Glycobiology Advance Access originally published online on January 9, 2008
Glycobiology 2008 18(3):242-249; doi:10.1093/glycob/cwm138
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Embryonic stem cells deficient in I β1,6-N-acetylglucosaminyltransferase exhibit reduced expression of embryoglycan and the loss of a Lewis X antigen, 4C9
2 Department of Biochemistry
3 Division of Disease Models, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya 468-8550
4 Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065
5 Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, 12 Araike, Nisshin, Aichi 470-0195, Japan
1 To whom correspondence should be addressed: Tel: +81-561-73-1111; Fax: +81-561-73-1142; e-mail: tmurama{at}dpc.aichi-gakuin.ac.jp
Received on November 15, 2007; revised on December 21, 2007; accepted on December 21, 2007
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Abstract |
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Embryoglycan is a class of branched high-molecular-weight poly-N-acetyllactosamines characteristically expressed in early embryonic cells and has been shown to be involved in the intercellular adhesion of early embryonic cells in vitro. Branching of poly-N-acetyllactosamine chains is performed by β1,6-N-acetylglucosaminylation of the galactosyl residue. We previously knocked out the gene encoding I β1, 6-N-acetylglucosaminyltransferase (IGnT), and the resultant deficient mice were born without any abnormality, although the mice exhibited various deficits in later life. In the present investigation, we produced embryonic stem (ES) cells from IGnT-deficient embryos. The mutant ES cells exhibited a reduced capability in embryoglycan synthesis. Thus, IGnT is a major enzyme involved in the branching of poly-N-acetyllactosamine chains in embryoglycan. Since ES cells are equivalent to multipotential cells of the embryonic ectoderm in early postimplantation embryos, this result indicates that an abundance of embryoglycan in these cells is not essential for normal embryogenesis. The IGnT-deficient ES cells continued to express SSEA-1, but lacked the expression of 4C9 antigen, although the epitope of 4C9 antigen was confirmed to be Lewis X by a transfection experiment. The result establishes the distinct nature of 4C9 antigenicity, which requires either Lewis X epitope on I-branch or clustering of Lewis X epitope, best accomplished by poly-N-acetyllactosamine branching.
6-Integrin was newly identified as a carrier of embryoglycan. The IGnT-deficient ES cells adhered to dishes coated with laminin, which is a ligand for 6-integrin, significantly less than wild-type ES cells, raising the possibility that embryoglycan in ES cells enhances 6-integrin-dependent adhesion in vitro. Key words: Embryonic stem cells / knockout mice / Lewis X / N-acetylglucosaminyltransferase / poly-N-acetyllactosamines
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Introduction |
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Cell-surface carbohydrates change dramatically during the early stages of mammalian embryogenesis (Muramatsu 1988
; Muramatsu T and Muramatsu H 2004). Notably, characteristic features of cell-surface carbohydrates have been found in multipotential cells of early embryos and in cells homologous to them, namely embryonic stem (ES) cells and teratocarcinoma stem cells called embryonal carcinoma (EC) cells (Muramatsu 1988; Muramatsu T and Muramatsu H 2004).
In the mouse, these cells express a carbohydrate antigen, SSEA-1, the epitope of which is Lewis X (Solter and Knowles 1978; Gooi et al. 1981; Fox et al. 1981). Furthermore, both in the mouse and human, these cells express a large amount of branched poly-N-acetyllactosamine of high molecular weight, called embryoglycan (Muramatsu et al. 1978, 1979, 1980; Muramatsu 1988; Muramatsu T and Muramatsu H 2004). Embryoglycan bears a number of cell-surface markers expressed in early embryonic cells (Muramatsu 1988). The major carrier of SSEA-1 in early embryonic cells is also embryoglycan (Ozawa et al. 1985). In EC cells lacking embryoglycan, cell adhesion and cell differentiation are not affected (Draber and Maly 1987), while embryoglycan-negative cells and embryoglycan-positive cells sort differently (Boubelik et al. 1996). The result suggests that embryoglycan plays regulatory roles in cell adhesion. This proposal is supported by the finding that beads with attached embryoglycan tend to self-aggregate (Kojima et al. 1994). Furthermore, direct evidence of a role of Lewis X epitope, presumably on embryoglycan, in intercellular recognition was shown by using cells deficient in cadherin (Handa et al. 2007). Although these in vitro data apparently establish a role of embryoglycan in intercellular recognition, it is not clear whether embryoglycan is essential for the process in early embryos in vivo. As is well known, important cellular processes are performed by redundant processes of different evolutional origins. Whether embryoglycan is required for embryogenesis can be answered by knocking out enzymes involved in its biosynthesis.
Narimatsu and co-workers knocked out fucosyltransferase IX (FUT9) (Kudo et al. 1998), which is involved in the synthesis of SSEA-1 in early embryos (Kudo et al. 2004). The loss of SSEA-1 was confirmed by immunohistochemical examination of preimplantation embryos. However, null mice are born without morphological abnormality. The result indicates that SSEA-1 epitope in embryoglycan is not essential for early embryogenesis. However, there remained a possibility that an epitope other than SSEA-1 plays essential roles in embryoglycan. Also, loss of SSEA-1 was documented in preimplantation embryos, but not in early postimplantation embryos.
The backbone of poly-N-acetyllactosamine chains is formed by concerted action of a β1,4-galactosyltransferase and a β1,3-N-acetylglucosaminyltransferase, while branches of poly-N-acetyllactosamine chains are mainly formed by I β1,6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen et al. 1993; Chen et al. 2000; Inaba et al. 2003). We have knocked out the IGnT gene; the null mice are born without morphological abnormalities but exhibit deficits in later life (Chen et al. 2005). The key issue is whether IGnT is the principal enzyme forming embryoglycan. If so, the function of embryoglycan in early embryogenesis may be dispensable. Another enzyme that forms branches in poly-N-acetyllactosamine is Core 2 β1,6-N-acetylglucosaminyltransferase-mucin type (Yeh et al. 1999).
The present investigation deals with the establishment of ES cells, which are considered to be equivalent to multipotential cells of early postimplantation embryos, from IGnT-deficient mice and examination of their properties to answer the above question. Furthermore, the antigenic properties of the deficient ES cells have established the difference between the two monoclonal antibodies in recognizing Lewis X. In addition, we raise the possibility that embryoglycan enhances the activity of 6-integrin in vitro.
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Results |
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Establishment and general properties of ES cells from IGnT-KO embryos
IGnT-KO ES cells were established by culturing preimplantation embryos of the IGnT-KO mice. They exhibited the morphology of ES cells (Figure 1A), and their tight adhesion to each other did not differ from that of typical ES cells such as D3 cells. Further evidence that the newly established cells were ES cells was obtained by analyzing the expressed genes. Oct 3/4, a marker of ES cells (Rosner et al. 1990
), was strongly expressed in IGnT-KO ES cells as in D3 cells (Figure 1B). In addition to Oct 3/4, Sox-2 (Yuan et al. 1995) and Rex-1 (Rogers et al. 1991), which are also markers of ES cells, were strongly expressed in IGnT-KO ES cells (Figure 1C). The growth rate of IGnT-KO ES cells was indistinguishable from that of D3 ES cells. No significant differences could be found morphologically in developmental potential between D3 and IGnT-KO ES cells. In both these cell lines, flat endoderm cells were found to be growing out from the stem cell clumps after 2 days of monolayer culture on a gelatin-coated dish in the absence of LIF. The suspension culture and transfer to gelatin-coated dishes induced differentiation of both ES cells and yielded endoderm cells, beating myocardial cells and neurons with extended neuritis, 5 to 7 days after the transfer in a similar manner.
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Reduction of embryoglycan in IGnT-KO ES cells
When D3 ES cells were cultured with radio-labeled fucose, extensively digested with proteinase K, and analyzed by Sephadex G-50 column chromatography, a large amount of radioactivity was eluted in the excluded volume (Figure 2A, closed square), indicating that ES cells are abundant in embryoglycan as in the case of EC cells and early embryonic cells (Muramatsu et al. 1978
, 1980). However, when IGnT-KO ES cells were analyzed, the radioactivity eluted in the excluded volume decreased significantly (Figure 2A, open square). The ratio of radioactivity in embryoglycan fraction (fractions 21–26) to that in total glycopeptides fraction (fractions 21–60) was 0.184 in D3 cells, while the value was 0.100 in IGnT-KO cells. Radioactive material labeled with galactose was abundant in the embryoglycan fraction in D3 cells (Figure 2B, closed square) and decreased in IGnT-KO ES cells (Figure 2C, closed square). The ratio of radioactivity in embryoglycan fraction to that in total glycopeptides fraction was 0.357 in D3 cells and 0.246 in IGnT-KO cells; the decrease was slightly less as compared to that in the fucose-labeled glycan. To further investigate whether labeled material in the embryoglycan faction from IGnT-KO cells was identical to that from D3 cells, galactose-labeled material in the embryoglycan fraction was digested with endo-β-galactosidase. The enzyme acts on linear portion of poly-N-acetyllactosamine, while branching of the chain hinders its action (Fukuda and Matsumura 1976). Thus, embryoglycan from EC cells was digested with the enzyme only to a limited extent, yielding arrays of products with different molecular weights (Muramatsu et al. 1979). This was also observed in embryoglycan from D3 ES cells (Figure 2B, open square). When galactose-labeled material in embryoglycan fraction from IGnT-KO cells was digested with the enzyme, more products were found in low molecular weight fraction (Figure 2C, open square). Thus, in IGnT-KO ES cells, branching of poly-N-acetyllactosamine was much decreased, while synthesis of poly-N-acetyllactosamine with few branches appeared to persist. In other words, IGnT is concluded to be a major enzyme involved in poly-N-acetyllactosamine branching in ES cells.
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Mode of expression of Lewis X antigens in IGnT-KO ES cells
IGnT-KO ES cells were stained by anti-SSEA-1 as in the case of D3 ES cells (Figure 3). However, IGnT-KO ES cells were not significantly stained with anti-4C9 (Figure 3). Epitope of 4C9 is Lewis X as in the case of SSEA-1 (Gooi et al. 1981
), as revealed through inhibition by a haptenic oligosaccharide, lacto-N-fucopentaose III (Yoshinaga et al. 1991) and inactivation by a fucosidase specific for 3/4GlcNAc linkage (Nomoto et al. 1986). We confirmed this by introducing human fucosyltransferase IX (hFUT9), which forms the Lewis X structure (Kudo et al. 1998; Kaneko et al. 1999), into COS-7 cells; the transfected cells newly gained 4C9 epitope (Figure 4). 4C9 antigen is located in embryoglycan, but not in lower molecular weight glycans as revealed by modified Farr's assay (Nomoto et al. 1986). Thus, the above results indicated that 4C9 antigenicity requires branching of poly-N-acetyllactosamine chains in ES cells, while SSEA-1 antigenicity is considered to be present in the linear portion of poly-N-acetyllactosamine, based on high susceptibility to endo-β-galactosidase (Childs et al. 1983). This result also confirms that the branching of poly-N-acetyllactosamine was much decreased in IGnT-KO ES cells.
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Identification of
6-integrin as a carrier of embryoglycanAs a step forward exploring new functions of embryoglycan using IGnT-KO ES cells, we analyzed glycoproteins which carry embryoglycan. Lotus tetragonolobus agglutinin (LTA) binds to Lewis X in EC cells (Kamada et al. 1987
). Thus we isolated glycoproteins capable of binding to LTA-agarose from cultured P19 EC cells. After deglycosylation by N-glycanase and SDS–PAGE, a major band of 110 kDa was found. The band was identified as 6-integrin after in-gel trypsin digestion and protein sequencing: two peptide sequences, LLLVGAPR and NLGTATLNIQ, matched those of mouse 6-integrin (residues 61–68 and 833–842, NP032423). Then, EC cells were labeled with fucose, and 6-integrin recovered by immunoprecipitation was digested with proteinase K and subjected to Sephadex G-50 column chromatography. A significant amount of the label was found in the excluded volume, indicating that 6-integrin carries embryoglycan (Figure 5A). This point was confirmed by analyzing glucosamine-labeled glycopeptides from 6-integrin. A large amount of the label was present in the excluded region (Figure 5B, closed square), and it was converted to a heterogeneous array of low molecular weight substances after digestion with endo-β-galactosidase (Figure 5B, open square). The presence of embryoglycan in 6-integrin from D3 ES cells was confirmed by conducting a galactose-labeling experiment as in EC cells (Figure 5C).
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IGnT-KO ES cells adhere to laminin-coated dishes only weakly
6-Integrins, especially 6β1 and 6β4, are known to be involved in the adhesion of cells to laminin (Ekbolm 1996; Mercurio et al. 2001). We found that IGnT-KO ES cells adhered only weakly to laminin-coated plates (Figure 6A). Six hours after plating, the number of IGnT-KO ES cells adhered to laminin-coated plates was much smaller than that of D3 ES cells (Figure 6B). Adhesion to gelatin-coated plates differed only slightly between the two ES cell lines. After 24 h, the tendency continued, although difference to adhesion to gelatin-coated plates was slightly increased.
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Discussion |
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We established an ES cell line from IGnT-KO embryos. The cells contained significantly reduced amounts of embryoglycan. ES cells are considered to be equivalent to embryonic ectoderm cells of early postimplantation embryos, which differentiate into various cell types forming the embryo (Evans and Hunter 2002
). Therefore, we conclude that IGnT is a major enzyme involved in the branching of poly-N-acetyllactosamine chains in embryoglycan and that an abundance of embryoglycan in the embryonic ectoderm of early postimplantation embryos is not essential for mouse embryogenesis. Our hypothesis is that the function of embryoglycan in the regulation of cell adhesion is shared by other molecules, which may be new in evolutional terms, and that carbohydrate structures formed by enzymes involved in embryoglycan synthesis play roles in later life (Chen et al. 2005), leading to conservation of abundance in embryoglycan. Similarly, mice deficient in FUT9, which lack SSEA-1 expression in preimplantation embryos, develop normally, but exhibit increased anxiety-like behavior in later life (Kudo et al. 2004, 2007): the Lewis X structure is not essential for early embryogenesis due to compensation by other cell-surface molecules including cadherins, but is preserved because of the role in adulthood.
By using LTA, which recognizes the Lewis X structure in EC cells, we newly identified 6-integrin as a carrier of embryoglycan. The β-chain to which an 6-chain of this integrin bound appeared to be β1: in the LTA-binding proteins from P19 cells, a minor band of 100 kDa contained both 6-and β1-chains, as shown by mass spectrometric analysis of the trypsin-digested products (data not shown). It remains to be elucidated either 6- or a β-chain carries embryoglycan. So far, the Lewis X structure has been identified in leukocyte integrins (Asada et al. 1991) and β1-integrin from neural stem cells (Yanagisawa et al. 2005). Furthermore, we found that the IGnT-KO ES cells exhibited weaker adhesion to laminin-coated plates, a process dependent on 6-integrin, than D3 ES cells, derived from the same mouse strain. Although the conclusion is derived from comparisons of the two cell lines, we emphasize that this is the only difference in the biological properties of the two cell lines. We have proposed that the Lewis X structure enhances integrin function, since 4C9 antibody inhibits cell-substratum adhesion of EC cells (Nomoto et al. 1986), and transfection with fucosyltransferase IV enhances integrin-dependent cell-substratum adhesion (Sudou et al. 1995). Thus, the present result is consistent with the view, and we propose that the branching structure in embryoglycan enhances the action of the Lewis X structure. However, the normal development of IGnT-deficient mice implies that the possible enhancement of the integrin activity is not of critical importance for development. N-Linked oligosaccharides are involved in the regulation of integrin activity (Gu and Taniguchi 2004). Notably, N-glycan in the β-propeller domain of the 5-subunit is required for heterodimerization of 5- and β1-chains (Isaji et al. 2006). Thus, the further study will be required at the level where the embryoglycan chain enhances an integrin action, if in fact it really does. An interesting hypothesis is that the association of an integrin with basigin and embigin, both of which are immunoglobulin superfamily members and enhance integrin action, is promoted by carbohydrate–carbohydrate interaction of embryoglycan (Muramatsu T and Muramatsu H 2004), and the I-branch structure is required for the putative carbohydrate–carbohydrate interaction. This hypothesis will explain why I-branches in embryoglycan enhance adhesion to laminin in vitro. On the other hand, IGnT-KO ES cells exhibited strong intercellular adhesion leading to cluster formation just as in the case of D3 ES cells, instead of the apparently established role of embryoglycan in intercellular adhesion. This is certainly due to the fact that the primary contributors to intercellular adhesion are cadherins.
The epitope of 4C9 antigen is Lewis X as shown by a hapten inhibition study (Yoshinaga et al. 1991) and confirmed here by a gene transfection experiment. We found that IGnT-KO ES cells continued to express SSEA-1, but failed to express 4C9, even though the epitope of the both antigens is Lewis X. We also noted that 4C9 antigenicity becomes weak in the cerebellum of IGnT-deficient mice (Chen et al. 2005). The epitope of SSEA-1 is considered to be in the linear portion of poly-N-acetyllactosamine (Childs et al. 1983). Thus, the present results provide convincing evidence that poly-N-acetyllactosamine branching is essential for 4C9 antigenicity in ES cells. Most probably, the Lewis X structure on I-branch is required for 4C9 antigenicity, but the possibility is not excluded that the clustering of Lewis X epitope, best accomplished by the branching of poly-N-acetyllactosamine, is sufficient for 4C9 antigenicity. The determination of the precise structure of embryoglycan with potent 4C9 antigenicity remains to be accomplished. Since only a fraction of embryoglycan shows the strong antigenicity of 4C9 (Nomoto et al. 1986), enrichment of the antigenic fraction will be required prior to structural studies. The structure of SSEA-1-carrying poly-N-acetyllactosamine has not been clarified in ES cells. The glycan from IGnTKO-ES cells will be a suitable material for the purpose because of the structural simplicity.
Both 4C9 and SSEA-1 serve as markers of primordial germ cells (Fox et al. 1981; Yoshinaga et al. 1991). However, the distribution of 4C9 is more restricted than that of SSEA-1; 4C9 has proved as an excellent marker of primordial germ cells (Tanaka et al. 2000;Yoshimizu et al. 2001; Kimura et al. 2003; Takeuchi et al. 2003). Furthermore, expression of 4C9 and the loss of binding site for Dolichos biflorus agglutinin serve as markers in lung carcinoma that correlated with poor prognosis of patients (Matsumoto et al. 1992). With a knowledge of the difference in epitope structures between 4C9 and SSEA-1, 4C9 antibody will be widely used in the research in reproductive biology and oncology.
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Establishment of ES-cell lines from IGnT-KO mice
IGnT-KO mice (Chen et al. 2005
) were maintained with the 129/SV background and used to establish an IGnT-KO ES cell line. Eighty-five 3.5-day embryos of the deficient mice (late morulas to early blastocysts) were cultured in an M16 medium (M7297, Sigma, St. Louis, MO). The day of plug detection was set at day 0 of embryonic development. After 2 days, expanded blastocysts were transferred to a 12-well dish (Falcon, 3043, Becton Dickinson Labware, Franklin Lake, NJ) and cultured on a feeder layer of mouse embryonic fibroblasts (SL-10 cells) treated with 15 µg/mL of mitomycin C in the ES cell medium for establishment, which consisted of a knockout Dulbecco-modified minimum essential medium (DMEM) (Invitrogen/GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (GIBCO), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 1000 U/mL of leukemia inhibitory factor (LIF) (ESG1107, Chemicon International, Temecula, CA), and 10% knockout serum replacement (Invitrogen/GIBCO 10828-028).
ES cell culture
Mouse D3 ES cells, which were derived from 129/SV mice (Doetschman et al. 1985) and provided by Dr. R. Kemler, and newly established IGnT-KO ES cells were cultured on SL-10 feeder cell layers in the ES growth medium consisting of knockout DMEM supplemented with 15% FBS, 2 mML-glutamine (GIBCO), 0.1 mM β-mercaptoethanol, and 1000 U/mL of LIF. Two to three days after plating, ES cells were treated with trypsin/EDTA and replated on SL-10 feeders. The medium was changed every day.
Differentiation of ES cells
D3 cells or IGnT-KO ES cells were cultured in the absence of embryonic fibroblasts (SL-10) in DMEM containing 15% FBS either in tissue culture dishes, or in suspension in bacterial dishes. For suspension culture, ES cells (4 x 105 cells) were plated on bacterial dishes (Falcon 1007). After 4 days, cell aggregates were transferred to tissue culture dishes (Falcon 3002) coated with 0.5% gelatin and continued to be cultured.
Cell adhesion assay
D3 cells or IGnT-KO ES cells were cultured on feeder layers for 2 days, treated with trypsin/EDTA, and cultured in the ES growth medium on 0.1% gelatin-coated dishes for 30 min to remove the feeder fibroblasts. Then ES cells (5 x 105/well) were plated on laminin-coated 6-well plates (BD BioCoat laminin cellware, BD Biosciences, Bedford, MA) or 0.5% gelatin-coated 6-well plates. After 6 or 24 h, the plates were washed gently with PBS two times and the number of ES cells attached to the substratum was counted.
Transfection with hFUT9 cDNA
COS-7 cells were maintained in DMEM supplemented with 10% FBS. For transfection, 4 x 105 cells were plated on 2-well chamber slides and cultured for 24 h in DMEM with 10% FBS. These cells were transfected with hFUT9 (Kaneko et al. 1999), which was kindly provided by Dr. H. Narimatsu, in the expression vector pAMo in the serum-free medium using LipofectAMINE PLUS (Invitrogen) for 3 h according to the manufacturer's directions and cultured in DMEM with 10% FBS for 40 h.
Immunocytochemistry
ES cells were treated with trypsin/EDTA for 5 min and were plated onto 0.1% gelatin-coated dishes at 37°C for 30 min, and unadhered ES cells were collected and cultured in the ES medium in 2-well chamber slides (Nalge Nunc International, Naperville, IL) coated with 0.5% gelatin overnight. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were blocked at room temperature for 45 min with 10% normal goat serum/1% BSA/0.1% Triton X-100/PBS (for Oct 3/4) or for 30 min with 3% BSA/PBS (for SSEA-1 and 4C9); reacted with mouse anti-SSEA-1 (Chemicon, Temecula, CA), rat anti-4C9 (Nomoto et al. 1986), or rabbit anti-Oct 3/4 (Neuromics, Northfield, MN); and incubated at 4°C overnight. The secondary antibodies used were tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG, fluorescein isothiocyanate (FITC)-conjugated goat antirat IgG, or FITC-conjugated rabbit antigoat IgG (Sigma). Fluorescence was detected and photographed with a confocal laser-scanning microscope (MRC-1024, Olympus, Tokyo, Japan). Staining of COS-7 cells to detect 4C9 expression was performed in an identical manner.
Evaluation of gene expression by RT-PCR
Total RNA was isolated from cell pellets using Isogen (Wako Chemicals, Osaka, Japan) according to the manufacturer's directions. Then, 1 µg was subjected to reverse transcription in a 20 µL reaction mixture containing 40 units of RNase inhibitor, 0.5 µg of oligo dT primers, and 50 units of SuperScriptTM II reverse transcriptase (Invitrogen). PCR amplification was performed using ExTaq DNA polymerase (Takara, Kyoto, Japan) for 35 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 1 min). The reaction mixture was incubated at 72°C for 10 min at the end of the reaction to ensure complete extension. The primers used for RT-PCR were as follows (forward and reverse): OCT 3/4 (5'-GGAGAGGTGAAACCGTCCCTAGG-3' and 5'-AGAGGA GGTTCCCTCTGAGTTGC-3'), REX-1 (5'-GGCCAGTCCAG AATACCAGA-3' and 5'-GAACTCGCTTCCAGAACCTG-3'), SOX-2 (5'-GTGGAAACTTTTGTCCGAGAC-3' and 5'-TGG AGTGGGAGGAAGAGGTAAC-3'), and IGnT (5'-GGAGTC CCTGGCTCCATGCCACC-3' and AGAGAGCTCGAGCCG GAGCTTGCTGCGGGTCAGA-3').
Sephadex G-50 column chromatography of glycopeptides labeled with radioactive sugars
D3 ES cells or IGnT-KO ES cells were cultured in the ES growth medium on 0.5% gelatin-coated dishes for 2 days, and their medium was replaced with 6 mL of the fresh ES growth medium containing radioactive sugars (L-[6-3H]-fucose; 2.2 MBq/ 10 cm dish, ICN Biomedicals, Costa Mesa, CA D-[1-14C]-galactose; 0.37 MBq/10 cm dish, specific activity; 1.66–2.22 GBq/mmol, NEC-302X, PerkinElmer, Boston). After 24 h, the cells were harvested using trypsin/EDTA and digested with 0.5 mg of proteinase K (Nacalai Tesque, Kyoto, Japan) in 0.5 mL of 0.1 M Tris–HCl buffer, pH 8.0 containing 0.1 M NaCl and 0.01 M CaCl2 with 20 µL of toluene at 37°C for 24 h. Then, 0.5 mg of proteinase K and 5 µL of 1 M CaCl2 were added and incubation was continued for another 24 h. After the final addition of 0.5 mg of proteinase K, the incubation was continued for a further 24 h. The digested material was clarified by centrifugation. The sample (0.5 mL) was mixed with Blue Dextran (Amersham Biosciences AB, Uppsala, Sweden) and 10 mg of galactose and was applied to a column of Sephadex G-50 (1.2 x 45 cm) equilibrated with 0.01 M Tris–HCl buffer, pH 8.0, containing 0.1 M NaCl. In the case of the galactose-labeled glycopeptides, the digest was treated with an equal volume of phenol, and the water layer was applied to a column of DEAE-Sephadex A-25 (0.5 x 5 cm) equilibrated with the above buffer. The unabsorbed fraction was dialyzed against distillated water, lyophilized, and applied to a column of Sephadex G-50 as mentioned above. Fractions (600 µL) were collected, and an aliquot of 200 µL was withdrawn for counting. Radioactive material in the embryoglycan fraction was digested with 50 munits of endo-β-galactosidase from Escherichia freundi (Seikagaku Kogyo, Tokyo, Japan) at 37°C for 15 h in 200 µL to 300 µL of reaction mixture after adjusting the pH to 5.3 by 0.2 M Na acetate buffer. The digested material was also analyzed by Sephadex G-50 column chromatography as described above.
Identification of 6-integrin as a carrier of the Lotus tetragonolobus agglutinin- binding site in P19 cells
P19 EC cells (Jones-Villeneuve et al. 1982) were provided by Dr. M. McBurney. The cells were cultured in DMEM with 10% FBS. The cells harvested from 1000 Falcon 3003 dishes by treatment with 0.02% EDTA in PBS (–) were homogenized with 500 mL of PBS (+) and centrifuged at 120,000 x g for 1 h. The precipitate was suspended in 300 mL of PBS (+) containing 1% Triton X-100. After centrifugation at 120,000 x g for 1 h, the supernatant was applied to a column of LTA-agarose (2 mL; Seikagaku Kogyo Co., Tokyo, Japan). The column was washed with 40 mL of PBS (+) containing 0.5% Triton X-100, and the bound proteins were eluted with 6 mL of PBS (+) containing 0.5% Triton X-100 with 0.1 M fucose. The eluate was concentrated by Centricon YM-10 (Milipore, Bedford, MA) to 500 µL and boiled for 3 min after addition of 26 µL of 10% SDS and 4 µL of 2-mercaptoethanol. Then 25 µL of 1 M Na phosphate buffer, pH7.5 was added, and the mixture was digested with 30 mU of N-glycanase F (Calbiochem, Darmstadt, Germany) at 37°C overnight. The digest was precipitated by trichloroacetic acid at a final concentration of 10%. The precipitate was subjected to SDS–PAGE using a 7.5% running gel. The major 110-kDa protein was digested by trypsin in the gel, and the peptides released were sequenced by protein sequencer after separation by high performance liquid chromatography as described previously (Muramatsu et al. 2000).
Analysis of glycopeptides from 6-integrin in P19 cells
P19 cells were cultured in DMEM containing 10% FBS in Falcon 3003 dishes. [3H]-Fucose (L-[6-3H]-fucose; 2.2 MBq/10 cm dish, ICN Biomedicals) or [14C]-glucosamine (D-[1-14C]-glucosamine hydrochloride, 0.56 MBq/10 cm dish, specific activity; 2.07 GBq/mmol, Amersham Bioscience) was added to each dish and cells were further cultured for 24 h. Labeled cells from four dishes were lysed in 4 mL of lysis buffer [20 mM Tris–HCl, pH 7.5, 0.3% CHAPS, 0.15 M NaCl, and protease inhibitor cocktail tablets (Roche, Mannheim, Germany)]. After centrifugation at 13,000 xg for 5 min, 6-integrin was immunoprecipitated by anti-VLA-66 antibody (Chemicon) and protein A Sepharose (Amersham Bioscience). The precipitate was digested with proteinase K and analyzed by Sephadex G-50 column chromatography as described in the previous section. In the case of glucosamine-labeled materials, the digest was boiled for 10 min to inactivate proteinase K and divided into two fractions. One fraction was digested with endo-β-galactosidase as described above. Each fraction was analyzed by Sephadex G-50 column chromatography as described above, except that fractions 800 µL were taken from fraction 21 and all of them were counted. 6-Integrin from D3 ES cells was isolated and analyzed by an identical manner from the cells labeled with [14C]-galactose.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interestReferences |
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The Ministry of Education, Culture, Sports, Science and Technology of Japan (14082202).
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Conflict of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interestReferences |
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None declared.
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Acknowledgements |
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We thank Dr. H. Narimatsu for the gift of hFUT9 cDNA.
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Abbreviations |
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DMEM, dulbecco-modified minimum essential medium; EC, embryonal carcinoma; ES, embryonic stem; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; hFUT9, human
1,3-fucosyltransferase IX; IGnT, I β1,6-N-acetylglucosaminyl- transferase; IGnT-KO, IGnT-deficient; LIF, leukemia inhibitory factor; LTA, Lotus tetragonolobus agglutinin; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TRITC, tetramethylrhodamine isothiocyanate |
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