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.     

Regulatory mutations in CHO cells induce expression of the mouse embryonic antigen SSEA-1

Two rare and dominant mutants of Chinese hamster ovary (CHO) cells, LEC11 and LEC12, express the mouse embryonic antigen SSEA-1. Parental CHO cells and the revertants, LEC11.R9 and LEC12.R10, do not express this antigen as detected by a sensitive radioimmunoassay with a monoclonal antibody to SSEA-1. The presence of the SSEA-1 determinant correlates with the apparent de novo expression of specific N-acetylglucosaminide alpha(1,3)fucosyltransferase activities not detected in parental or revertant cell extracts. Several differences in the enzymes substrate specificities and their products have been identified. The combined data suggest that LEC11 and LEC12 mutants result from regulatory mutations affecting different fucosyltransferase genes.     

The epitope of mouse embryonic antigen(s) recognized by monoclonal antibody TEC-02 is a carbohydrate carried by high-molecular-weight glycoconjugates

The expression and properties of mouse embryonic antigens, recognized by monoclonal antibody TEC-02, were analyzed in teratocarcinoma-derived cell lines. TEC-2 antigens were found in the majority of the parietal endoderm cells PYS-2 and in a fraction of cultured embryonal carcinoma cells but not in other cell lines tested. During the course of retinoic acid-induced differentiation of embryonal carcinoma cells F9, the expression of TEC-2 was transiently increased. Immunolabeling of extracts from F9 and PYS-2 cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydisperse glycoconjugates of high molecular weight (mostly greater than 100,000). The TEC-2 epitope was shown to be carbohydrate which in F9 cells might be attached to the same carrier as another developmentally regulated carbohydrate epitope TEC-1. The TEC-2 antigens, isolated by indirect immunoprecipitation, were degraded by extensive pronase digestion or mild alkaline treatment to mostly large products. Immunostaining of glycolipid standards suggested that TEC-2 epitope involves the GalNAc beta 1----4Gal beta 1----4R sequence. Combined data indicate that TEC-2 is a new developmentally regulated carbohydrate epitope carried in embryonal carcinoma cells predominantly on glycoprotein-bound large carbohydrates.     

Synthesis of cancer-associated glycoantigens: stage-specific embryonic antigen 3 (SSEA-3)

The synthesis of the tumor-associated carbohydrate antigens SSEA-3 and Gb3 in a semi-convergent fashion using building blocks bearing a S-thiazolinyl (STaz) moiety is reported. Complete stereoselective control of a difficult alpha-(1-->4)-galactosylation and high overall yields were achieved.     

The glycosidic antigen recognised by a novel monoclonal antibody, 75.12, is developmentally regulated on mouse embryonal carcinoma cells

Monoclonal antibody 75.12 raised against the human ovarian teratocarcinoma cell line PA1 detects a 'Y' or iso-leb glycosidic structure. Using the 75.12 antibody we have established that the Y antigen is expressed on some but not all mouse embryonal carcinoma (EC) lines. The Y or 75.12 antigen-positive EC cell lines F9 and PCC4 cease to express the antigen after differentiation induced with retinoic acid and this decreased expression parallels the morphological differentiation of the EC cells. These results support not only the idea that carbohydrate structures present on embryonic cells undergo marked alteration during differentiation, but also that established mouse EC cells may differ in their differentiation states.     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

Synthesis and NMR study of a heptasaccharide, epitope of the stage-specific embryonic antigen-1 (SSEA-1)

This paper describes an efficient synthesis of the beta-2-trimethylsilylethyl glycoside of lacto-N-fucoheptaose based on a highly stereo- and regioselective glycosylation between a Lewis(x) trisaccharidic donor and a tetraol tetrasaccharidic acceptor. The title compound was characterized by high-resolution NMR spectroscopy.     

Monoclonal antibody directed to the stage-specific embryonic antigen (SSEA-1) reacts with branched glycosphingolipid similar in structure to Ii antigen

A monoclonal antibody reacting with early mouse embryos and murine embryonal carcinoma cells (F9) defines the stage-specific embryonic antigen (SSEA-1). We now report that the antigen (SSEA-1) is a complex glycolipid with the branched lacto-N-glycosyl series. Antibody to SSEA-1 reacts strongly with the branched H₄-glycosphingolipid but not with other various glycolipids so far tested. This reactivity was abolished by endo-β-galactosidase treatment. The homogeneous H₄-glycolipid not only reacted with the monoclonal antibody to SSEA-1 but also with antibody to I-(Ma), i-(Dench) and with anti-H specific lectin. Chemical analysis, including methylation, also indicates that the glycolipid antigen had a close resemblance to I-antigen.     

The beta 1,3-galactosyltransferase beta 3GalT-V is a stage-specific embryonic antigen-3 (SSEA-3) synthase

We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.     

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.     

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.     

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.     

Immunohistochemical localization of the early embryonic antigen (SSEA-1) in postimplantation mouse embryos and fetal and adult tissues

Distribution of the stage-specific embryonic antigen (SSEA-1) was studied in postimplantation murine embryos, fetuses, and adult mice by immunohistochemical techniques. SSEA-1 was also localized on the stem cells of differentiating solid teratocarcinomas and on the surface of core cells of solid embryoid bodies. At the egg cylinder stage the antigen is restricted to embryonic ectoderm and visceral endoderm. During subsequent development SSEA-1 becomes localized to portions of the brain and primordial germ cells. In addition some sites of the urogenital anlage are SSEA-1 positive. In adult mice, the epithelium of the oviduct, the endometrium, and the epididymis are the cells most reactive with the monoclonal antibody to SSEA-1; although some areas of the brain and kidney tubules are weakly positive. Study of this antigenic determinant might disclose some previously unexpected cell lineage relationships and/or might elucidate events necessary for reproduction.     

Establishment of a pluripotent embryonal carcinoma cell line not expressing SSEA-1 and ECMA-7 phenotypes

A murine embryonal carcinoma (EC) cell line heterozygous for t0 recessive lethal mutation has been established from an embryo-derived transplantable teratocarcinoma TC1Ph of the genotype (129-T/t0 X C3H/Di)t0/+. The EC cell line, designated EC1Ph, and two cloned sublines, EC1Ph/a and EC1Ph/b, maintain the diploid karyotype (40, XY) and give rise to teratocarcinomas with differentiated derivatives of EC cells after inoculation into syngeneic recipients. The cloned sublines express low or zero amounts of SSEA-1 and ECMA-7 stage-specific antigens. At some passages, the EC1Ph line and the cloned subline EC1Ph/b express a significant quantity of class I H-2 antigens. This unusual EC phenotype resembles that of human teratocarcinoma cell lines.     

Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells.

The LINE-1 repeat family is interspersed throughout mammalian genomes and is thought to be the result of duplicative transposition of LINE-1 sequences via an RNA intermediate. This report describes a ribonucleoprotein particle with LINE-1 RNA in the mouse embryonal carcinoma cell line F9. This ribonucleoprotein particle is a potential intermediate in the transposition of LINE-1 in the mouse genome.     

PCC4, a new cell surface antigen common to multipotential embryonal carcinoma cells, spermatozoa, and mouse early embryos

Cell surface antigens of a multipotential embryonal carcinoma (EC) line, PCC4, have been investigated. As do other EC cells, these cells express the F9 antigen but not the H-2 or Ia antigens. In addition, these cells express another antigen called PCC4. This antigen is present on the multipotential EC cells tested, on spermatozoa, and on the inner cell mass of newly implanted blastocyst.     

High-molecular-weight glycoproteins are the major carriers of the carbohydrate differentiation antigens I, i and SSEA-1 of mouse teratocarcinoma cells.

The carriers of the carbohydrate differentiation antigens I, i and SSEA-1 were investigated in embryonal carcinoma cell lines of mouse and differentiated cell lines derived from them. Glycoproteins were studied by immunostaining ('Western blotting') of total cell lysates and immunoprecipitation from lysates of galactose oxidase/NaB3H4-labelled cells; glycolipids were investigated by immunostaining of thin layer chromatograms. The antigenic activities detected by immunofluorescence of cell smears were reflected in the antigenicities of high-molecular-weight glycoproteins. These were polydisperse and markedly susceptible to digestion with endo-beta-galactosidase. Only the I antigen was detected on minor glycolipids. These observations indicate that glycoproteins rather than glycolipids are the major carriers of carbohydrate differentiation antigens I, i and SSEA-1 in the teratocarcinoma cell lines.