SSEA-1, a stage-specific embryonic antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells

Molecules carrying SSEA-1 were isolated from [3H]galactose-labeled embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. The antigenic molecules were degraded by extensive pronase digestion or mild alkaline treatment, and the majority of the products thus formed were so large as to be excluded from a column of Sephadex G-50. Therefore, the major carbohydrate constituent of the antigenic molecule was embryoglycan, the glycoprotein-bound large glycan in early embryonic cells. Furthermore, the binding of isolated embryoglycan with anti-SSEA-1 was directly shown by a modified Farr's assay. From these results we concluded that SSEA-1 determinant was carried by the large glycan.     

Embryonic stem cells deficient in I beta1,6-N-acetylglucosaminyltransferase exhibit reduced expression of embryoglycan and the loss of a Lewis X antigen, 4C9

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 beta1,6-N-acetylglucosaminylation of the galactosyl residue. We previously knocked out the gene encoding I beta1, 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. Alpha6-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 alpha6-integrin, significantly less than wild-type ES cells, raising the possibility that embryoglycan in ES cells enhances alpha6-integrin-dependent adhesion in vitro.     

Embryoglycan: a highly branched poly-<Emphasis Type="Italic">N</Emphasis>-acetyllactosamine in pluripotent stem cells and early embryonic cells

Embryonal carcinoma cells, stem cells of teratocarcinomas, are pluripotent stem cells and also prototypes of embryonic stem cells. Embryonal carcinoma cells contain large amounts of a highly branched poly-N-acetyllactosamine called embryoglycan, which has a molecular weight of approximately 10,000 or greater, and is asparagine-linked. This glycan was found by analyses of fucose-labeled glycopeptides, and its characteristics were established by biochemical analyses. The content of embryoglycan progressively decreases during the in vitro differentiation of embryonal carcinoma cells. Embryoglycan is also abundant in mouse embryonic stem cells and preimplantation mouse embryos, and decreases during embryogenesis. Embryoglycan carries a number of carbohydrate markers of murine pluripotent stem cells. Lewis x markers, such as SSEA-1, 4C9 antigen, and binding sites for Lotus tetragonolobus agglutinin are of particular importance. 4C9 antigenicity requires clustering of Lewis x, best accomplished by poly-N-acetyllactosamine branching, whereas SSEA-1 does not. Although in vivo evidence is lacking, these epitopes have been suggested to participate in cell-to-cell and cell-to-substratum adhesion. Other markers on embryoglycan include α-galactosyl antigens such as ECMA-2, and binding sites for Dolichos biflorus agglutinin, the epitope of which is considered to be identical to Sd^a antigen, namely, GalNAcβ1–4(NeuAcα2–3)Galβ1–4GlcNAc. While embryoglycan is also present in human teratocarcinoma cells, the carbohydrate markers characterized in human pluripotent stem cells to date are largely carried by glycolipids and keratan sulfate. Information on embryoglycan and markers carried by it may assist in the development of new markers of human pluripotent stem cells and their progenies.     

The glycoprotein-bound large carbohydrates from embryonal carcinoma cells carry receptors for several lectins recognizing N-acetylgalactosamine and galactose

When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.     

Molecular properties of ECMA 2 and ECMA 3 antigens defined by monoclonal antibodies against embryonal carcinoma cells

ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other alpha-galactosyl antigens, namely blood group B and P1 antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.     

1H-NMR spectroscopy of the glycoprotein-bound large carbohydrates from embryonal carcinoma cells

400 MHz NMR spectrum was recorded for the glycoprotein -bound large carbohydrates (embryoglycan) isolated from F9 embryonal carcinoma cells. Two intense signals at 4.13 ppm and 4.69 ppm were assigned to be H-4 of galactosyl residues substituted at C-3 and H-1 of G1cNAc beta 1----3, respectively. The result is consistent with the proposal that the fundamental building unit of the large glycan is G1cNAc beta 1----3Ga1 beta. Furthermore, the spectral data confirmed a conclusion obtained by glycosidase digestion that fucosyl residues are linked mostly to N-acetylglucosamine rather than galactose.     

Cell-surface changes during in vitro differentiation of pluripotent embryonal carcinoma cells

When aggregates of HM-1 embryonal carcinoma (EC) cells were exposed to 10(-6) M retinoic acid for 2 days and cultured in medium lacking retinoic acid, they differentiated to nerve cells, endoderm cells, and myoblasts. Cells 2 days after initial exposure to retinoic acid were not significantly different from the parental EC cells, as judged by cell-surface architecture and by reactivity to lectins. On the fourth day, the surface of the aggregates was covered with two kinds of cells distinguishable from the parental cells. The round cells with short villi seemed to be precursors to endoderm cells. Receptors for Dolichos biflorus agglutinin (DBA) newly appeared and receptors for peanut agglutinin (PNA) were still expressed on their surfaces. The other cells, which were round cells with a few processes, might be precursors to nerve cells. PNA receptors had disappeared from their surfaces, and DBA receptors were not expressed. On the sixth day of differentiation, possible precursors to myoblasts were detected; they were flat cells with smooth surfaces. These cells lacked cell-surface receptors for the two lectins, while the precursor cells and the myoblasts excreted intercellular fibers reacting with PNA. HM-1 cells synthesized much embryoglycan, the structure of which was similar to that of the glycan isolated from quasinullipotent F9 cells. The only difference was that the glycan from HM-1 cells lacked DBA binding sites. Synthesis of fucosylated embryoglycan mainly decreased between the second and fourth day of differentiation. As above, cell-surface changes occurred mainly between the second and fourth day. The period seems to be important in determining the fate of the cells, since endoderm cells were scarcely seen among differentiated cells which had been continuously exposed to 10(-6) M retinoic acid during the period.     

Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.     

Identification of free glycan chain liberated by de-N-glycosylation of the cortical alveolar glycopolyprotein (hyosophorin) during early embryogenesis of the Medaka fish, Oryzias latipes

In Medaka embryos (at the stages of blastulation to organogenesis), we found the presence of free glycan of which structure is identical with the multiantennary N-linked sugar chain of L-hyosophorin molecules which were originally present in the cortical alveoli of the unfertilized eggs in their precursor high molecular form. The free glycan-enriched fraction was separated from L-hyosophorin by chromatography on DEAE-Sephadex A-25 and Sephadex G-50 after removal of the sialic acid residues with exo-sialidase. Composition analysis, 400-MHz 1H NMR spectroscopy, and pyridylamination-hydrazinolysis-nitrous acid deamination of the free glycan showed the presence of di-N-acetylchitobiosyl structure at the reducing end, suggesting that the free glycan chain was derived from L-hyosophorin by the action of a specific peptide:N-glycosidase (PNGase). When we combine the previous finding of the hyosophorin-derived unique pentaantennary free glycan chain in the flounder embryos [A. Seko et al. (1989) J. Biol. Chem. 264, 15922-15929], it is anticipated that PNGase-catalyzed de-N-glycosylation of L-hyosophorin would be required at a certain stage of embryogenesis for L-hyosophorin to play a yet undefined functional role during early development.     

The Effects of lithium and Zinc Ions on the Pattern of Acidic Glycans in the Sea Urchin (Hemicentrotus pulcherrimus) Embryo

Among several acidic glycan components found in Hemicentrotus embryos, the "F"- and "S"-components were specifically affected by treatment with Li⁺and Zn²⁺, respectively. The amount of the "F"-component in Li⁺-treated embryos was about 60% that in normal embryos. This fact was in accordance with the reduced alcian blue staining of the surfaces in Li⁺-treated embryos. Moreover, the "F"-component in Li⁺-treated embryos appeared to be composed of two subcomponents, while in normal and Zn²⁺-treated embryos it appeared to be single. The "S"-component in Zn²⁺-treated embryos was about 8% that in normal embryos. According to histochemistry with a lectin probe, it was found that UEA-I was much more strongly associated with a hyaline layer in Li⁺-treated than in normal and Zn²⁺-treated embryos. Li⁺-treated embryos developed into exogastrulas, which were divided by a constriction into two parts; an animal half which stained intensely with alcian blue, and a vegetal half which stained poorly. On the other hand, Zn²⁺-treated embryos remained as permanent blastulas. Considering the above, it is suggested that change in the acidic glycan pattern leads to alterations in the morphogenesis of sea urchin embryos.     

The effects of collagen synthesis inhibitory drugs on somitogenesis and myogenin expression in cultured chick and mouse embryos

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.     

Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells

Specific glycan expression is an essential characteristic of developing tissues. Our molecular characterization of a mutation that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. Embryos homozygous for the TM3 balancer chromosome lack neural HRP-epitope expression. Genetic and molecular mapping of the relevant locus reveals that Tollo/Toll-8, a member of the Toll-like receptor family, is altered on the TM3 chromosome. In wild-type embryos, Tollo/Toll-8 is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo/Toll-8 into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. The portfolio of expressed oligosaccharides in a cell reflects its identity and also influences its interactions with other cells and with pathogens. Therefore, the ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors.     

Preliminary observations on the synthesis of glycosaminoglycans in the sea urchin embryo.

A method based on the degradation by enzymes and nitrous acid of isotopically labelled glycosaminoglycans has been employed to study the synthesis of these compounds in normal, animalized and vegetalized sea urchin embryos. According to standard criteria, these organisms synthesize dermatan sulfate, heparan sulfate, hyaluronate and keratan sulfate. The hyaluronate seems to be slightly sulfated, it may thus be mucoitin sulfate. The preliminary results obtained suggest a conspicuous difference between animalized and vegetalized embryos: the synthesis of dermatan sulfate is suppressed in the former, while proceeding normally in the latter. The synthesis of heparan sulfate is not affected by our experimental conditions, but the isotope incorporation in hyaluronate and in keratan sulfate is decreased, more in the vegetalized than in the animalized embryos.     

Characterization of protein glycosylation in Francisella tularensis subsp. holarctica: identification of a novel glycosylated lipoprotein required for virulence

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

Recent advances in glycan synthesis

Carbohydrates play important roles in life science, but their synthesis is always hampered by their complicated chemical structures. Scientists have never stopped trying to solve the problem of glycan synthesis from various aspects. Here a brief overview of recent progress in glycan synthesis, including chemical approaches, chemoenzymatic approaches, and automated synthesis, will be discussed, focusing on the efficiency of new glycosylation methods, the stereoselectivity of coupled products, and their applications in the assembly of complex glycan chains.     

糖芯片技术在肿瘤研究中的应用

糖基化修饰是蛋白质常见的翻译后修饰之一,通过与糖结合蛋白如凝集素、抗体等相互作用调节肿瘤细胞侵袭、转移的能力及肿瘤异质性。通过化学合成法、化学-酶合成法或释放天然聚糖构建的糖芯片是分析聚糖与糖结合蛋白相互作用的重要工具。文中综述了常见的点制糖芯片的技术及糖芯片在癌症疫苗、单克隆抗体及诊断标志物中的广泛运用。由于肿瘤发生的各个环节都伴随着聚糖结构的改变,利用糖芯片探究肿瘤细胞特异表达的聚糖所参与的生理病理过程具有重大意义。     

Complex <Emphasis Type="Italic">N</Emphasis>-glycans or core 1-derived <Emphasis Type="Italic">O</Emphasis>-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le<Superscript>Y</Superscript> in the preimplantation mouse embryo

The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and Le^Y was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or Le^Y on mouse embryos.     

Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wild-type or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing alpha-mannosidase II and core fucosyl-transferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.     

Carbohydrate antigens expressed on stem cells and early embryonic cells

Lewis X antigen (Le(X)) is a marker of embryonic stem cells, embryonal carcinoma cells and multipotential cells of early embryos in the mouse. Le(X) is carried by branched, high-molecular weight poly-N-acetyllactosamines (embryoglycan). While embryoglycan is present in human embryonal carcinoma cells, Le(X) is not expressed in human embryonic stem cells, embryonal carcinoma cells or inner cell mass cells. Instead, these cells express SSEA-3 and SSEA-4, both of which are carried by globo-series glycolipids. Le(X) is a marker of primordial germ cells or multipotential stem cells derived from primordial germ cells both in the mouse and human. In other species of vertebrates, Le(X) is widely expressed in early embryonic cells and primordial germ cells, but the mode of expression is not completely conserved among species. Le(X) is expressed in neural stem cells from both humans and mice. Hematopoietic stem cells are not reported to express the above carbohydrate markers. A marker of these cells is CD34, a membrane-bound sialomucin. Another sialomucin, CD164 (MGC-24v) is expressed in hemotopoietic progenitor cells. As a function of Le(X) in stem cells, the promotion of integrin action is proposed, based on analyses of glycoproteins with the marker, cDNA transfection experiments and the inhibitory effects of an anti-Le(X) antibody. Most probably, Le(X) antigen as well as poly-N-acetyllactosamines play roles in the interactions on the same membrane. On the other hand, O-linked oligosaccharides on CD34 and CD164 are probably involved in the regulation of cell adhesion and proliferation via intercellular recognition.