Eagle’s me- were required for this procedure. dium (Sigma-Aldrich) supplemented with 15% fetal bovine serum Glycoblotting of the sample mixtures containing whole cell N- (FBS; Biological Industries, Kibbutz Beit Haemek, Israel) and mini- glycans by means of BlotGlyco H bead was performed according to mum Eagle’s medium supplemented with 10% FBS and penicillin- the procedure described previously (26). BlotGlyco H beads (500 l) streptomycin (Invitrogen), respectively, at 37 °C in an atmosphere of (10 mg/ml suspension; Sumitomo Bakelite Co., Ltd.) were aliquoted 5% CO . P19C6 cells were differentiated as described by Tang and onto a well of a MultiScreen Solvinert filter plate (Millipore, Billerica, co-workers (29). In brief, P19 cells were cultured by the hanging drop MA). Peptide-N-glycosidase F-digested samples were dissolved with method and allowed to aggregate in bacterial grade Petri dishes at a 20 l of water and applied to the well followed by the addition of 180 cell density of 1 10 cells/ml in the presence of 1 M retinoic acid l of 2% acetic acid in ACN. The plate was incubated at 80 °C for 45 (RA) (Sigma) in minimum Eagle’s medium supplemented with 10% FBS. After 4 days of aggregation, cells were collected in “aggregate,” min to capture total glycans in sample mixtures specifically onto 524 Molecular & Cellular Proteomics 9.3 Glycoblotting-based Cellular Glycomics of ESCs FIG.1. Basic protocol of high throughput and quantitative cellular glycomics based on glycoblotting method using BlotGlyco H bead. , inner diameter; PNGaseF, peptide-N-glycosidase F. beads via stable hydrazone bonds. The plate was washed by 200 l changes of glycan expression levels were calculated as differentiated of 2 M guanidine HCl in ammonium bicarbonate followed by washing versus undifferentiated. Student’s t test was used to calculate the with the same volume of water and 1% triethylamine in methanol statistical difference of cell status, namely differentiated versus undif- (MeOH). Each washing step was performed twice, respectively. Un- ferentiated. The glycan structures were speculated using GlycoMod reacted hydrazide functional groups on beads were capped by incu- Tool and GlycoSuite. bation with 10% acetic anhydride in MeOH for 30 min at room RT-PCR—RNA was extracted from 1.5–5 10 P19CL6 cells using temperature. Then the solution was removed by vacuum, and then the an “R&D Quick” kit (Dainippon Sumitomo Pharma Co., Ltd., Osaka, bead was serially washed by 2 200 lof10mM HCl, MeOH, and Japan) according to the manufacturer’s instructions. RT-PCR was per- dioxane, respectively. On-bead methyl esterification of carboxyl formed using the SuperScriptIII One-Step RT-PCR System with Plati- groups in sialic acids was carried out by incubation with 150 mM num Taq DNA polymerase (Invitrogen) to reverse transcribe and amplify 3-methyl-1-p-tolyltriazene in dioxane at 60 °C to dryness. It usually cDNAs coding the proteins described below. The primer sequences took 90 min in a conventional oven. Then the bead was serially were as follows: -actin, 3-TGTGATGGTGGGAATGGGTCGG-5 and washed by 200 l of dioxane, water, MeOH, and water. The glycans 5-TTTGATGTCACGCACGATTTCC-3; -cardiac myosin heavy chain, blotted on beads were subjected to the trans-iminization reaction with 3-CTGCTGGAGAGGTTATTCCTCG-5 and 5-GGAAGAGTGAGCG- aoWR (aminooxy-functionalized peptide reagent) for 45 min at 80 °C. GCGCATCAAGG-3; and -cardiac myosin heavy chain, 3-TGCAAAG- WR-tagged glycans were eluted by adding 100 l of water and then GCTCCAGGTCTGAGGGC-5 and 5-GCCAACACCAACCTGTCCA- TM purified by a Mass PREP hydrophilic interaction chromatography AGTTC-3). (HILIC) Elution Plate (Waters) according to the manufacturer’s Immunohistochemistry—Sixteen days after incubation with or with- description. out 1% DMSO, P19CL6 cells were collected as described above and The purified N-glycans were 10-fold concentrated by SpeedVac dissociated intensively by EDTA/PBS containing 0.05% trypsin. After followed by direct dissolution with 2,5-dihydroxylbenzoic acid (10 washing, cells were dissolved in PBS and smeared onto silane- mg/ml in 30% ACN) and were crystallized. Then the analytes were coated slide glass (SUPERFROST, Matsunami Glass, Osaka, Japan). subjected to MALDI-TOF-MS analysis using an Ultraflex time-of-flight Cells were fixed in cold acetone and treated with MeOH containing 3% mass spectrometer III (Bruker Daltonics, Billerica, MA) in reflector, H O for 10 min at room temperature to quench internal peroxidase 2 2 positive ion mode typically summing 1000 shots. The detected N- activity. Nonspecific binding on cells was blocked by incubation with glycan peaks in MALDI-TOF-MS spectra were picked using the soft- CAS-Block (Zymed Laboratories Inc.) for 10 min at room temperature. ware FlexAnalysis version 3 (Bruker Daltonics) in independently per- Cells were then incubated with MF20 (1:800) (The monoclonal antibody formed experiments in P19C6, P19CL6, and ESCs, respectively. The developed by Donald A. Fischman was obtained from the Developmen- intensity of the isotopic peaks of each glycan was normalized to 15 tal Studies Hybridoma Bank developed under the auspices of the pmol of internal standard (A2 amide glycan) in each status. The NICHD, National Institutes of Health and maintained by The University Molecular & Cellular Proteomics 9.3 525 Glycoblotting-based Cellular Glycomics of ESCs FIG.2. Differentiation of P19CL6 cells to cardiomyocytes in presence of DMSO. A, RT-PCR analysis for the confirmation of cellular differentiation to cardiac muscle. M, DNA size marker (bp); lanes 1, 5, and 9, -actin (503 bp), lanes 2, 6, and 10, -cardiac myosin heavy chain (302 bp); lanes 3, 7, and 11, -cardiac myosin heavy chain (205 bp); lanes 4, 8, and 12, embryonic skeletal muscle (151 bp). B, immunocytochemis- try for the confirmation of P19CL6 cell differentiation to cardiac muscle. Pri- mary antibody, MF20 (mouse mono- clonal anti-sarcomere myosin); second- ary antibody, HRP polymer-conjugated IgG; chromogenic substrate, diamino- benzidine. The bar represents 1.0 cm. of Iowa, Department of Biological Sciences, Iowa City, IA.), which is a cess in mass spectrometry. Given that human ESCs and iPS sarcomeric myosin-specific monoclonal antibody, overnight at 4 °C. cells currently being developed are produced by using an- They were then washed by PBS and incubated with HRP-labeled sec- imal-derived materials such as serum and feeder layers or ondary antibody (Zymed Laboratories Inc.) for 10 min at room temper- even fractionated glycoproteins, it is important to address ature. After washing, diaminobenzidine solution was added, and the reaction was stopped by washing with water. In the case of P19C6, potential contamination by introduction of non-human sialic anti-mouse neurofilament 160 was used as the primary antibody, and acid Neu5Gc into human stem cell lines proposed for ther- the other part of the procedure was performed similarly. apeutic applications in humans (13, 21). The reaction con- ditions and all procedures are carefully optimized by using RESULTS not only mouse embryonic cells but also various human Concept—Our strategy of a glycoblotting-based rapid and cancer cell lines to maximize efficacy of N-glycan enrich- quantitative glycomics designed for whole cellular N-glycans/ ment and reproducibility of data acquisition. To facilitate free oligosaccharides using BlotGlyco (BlotGlyco H) beads is quantitative N-glycan profiling analysis, the expression level diagrammed in Fig. 1. The specific steps of this optimized of individual N-glycans was normalized and represented by protocol involve (i) enzymatic release of entire N-glycans from using a standardized unit (pmol/200 g of cellular proteins). cellular glycoprotein fractions, including both cell surface We note that the present protocol is readily feasible for any and endogenous glycoproteins, (ii) glycoblotting (chemos- type of mammalian cells, and the efficiency of glycoblotting elective enrichment) by BlotGlyco beads, (iii) on-bead deri- is not dependent on cell type when the required cell num- vatization and labeling with a reagent to enhance MS sen- bers (for example, confluence on a 6-cm dish; 5 10 sitivity by trans-iminization, and (iv) subsequent quantitative cells) can be prepared. Rapid and quantitative analysis of mass spectrometry-based glycomics and typing subgroups major N-glycans (60–80 major glycoforms) enriched by the of characteristic glycoforms, namely glycotypes, in the glycoblotting method provided us with reliable and satisfac- presence of an internal standard. The key difference when tory information for investigating structural changes in entire compared with other published approaches for cellular gly- N-glycans and comparing the ratio of significant glycoforms comics is specific chemistry-based enrichment of entire by bar coding with characteristic subtypes, namely stage- N-glycans by a commercially available high density hydra- specific embryonic glycotypes, during dynamic cell differ- zide bead (BlotGlyco) that allows for high throughput and entiation and proliferation. quantitative glycan profiling. Through on-bead chemical Monitoring Entire N-Glycan Expression during Mouse P19 protection of carboxyl groups, both human sialic acid Cell Differentiations—Herein we selected the mouse P19 sub- Neu5Ac and non-human sialic acid Neu5Gc are stabilized to clone to evaluate efficiency and versatility of our glycoblot- prevent the significant cleavage at the sensitive O-glycoside linkage of sialosides during the high energy ionization pro- ting-based strategy. P19C6 cells are mouse embryonic car- 526 Molecular & Cellular Proteomics 9.3 Glycoblotting-based Cellular Glycomics of ESCs FIG.3. Large scale N-glycan analysis during P19CL6 cell differentiation. A, MALDI-TOF-MS of whole N-glycans of undifferentiated and differentiated cells. IS, internal standard. B, quantitative and total glycomics of undifferentiated and differentiated cells. C, magnification to visualize increased glycans (*, p 0.01; **, p 0.05). D, magnification to visualize decreased glycans (*, p 0.01; **, p 0.05). Error bars mean standard deviations. E, bar coding analysis. Intens., intensity; a.u., arbitrary units. cinoma cells and serve as a common model for studying from P19 cells, efficiently differentiate into beating cardio- neuronal differentiation after RA inducement (32). P19 cells myocytes by treatment with 1% DMSO (27, 34). treated with lower level RA or DMSO differentiate into muscle Differentiation of P19CL6 Cells to Cardiomyocytes—In the (33), and P19CL6 cells, a well established subclone derived present study, we carefully characterized whole N-glycans of Molecular & Cellular Proteomics 9.3 527 Glycoblotting-based Cellular Glycomics of ESCs FIG.3—continued 528 Molecular & Cellular Proteomics 9.3 Glycoblotting-based Cellular Glycomics of ESCs TABLE I TABLE I—continued Glycoforms detected during P19CL6 cell differentiation Peak no. CL6 m/z Composition Hex, hexose; dHex, deoxyhexose; HexNAc, N-acetylhexosamine. 59 3090.19 Hex (HexNAc) dHex (NeuAc) 6 6 1 1 Peak no. CL6 m/z Composition 60 3192.22 Hex (HexNAc) dHex (NeuAc) 6 5 1 2 61 3351.28 Hex (HexNAc) (NeuAc) 1 1178.50 Hex (HexNAc) 6 5 3 2 2 62 3395.30 Hex (HexNAc) dHex (NeuAc) 2 1324.55 Hex (HexNAc) dHex 6 6 1 2 2 2 1 63 3497.34 Hex (HexNAc) dHex (NeuAc) 3 1340.55 Hex (HexNAc) 6 5 1 3 3 2 64 3557.36 Hex (HexNAc) dHex (NeuAc) 4 1486.61 Hex (HexNAc) dHex 7 6 1 2 3 2 1 65 3700.42 Hex (HexNAc) dHex (NeuAc) 5 1502.60 Hex (HexNAc) 6 6 1 3 4 2 66 3862.47 Hex (HexNAc) dHex (NeuAc) 6 1543.63 Hex (HexNAc) 7 6 1 3 3 3 67 4021.52 Hex (HexNAc) (NeuAc) 7 1664.65 Hex (HexNAc) 7 6 4 5 2 68 4167.58 Hex (HexNAc) dHex (NeuAc) 8 1689.69 Hex (HexNAc) dHex 7 6 1 4 3 3 1 9 1746.71 Hex (HexNAc) 3 4 10 1826.71 Hex (HexNAc) 6 2 11 1848.74 Hex (HexNAc) (NeuAc) 3 4 1 P19C6 cells with or without inducement into neural cells as 12 1851.74 Hex (HexNAc) dHex 4 3 1 well P19CL6 cells differentiated into cardiomyocytes. After 13 1892.76 Hex (HexNAc) dHex 3 4 1 1% DMSO treatment, P19CL6 cells formed a monolayer at 14 1908.76 Hex (HexNAc) 4 4 15 1949.79 Hex (HexNAc) day 3 and multilayers on day 5 and started beating synchro- 3 5 16 1988.76 Hex (HexNAc) 7 2 nously at day 16 of differentiation as reported (27), whereas 17 2010.79 Hex (HexNAc) (NeuAc) 4 3 1 control cells did not show any changes at the same period of 18 2013.79 Hex (HexNAc) dHex 5 3 1 culture. The differentiation into cardiac muscle was confirmed 19 2029.79 Hex (HexNAc) (NeuAc) 6 3 1 by conventional RT-PCR detecting mRNA coding cardiomyo- 20 2054.82 Hex (HexNAc) dHex 4 4 1 21 2070.81 Hex (HexNAc) cyte-specific proteins and immunocytochemistry (Fig. 2, A 5 4 22 2095.84 Hex (HexNAc) dHex 3 5 1 and B). As shown in Fig. 2A, - and -cardiac myosin heavy 23 2111.84 Hex (HexNAc) 4 5 chains, the markers of cardiomyocytes (35), were specifically 24 2150.81 Hex (HexNAc) 8 2 detected in the DMSO-treated 16-day cultured P16CL6 cells 25 2156.85 Hex (HexNAc) dHex (NeuAc) 4 3 1 1 and mouse cardiac muscles as a positive control. In addition, 26 2172.84 Hex (HexNAc) (NeuAc) 5 3 1 27 2213.87 Hex (HexNAc) (NeuAc) mouse monoclonal antibody MF20 reacted specifically with 4 4 1 28 2216.87 Hex (HexNAc) dHex 5 4 1 differentiated cardiomyocytes (Fig. 2B), indicating that differ- 29 2257.90 Hex (HexNAc) dHex 4 5 1 entiated cells readily express sarcomeric myosin. 30 2312.86 Hex (HexNAc) 9 2 Whole N-glycans of P19CL6 cells (undifferentiated cells) 31 2334.90 Hex (HexNAc) (NeuAc) 6 3 1 and differentiated cells on day 18 (n 6, six dishes each for 32 2359.93 Hex (HexNAc) dHex1(NeuAc) 4 4 1 33 2362.93 Hex (HexNAc) dHex both cells) were analyzed and identified for the first time by 5 4 2 34 2375.92 Hex (HexNAc) (NeuAc) 5 4 1 means of glycoblotting-based high throughput MALDI-TOF 35 2403.95 Hex (HexNAc) dHex 4 5 2 mass spectrometry (Fig. 3A). As summarized in Table I, 67 36 2416.95 Hex (HexNAc) (NeuAc) 4 6 1 kinds of glycoforms were detected and quantified reproduc- 37 2419.95 Hex (HexNAc) dHex 5 5 1 ibly in both cases. When the full portraits of N-glycan diversity 38 2521.98 Hex (HexNAc) dHex (NeuAc) 5 4 1 1 39 2537.98 Hex (HexNAc) (NeuAc) of both cells were represented quantitatively (Fig. 3B), it 6 4 1 40 2540.98 Hex (HexNAc) dHex 7 5 1 seems likely that high mannose type N-glycans (peak num- 41 2563.01 Hex (HexNAc) dHex (NeuAc) 4 5 1 1 bers 1(M2), 3(M3), 5(M4), 7(M5), 10(M6), 16(M7), 24(M8), and 42 2566.01 Hex (HexNAc) dHex 5 5 2 30(M9)) are major components throughout cardiomyocytic 43 2579.00 Hex (HexNAc) (NeuAc) 5 5 1 differentiation. However, it was also clearly suggested that 44 2651.33 Internal standard 45 2668.04 Hex (HexNAc) dHex (NeuAc) expression levels of 26 N-glycans were significantly accom- 5 4 2 1 46 2681.03 Hex (HexNAc) (NeuAc) 5 4 2 panied by cellular differentiation; 19 N-glycans, most of which 47 2684.03 Hex (HexNAc) dHex (NeuAc) 6 4 1 1 are monofucosylated glycoforms, were increased concert- 48 2725.06 Hex (HexNAc) dHex (NeuAc) 5 5 1 1 edly, whereas seven difucosylated N-glycans decreased. 49 2741.06 Hex (HexNAc) (NeuAc) 6 6 1 Changes in two glycoforms (peak numbers 40 and 53) were 50 2785.08 Hex (HexNAc) dHex 6 6 1 51 2827.09 Hex (HexNAc) dHex (NeuAc) especially significant (p 0.001); their expression level was 5 4 1 2 52 2830.09 Hex (HexNAc) dHex (NeuAc) 6 4 2 1 increased 16-fold (peak number 40) and decreased 30-fold 53 2871.12 Hex (HexNAc) dHex (NeuAc) 5 5 2 1 (peak number 53) according to differentiation, respectively. 54 2887.11 Hex (HexNAc) dHex (NeuAc) 6 5 1 1 Fig. 3, C and D, highlights dramatically changed N-glycan 55 2928.14 Hex (HexNAc) dHex (NeuAc) 5 6 1 1 structures during cell differentiation; namely monofucosylated 56 3030.17 Hex (HexNAc) dHex (NeuAc) 5 5 1 2 57 3033.17 Hex (HexNAc) dHex (NeuAc) N-glycans were drastically increased, whereas difucosylated 6 5 2 1 58 3046.17 Hex (HexNAc) (NeuAc) 6 5 2 N-glycans decreased in comparison with the expression level of whole cellular N-glycans. Molecular & Cellular Proteomics 9.3 529 Glycoblotting-based Cellular Glycomics of ESCs FIG.4. Differentiation of P19C6 cells to neural cells. A, immunocytochemistry for the confirmation of neural differentiation. Primary antibody, mouse monoclonal anti-neurofilament 160; secondary antibody, HRP polymer-conjugated IgG; chromogenic substrate, diamino- benzidine. The bar represents 200 m. B, MALDI-TOF-MS of whole N-glycans of undifferentiated and differentiated cells. IS, internal standard. * represents peak number 27. C, quantitative and total glycomics of undifferentiated and differentiated cells. IS, internal standard. * represents peak number 27. D, magnification to visualize increased glycans. E, bar coding analysis. 530 Molecular & Cellular Proteomics 9.3 Glycoblotting-based Cellular Glycomics of ESCs FIG.4—continued The significance of the dramatic changes in the expression mannose type (HM), monofucosylated type (MF), difucosy- level of two distinct glycotypes uncovered by quantitative lated type (DF), and others (O) (Fig. 3E). It is clear that the total glycomics of entire cellular N-glycans was revealed much N-glycan expression level was significantly increased from more simply by bar coding individual glycotypes such as high undifferentiated (68.5 pmol/200 g of protein) to the interme- Molecular & Cellular Proteomics 9.3 531 Glycoblotting-based Cellular Glycomics of ESCs TABLE II TABLE II—continued Glycoforms detected during P19C6 cell differentiation Peak no. m/z Composition Hex, hexose; dHex, deoxyhexose; HexNAc, N-acetylhexosamine. 59 2741.06 Hex (HexNAc) (NeuAc) 6 6 1 Peak no. m/z Composition 60 2766.09 Hex (HexNAc) dHex (NeuAc) 4 6 1 1 61 2785.08 Hex (HexNAc) dHex 1 1137.47 Hex (HexNAc) 6 6 1 3 1 62 2827.09 Hex (HexNAc) dHex (NeuAc) 2 1178.50 Hex (HexNAc) 5 4 1 2 2 2 63 2830.09 Hex (HexNAc) dHex (NeuAc) 3 1299.52 Hex (HexNAc) 6 4 2 1 4 1 64 2887.11 Hex (HexNAc) dHex (NeuAc) 4 1324.55 Hex (HexNAc) dHex 6 5 1 1 2 2 1 65 2928.14 Hex (HexNAc) dHex (NeuAc) 5 1340.55 Hex (HexNAc) 5 6 1 1 3 2 66 3030.17 Hex (HexNAc) dHex (NeuAc) 6 1461.57 Hex (HexNAc) 5 5 1 2 5 1 67 3033.17 Hex (HexNAc) dHex (NeuAc) 7 1486.61 Hex (HexNAc) dHex 6 5 2 1 3 2 1 68 3046.17 Hex (HexNAc) (NeuAc) 8 1502.60 Hex (HexNAc) 6 5 2 4 2 69 3090.19 Hex (HexNAc) dHex (NeuAc) 9 1543.63 Hex (HexNAc) 6 6 1 1 3 3 70 3192.22 Hex (HexNAc) dHex (NeuAc) 10 1623.63 Hex (HexNAc) 6 5 1 2 6 1 71 3338.28 Hex (HexNAc) dHex2(NeuAc) 11 1664.65 Hex (HexNAc) 6 5 2 5 2 72 3351.28 Hex (HexNAc) (NeuAc) 12 1689.69 Hex (HexNAc) dHex 6 5 3 3 3 1 73 3395.30 Hex (HexNAc) dHex (NeuAc) 13 1705.68 Hex (HexNAc) 6 6 1 2 4 3 74 3497.34 Hex (HexNAc) dHex (NeuAc) 14 1746.71 Hex (HexNAc) 6 5 1 3 3 4 75 3557.36 Hex (HexNAc) dHex (NeuAc) 15 1785.68 Hex (HexNAc) 7 6 1 2 7 1 76 3862.47 Hex (HexNAc) dHex (NeuAc) 16 1826.71 Hex (HexNAc) 7 6 1 3 6 2 17 1851.74 Hex (HexNAc) dHex 4 3 1 18 1892.76 Hex (HexNAc) dHex 3 4 1 diate cells at day 8 (219.4 pmol/200 g of protein) and differ- 19 1908.76 Hex (HexNAc) 4 4 entiated cells at day 16 (239.5 pmol/200 g of protein). It was 20 1949.79 Hex (HexNAc) 3 5 21 1988.76 Hex (HexNAc) revealed that the ratio of glycotype MF was increased from 15 7 2 22 2010.79 Hex (HexNAc) (NeuAc) 4 3 1 to 34%, and at least 11 glycoforms could be assigned as 23 2013.79 Hex (HexNAc) dHex 5 3 1 newly generated N-glycans involved in the glycotype MF after 24 2029.79 Hex (HexNAc) (NeuAc) 6 3 1 differentiation, whereas the others, glycotype HM, glycotype 25 2054.82 Hex (HexNAc) dHex 4 4 1 DF, and glycotype O, showed no significant change both in 26 2070.81 Hex (HexNAc) 5 4 27 2095.84 Hex (HexNAc) dHex the ratio and the number of glycoforms. When P19CL6 cells 3 5 1 28 2111.84 Hex (HexNAc) 4 5 were subjected to culture continuously until day 16 without 29 2150.81 Hex (HexNAc) 8 2 DMSO induction, there was no notable change in entire N- 30 2172.84 Hex (HexNAc) (NeuAc) 5 3 1 glycan expression during this period. Surprisingly, these cells 31 2175.84 Hex (HexNAc) dHex 6 3 1 exhibited N-glycan profiles quite similar to each other in terms 32 2200.88 Hex (HexNAc) dHex 4 4 2 33 2213.87 Hex (HexNAc) (NeuAc) of not only the ratio of the above four glycotypes but also the 4 4 1 34 2216.87 Hex (HexNAc) dHex 5 4 1 numbers of glycoforms identified in the individual glycotypes. 35 2257.90 Hex (HexNAc) dHex 4 5 1 Differentiation of P19C6 Cells and ESCs to Neural Cells— 36 2312.86 Hex (HexNAc) 9 2 Versatility of the present concept and protocol was demon- 37 2334.90 Hex (HexNAc) (NeuAc) 6 3 1 strated by using P19C6 cells that differentiate into neural cells 38 2359.93 Hex (HexNAc) dHex (NeuAc) 4 4 1 1 39 2362.93 Hex (HexNAc) dHex by RA inducement in which differentiation can be confirmed 5 4 2 40 2375.92 Hex (HexNAc) (NeuAc) 5 4 1 conventionally by immunocytochemistry using mouse mono- 41 2403.95 Hex (HexNAc) dHex 4 5 2 clonal antibody (anti-neurofilament 160 monoclonal antibody) 42 2419.95 Hex (HexNAc) dHex 5 5 1 as shown in Fig. 4A. Fig. 4B shows MALDI-TOF mass spectra 43 2460.98 Hex (HexNAc) dHex 4 6 1 of whole N-glycans enriched by glycoblotting at three distinct 44 2474.92 Hex (HexNAc) 10 2 45 2480.95 Hex (HexNAc) dHex (NeuAc) stages observed during P19C6 cell differentiation to neural 6 3 1 1 46 2508.99 Hex (HexNAc) dHex 5 4 3 cells, namely undifferentiated, aggregate (day 4), and differ- 47 2521.98 Hex (HexNAc) dHex (NeuAc) 5 4 1 1 entiated neural cells (day 9). A total of 75 N-glycans were 48 2537.98 Hex (HexNAc) (NeuAc) 6 4 1 identified; they are summarized in Table II and represented in 49 2563.01 Hex (HexNAc) dHex (NeuAc) 4 5 1 1 a quantitative manner in Fig. 4C. In the case of P19C6 cell 50 2566.01 Hex (HexNAc) dHex 5 5 2 51 2579.00 Hex (HexNAc) (NeuAc) differentiation into neural cells, it was revealed that 12 glyco- 5 5 1 52 2582.00 Hex (HexNAc) dHex 6 5 1 forms (peak numbers 20, 27, 28, 35, 41, 43, 53, 60, 65, 66, 69, 53 2623.03 Hex (HexNAc) dHex 5 6 1 and 73) were identified as bisect type (BS) N-glycans among 54 2651.33 Internal standard 13 N-glycans that increased more than 5-fold after differenti- 55 2668.04 Hex (HexNAc) dHex (NeuAc) 5 4 2 1 ation (Fig. 4D). The merit of bar coding analysis based on the 56 2681.03 Hex (HexNAc) (NeuAc) 5 4 2 57 2684.03 Hex (HexNAc) dHex (NeuAc) characteristic subtypes is clear because profiling by focusing 6 4 1 1 58 2725.06 Hex (HexNAc) dHex (NeuAc) 5 5 1 1 on glycotype BS clearly shows a drastic increase of this subtype (from 3 to 12%) compared with glycotype HM (from 532 Molecular & Cellular Proteomics 9.3 Glycoblotting-based Cellular Glycomics of ESCs FIG.5. Differentiation of mouse ESCs to neural cells. A, MALDI-TOF-MS spectra during cell differentiation. B, quantitative glycan profiling during ESC differentiation. C, bar coding analysis. D, novel glycan biomarkers for identifying and monitoring the processes of mouse neural cell differentiation. Intens., intensity; a.u., arbitrary units; d, day. Molecular & Cellular Proteomics 9.3 533 Glycoblotting-based Cellular Glycomics of ESCs FIG.5—continued 63 to 59%) and glycotype O (from 34 to 29%) (Fig. 4E). On the 46%) (Fig. 5C). During the differentiation from neural stem contrary, L-fucose-focused bar coding analysis used in the case sphere into neural stem cells, it seems likely that an increase of the P19CL6/cardiomyocytes system did not show any mean- of glycotype HM (from 45 to 61%) and a decrease of gly- ingful N-glycans expression change during differentiation: gly- cotype BS (from 10 to 5%) occurred concurrently, although cotype HM, from 63 to 59%; glycotype DF, from 6 to 4%; the reason is not clear. Surprisingly, it was revealed that glycotype MF, from 21 to 30%; and glycotype O, from 10 to 7%. neural cells differentiated from ESCs also exhibited up- Our interest was next directed toward alteration of N- regulated high level expression of the same three glyco- glycan expression of mouse ESC differentiation to neural forms (Fig. 5D; peak numbers 37, 48, and 58 in Fig. 5B)as cells. We considered that neural cells differentiated from those observed in P19C6 differentiation (peak numbers 27, mouse ESCs should also exhibit a structural alteration in the 35, and 41 in Fig. 4D). We could not detect any Neu5Gc stage-specific glycotypes, an increase of glycotype BS, residue in whole N-glycans identified in the present study, similar to those observed in P19C6 cell differentiation. Fig. and the expression levels of any N-glycans containing 5, A and B, and Table III show the results of MALDI-TOF-MS Neu5Ac residue(s) did not accompany P19 series cell dif- of all typical cellular stages during mouse ESC differentia- ferentiation. This means that bar coding analysis focusing tion into neural cells. As expected, bar coding analysis by on the expression level of Neu5Ac did not work for identi- three glycotypes used in the P19C6 cells demonstrated the fying stage-specific embryonic glycotypes in these cell significant increase of glycotype BS (10% at neural stem lines. Our preliminary result (n 3) indicates clearly the sphere or 5% at neural stem cell to 14% at neural cell day importance of the stage-specific embryonic glycotype BS 7) in comparison with glycotype HM (45% at neural stem as a new class of biomarkers for identifying and monitoring processes of mouse ESC differentiation into neural cells, sphere or 61% at neural stem cell to 40%) and glycotype O although the effect of feeder cells and other various factors (45% at neural stem sphere or 34% at neural stem cell to 534 Molecular & Cellular Proteomics 9.3 Glycoblotting-based Cellular Glycomics of ESCs TABLE III TABLE III—continued Glycoforms detected during ESC differentiation Peak no. ESC m/z Composition Hex, hexose; dHex, deoxyhexose; HexNAc, N-acetylhexosamine. 60 2460.98 Hex (HexNAc) dHex 4 6 1 Peak no. ESC m/z Composition 61 2474.92 Hex (HexNAc) 10 2 62 2480.95 Hex (HexNAc) dHex (NeuAc) 1 934.39 Hex 6 3 1 1 63 2508.99 Hex (HexNAc) dHex 2 1096.44 Hex 5 4 3 64 2521.98 Hex (HexNAc) dHex (NeuAc) 3 1137.47 Hex (HexNAc) 5 4 1 1 3 1 65 2537.98 Hex (HexNAc) (NeuAc) 4 1178.50 Hex (HexNAc) 6 4 1 2 2 66 2540.98 Hex (HexNAc) dHex 5 1258.49 Hex 7 5 1 67 2566.01 Hex (HexNAc) dHex 6 1299.52 Hex (HexNAc) 5 5 2 4 1 68 2651.33 Internal standard 7 1324.55 Hex (HexNAc) dHex 2 2 1 69 2668.04 Hex (HexNAc) dHex (NeuAc) 8 1340.55 Hex (HexNAc) 5 4 2 1 3 2 70 2681.03 Hex (HexNAc) (NeuAc) 9 1420.55 Hex 5 4 2 71 2684.03 Hex (HexNAc) dHex (NeuAc) 10 1461.57 Hex (HexNAc) 6 4 1 1 5 1 72 2712.79 Hex (HexNAc) (NeuGc) 11 1486.61 Hex (HexNAc) dHex 5 4 2 3 2 1 73 2827.09 Hex (HexNAc) dHex (NeuAc) 12 1502.60 Hex (HexNAc) 5 4 1 2 4 2 13 1543.63 Hex (HexNAc) 3 3 14 1582.60 Hex 15 1623.63 Hex (HexNAc) of individual culture conditions used on the ratio of these 6 1 16 1645.66 Hex (HexNAc) dHex 2 2 1 glycotypes must be examined carefully. 17 1664.65 Hex (HexNAc) 5 2 18 1689.69 Hex (HexNAc) dHex 3 3 1 DISCUSSION 19 1705.68 Hex (HexNAc) 4 3 For most mammalian cell types, it is not known which 20 1746.71 Hex (HexNAc) 3 4 21 1785.68 Hex (HexNAc) proteins are expressed at each cellular stage and how these 7 1 22 1826.71 Hex (HexNAc) 6 2 protein expression patterns change quantitatively upon differ- 23 1848.74 Hex (HexNAc) (NeuAc) 3 3 1 entiation and proliferation. Flow cytometry and immunohisto- 24 1851.74 Hex (HexNAc) dHex 4 3 1 chemistry have been generally used for the identification of 25 1867.73 Hex (HexNAc) 5 3 cell surface proteins such as cell differentiation markers. 26 1892.76 Hex (HexNAc) dHex 3 4 1 27 1908.76 Hex (HexNAc) However, it is not currently possible to profile a global view of 4 4 28 1947.73 Hex (HexNAc) 8 1 the cell surface protein landscape due to the limitation of 29 1949.79 Hex (HexNAc) 3 5 feasible antibodies with validated specificity and affinity 30 1988.76 Hex (HexNAc) 7 2 strength and the difficulty in the development of multiplexed 31 2010.79 Hex (HexNAc) (NeuAc) 4 3 1 assays for identifying sets of cell surface proteins in a single 32 2013.79 Hex (HexNAc) dHex 5 3 1 33 2029.79 Hex (HexNAc) experiment. 6 3 35 2054.82 Hex (HexNAc) dHex 4 4 1 Large scale proteomics analysis by two-dimensional gel 36 2070.81 Hex (HexNAc) 5 4 electrophoresis MALDI-TOF-MS suggested that only 17 pro- 37 2095.84 Hex (HexNAc) dHex 3 5 1 teins (0.7% of total detected 2200 proteins) with different 38 2111.84 Hex (HexNAc) 4 5 expression patterns may be involved in the DMSO-induced 39 2150.81 Hex (HexNAc) 8 2 40 2156.85 Hex (HexNAc) dHex (NeuAc) cardiac differentiation of P19CL6 cells (36). They also re- 4 3 1 1 41 2172.84 Hex (HexNAc) (NeuAc) 5 3 1 ported that real time PCR data showed discrepancies from 42 2175.84 Hex (HexNAc) dHex 6 3 1 that of proteomics in at least three kinds of proteins that 43 2191.84 Hex (HexNAc) 7 3 reflected the importance of posttranslational modifications in 44 2200.88 Hex (HexNAc) dHex 4 4 2 expressed proteins. On the other hand, it was also reported 45 2213.87 Hex (HexNAc) (NeuAc) 4 4 1 46 2216.87 Hex (HexNAc) dHex that only 0.8% of total detected proteins (28 proteins of 5 4 1 47 2232.92 Hex (HexNAc) 6 4 3500 proteins) were increased or decreased during the 48 2257.90 Hex (HexNAc) dHex 4 5 1 8-day differentiation of P19 cells to neural cells (34, 37). 49 2273.90 Hex (HexNAc) 5 5 Consequently, it was concluded that changes in the expres- 50 2298.92 Hex (HexNAc) dHex 3 6 1 sion level of detected proteins are not helpful for identifying or 51 2312.86 Hex (HexNAc) 9 2 52 2318.90 Hex (HexNAc) dHex (NeuAc) monitoring the processes of cellular differentiation of both 5 3 1 1 53 2334.90 Hex (HexNAc) (NeuAc) 6 3 1 P19 and P19C6 cells. Compared with the results of proteome- 54 2359.93 Hex (HexNAc) dHex (NeuAc) 4 4 1 1 based analysis, the high potential of the glycome-based ap- 55 2375.92 Hex (HexNAc) (NeuAc) 5 4 1 proach is clear because our results revealed for the first time 56 2378.92 Hex (HexNAc) dHex 6 4 1 that 28% of glycoforms (19 N-glycans of 67 total N-glycans; 57 2394.92 Hex (HexNAc) 7 4 58 2403.95 Hex (HexNAc) dHex peak numbers 13, 20, 21, 22, 25, 28, 29, 32, 34, 37, 38, 39, 40, 4 5 2 59 2419.95 Hex (HexNAc) dHex 5 5 1 41, 47, 48, 54, 60, and 63) were increased, and 10% (7 of 67) were decreased during P19CL6 cell differentiation to car- Molecular & Cellular Proteomics 9.3 535 Glycoblotting-based Cellular Glycomics of ESCs diomyocytes. Furthermore, the expression level of 31 N-gly- and iPS cell lines established by different laboratories and to cans involved in the glycotype MF was up-regulated to 34% make these resources readily available to the scientific com- (78.2 pmol/200 g of protein) against total detected N-glycan munity as soon as possible. expression (239.5 pmol/200 g of protein), whereas undiffer- * This work was supported in part by a grant for “Innovative pro- entiated cells expressed only 15% glycotype MF (20 glyco- gram for future drug discovery and medical care” from the Japan forms). Interestingly, differentiated cardiomyocytes lost most Science and Technology Agency and the Ministry of Education, Cul- Lewis X trisaccharide (SSEA-1) and sialyl-Lewis X tetrasac- ture, Science, Sports, and Technology of Japan. To whom correspondence should be addressed. 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Journal
Molecular & Cellular Proteomics – American Society for Biochemistry and Molecular Biology
Published: Mar 1, 2010