Cell Differentiation, 16 (1985) 169-173 169 Elsevier Scientific Publishers Ireland, Ltd.

CDF 00296

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

embryonal carcinoma cells

Masayuk i Ozawa 1, Takash i M u r a m a t s u 1 and D a v o r Solter 2

J Department of Biochemistry, Kagoshima University School of Medicine, Kagoshima, 890 Japan and e The Wistar Institute, Philadelphia, PA 19104, U.S.A.

(Accepted 17 December 1984)

Molecules carrying SSEA-I 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-filL Therefore, the major carbohydrate constituent of the antigenic molecule was embryoglycan, the glycoprotein-bound large glyean 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-I determinant was carried by the large glycan.

SSEA-I; embryonic antigens; mouse embryos; embryonal carcinoma cells; glycoprotein; embryoglycan

Introduction

Stage-specific embryonic antigen-1 (SSEA-1) is a cell-surface antigen defined by a monoclonal IgM antibody (Solter and Knowles, 1978). The antigen is expressed in preimplantation mouse em- bryos beginning at the 8-cell stage and, also, in the postimplantation embryos in embryonic ectoderm and visceral endoderm. In adult mice the antigen is detected only in a restricted region. Embryonal carcinoma cells, stem cells of teratocarcinomas, also express the antigen and lose it during their in vitro differentiation. Thus, SSEA-1 has been used as an excellent cell surface marker to monitor early stages of embryogenesis (Solter and Knowles, 1978; Solter et al., 1979; Fox et al., 1981; Grover et al., 1983).

SSEA-1 is a carbohydrate antigen and is de-

termined by GalB1 ~ 4(FucM ~ 3)GlcNAc link- age (Gooi et al., 1981). Although SSEA-1 in hu- man erythrocytes is glycolipid in nature (Kannagi et al., 1982), the antigen in embryonal carcinoma cells was carried by a high-molecular-weight com- plex, whose nature appears to be that of glycopro- teins (Andrews et al., 1982). More recently, Childs et al. (1983) have shown that SSEA-I from em- bryonal carcinoma cells migrates as glycoproteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In embryonal carcinoma cells, a significant fraction of glycopro- tein-bound carbohydrates are unusually large, with molecular weight more than 10000 (Muramatsu et al., 1978; H. Muramatsu et al., 1983). Abundance of the fucosylated form of the glycan can be taken as an index of early embryonic phenotype. Fur- thermore, non-reducing sugars of the glycan ap-

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pear to be altered during the course of embryogen- esis (T. Muramatsu et al., 1983). In the present study, we investigated whether the large glycan called embryoglycan carries the developmentally regulated antigen, SSEA-1.

Materials and Methods

Cell labeling and indirect immunoprecipitation

F9 embryonal carcinoma cells were labeled with 6-[SH]galactose (8.2 Ci /mmol , Amersham) for 20-24 h as described elsewhere (Muramatsu et al., 1979b). The labeled cells (1.8-2.4 × 107) were ex- tracted with 3 ml of 2% (w/v) Triton X-100 in Tris-buffered saline (TBS; 0.15 M NaC1 in 10 mM Tris-HC1 buffer, pH 7.6, containing 50 /~g/ml phenylmethylsulfonyl fluoride). After centrifuga- tion at 110000 × g for 30 min, the supernatant was mixed with 0.3 ml of 10% suspension of formalin-killed Staphylococcus aureus in 2% Triton X-100 in TBS containing 0.5% (w/v) bovine serum albumin (BSA) and was left to stand for 30 min at 4°C. The supernatant, which contained 4.7 × 106 cpm/ m l of the radioactivity precipitable by the addition of 5 ml ethanol, was saved after centrifu- gation at 2000 x g for 20 min. A fraction of the supernatant (1 ml) was mixed with 400 #1 of the anti-SSEA-1 monoclonal antibody (ascitic fluid diluted 20-fold with PBS (Dulbecco and Vogt, 1954)) and incubated at room temperature for 30 min. In the control run, anti-FT-1 mouse IgM monoclonal antibody (Kasai et al., 1984) (ascitic fluid diluted in the same way) was used. Twenty ~1 of anti-mouse IgM serum (Miles) was then added and incubation was continued for 1 h. The im- mune complex was precipitated by the addition of 200/~1 of the 10% suspension of S. aureus. After 1 h at 4°C, 5 ml of TBS containing 2% Triton X-100 was added, and the immune complex was collected by centrifugation at 2000 x g for 20 rain and washed with 5 ml of TBS containing 0.1% Triton X-100 and then with 5 ml of PBS. The pellet was resuspended in the solubilization buffer containing 2% (w/v) sodium dodecyl sulfate (SDS) and boiled for 3 rain. After centrifugation at 2000 x g for 20

rain, the supernatant was collected, counted and analyzed as described below.

Alkaline NaBH 4 treatment

The immune precipitate dissolved in SDS (5000-10000 cpm in 20-30 /~1) was mixed with 0.1 ml of BSA (1 mg/ml). Then 1 ml of 99% ethanol was added, and the mixture was kept at 4°C overnight. The precipitate was collected by centrifugation at 2000 × g for 10 rain. The ethanol precipitate was dissolved in 1 ml of 0.05 N NaOH containing 1 M NaBH 4 and incubated at 50°C for 16 h (Ogata and Lloyd, 1982).

Preparation of embryoglycan

Embryoglycan labeled with 6-[3H]galactose was prepared as described previously from F9 cells cultured in vitro (Muramatsu et al., 1979b).

Farr's assay

Binding ability of monoclonal antibodies with the radioactively labeled embryoglycan isolated from F9 cells was measured by Farr's assay (Minden and Farr, 1973) with the following mod- ifications. Ten #1 of [3H]galactose-labeled em- bryoglycan (1 x 10 4 cpm) was incubated with 100 /~1 of the anti-SSEA-1 antibody or anti-FT-1 anti- body at 37°C for 30 min and then at 4°C for 30 rain. To the above solution, 10 /zl of rabbit non- immune serum, which was preabsorbed with em- bryoid bodies of teratocarcinoma OTF6050 at the ratio of 4 :1 and diluted 2-fold with PBS, was added as a carrier. Then, the immune complex was precipitated by adding 120 ~1 of saturated am- monium sulfate solution, collected by centrifuga- tion at 1500 x g for 15 min and washed with 50% saturated ammonium sulfate solution. Radioactiv- ity in the precipitate was counted.

Results and Discussion

[3H]galactose-labeled SSEA-1 was isolated by detergent solubilization followed by indirect im- munoprecipitation. From 4.7 x 10 6 cpm of the

e thano l -p rec ip i t ab le radioact iv i ty , 1.8 × 105 cpm of the rad ioac t iv i ty was recovered into the im- munoprec ip i t a te . In the cont ro l run where the non- reac t ing monoc lona l an t i body was used, ra- d ioact iv i ty p rec ip i t a ted was only 8 × 103 cpm. The isola ted SSEA-1 migra ted as h igh-molecular -weight g lycopro te ins upon Sephacryl S-300 co lumn chro- m a t o g r a p h y (Fig. 1A) and upon S D S - P A G E (Fig. 1 E). The appa ren t molecular weight of the ant igen was es t imated to be more than 120000 upon S D S - P A G E (Fig. 1E). When the ant igen was t rea ted with 0.05 N N a O H , 1 M N a B H 4 at 50°C for 16 h, it was depo lymer ized (Fig. 1A), but the size of the p roduc t was most ly large: it was e lu ted near the exc luded volume upon a G-50 co lumn (Fig. 2). The condi t ion of the mild alkal ine t reat- ment is such that O-glycosidic l inkages be tween the ca rbohydra t e s and pro te in are cleaved, and even in the case of N-glycosidic l inkages, only m i n i m u m amount s of pep t ides remain a t tached to the ca rbohydra tes . Fur the rmore , p ronase digest ion also depo lymer ized the ant igen to the size of the a lka l ine -NaBH4- t rea ted one (Fig. 1B). C o m b i n e d t r ea tment with p ronase and a lka l i ne -NaBH 4 did not further depo lymer ize the ant igen (Fig. 1B). F r o m these results, we reached two conclusions. Firs t , SSEA-1 is cer ta in ly carr ied by glycoprote ins . Sensi t ivi ty of the ant igen to pronase or mild al- ka l ine t rea tment exc luded the poss ib i l i ty that the ant igen is a po lysacchar ide devoid of p ro te in moie ty ( Iwakura et al., 1983) or macrog lyco l ip id (Dej te r -Juszynski et al., 1978). Second, the carbo- hydra te moie ty of the ant igenic g lycopro te in is most ly the large one excluded f rom a Sephadex G-50 column, namely embryoglycan .

Next we invest igated whether the large glycan indeed carries the ant igenic site of SSEA-1. [3H]galactose_labeled embryog lycan isola ted from cell homogena te was mixed with ant i -SSEA-1, and the immune complex was p rec ip i t a t ed with am- m o n i u m sulfate. A r o u n d 20% of the glycan was p rec ip i t a t ed by the p rocedure (Fig. 3). On the o ther hand, control an t ibody prec ip i t a ted only 3.1% of the glycan. Therefore, a f ract ion of em- b ryog lycan is es tabl i shed to car ry SSEA-1 activity. The fact that only a l imited por t ion of the glycan was specif ical ly p rec ip i t a ted may reflect hetero- genei ty in the non- reduc ing s t ructure of em- bryoglycan.

171

A I 100C TOP -

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20 30 40 50

Fig. 1. Analysis of SSEA-1 recovered by indirect im- munoprecipitation. (A, B) Sephacryl S-300 column chromatog- raphy. The [3H]galactose-labeled antigen was analyzed by gel filtration on a column of Sephacryl S-300 superfine (1.5 × 89 cm) which was equilibrated with 0.01 M Tris-HCl buffer, pH 8.0, containing 0.15 M NaCI and 0.1% SDS. Fractions of 3.0 ml were collected. BD and Gal represent the elution positions of blue dextran and galactose, respectively. The standard sub- stances, IgG (molecular weight 150000), bovine serum albumin (molecular weight 67000), and cytochrome C (molecular weight 12400) were eluted around fractions 26, 29, and 37, respec- tively. (A) • O, the intact antigen; • t the anti- gen treated with 0.05 N NaOH/1 M NaBH 4 as described in Materials and Methods. (B) • o, the antigen extensively digested with pronase as described by Ozawa et al. (1982); • i , the antigen first treated with 0.2 N NaOH/0.4 M NaBH 4 at 37°C for 24 h, pH of the reaction mixture was brought to 8.0 with glacial acetic acid and then extensively digested with pronase (10 mg) at 37°C for 24 h under toluene layer. (C-E) SDS-PAGE of SSEA-1. The antigens isolated by immunoprecipitation from [3H]galactose-labeled extract of F9 cells were analyzed by SDS-PAGE on a 3.5% acrylamide gel according to Fairbanks et al. (1971) under reducing conditions. (C) The Triton X-100 extract of F9 cells after ethanol precipita- tion (140000 cpm); (D) immunoprecipitates using anti-FT-1 (800 cpm); (E) immunoprecipitates using anti-SSEA-1 (18000 cpm). Radioactivity on the slab gel was detected by fluorogra- phy after impregnating the gel with En3Hance (New England Nuclear). The duration of film exposure was 7 days. Molecular weight markers were thyroglobulin (330000) and BSA (67000).

Resul ts thus far descr ibed de mons t r a t e d that embryog lycan is a carr ier of the SSEA-1 de termi- nant . In the immunopre c ip i t a t e of SSEA-1, no glycol ipids migra t ing near the dye front were de- tec ted upon S D S - P A G E (Fig. 1). However , it is poss ib le that g lycol ip ids also car ry the ant igenic site, but were not recovered in immunoprec ip i t a t e s

20 v

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z lO

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BD Gaf

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~" 500 F- t ) ,< 0 D IZ

30 40 50

FRACTION NUMBER

Fig. 2. Sephadex G-50 column chromatography of [3H]galac- tose-labeled SSEA-I treated with 0.05 N NaOH/1 M NaBH4. The antigen was alkaline-treated as described in Materials and Methods, and then applied to a column of Sephadex G-50, fine (1.6x69 cm) equilibrated with 50 mM ammonium acetate buffer, pH 6.0. The column was eluted with the same buffer, and fractions of 2 ml were collected. BD and Gal represent the elution positions of blue dextran and galactose, respectively,

after indirect immunoprecipitation. Embryoglycan has been shown to carry other

developmentally regulated cell surface markers, such as receptors for Lotus tetragonolobus ag- glutinin, peanut agglutinin (Muramatsu et al., 1979a), Dolichos biflorus agglutinin (Muramatsu et al., 1981), and TC antigen (Ozawa et al., 1982). Thus, the present result establishes that the glycan is the important carrier of developmentally regu- lated cell surface markers found in embryonal carcinoma cells.

Acknowledgements

We thank Dr. M. Kasai for the gift of mono- clonal anti-FT-1 and Miss Kumiko Sato for her expert secretarial assistance. This work has been supported by Grants from the Ministry of Educa- tion, Science and Culture, Japan, Ministry of Health and Welfare, Japan, Grant HD 12487 from the National Institutes of Health, U.S.A. and Grant PCM 81-18801 from the National Science Foun- dation, U.S.A.

Fig. 3.

t i t

lO lO 2 lO 3 ANTIBODY DILUTION

Precipitation

172

I

10 4

of embryoglycan by anti-SSEA-1. [3H]galactose-labeled embryoglycan prepared from F9 cells was mixed with the indicated concentrations of antibodies, and the immune complex was collected and washed as described in Materials and Methods. Results were expressed as percent of the radioactivity precipitated by the antibodies. • • , anti-SSEA-1; • • , anti-FT-1.

References

Andrews, P.W., B.B. Knowles, G. Cossu and D. Solter: Teratocarcinoma and mouse embryo cell surface antigens: Characterization of the molecule(s) carrying the SSEA-1 antigenic determinant. In: Teratocarcinoma and Embryonic Cell Interactions, eds. T. Muramatsu, G. Gachelin, A.A. Moscona and Y. lkawa (Japan Scientific Soc. Press, Tokyo)/Academic Press, New York) pp. 103 119 (1982).

Childs, R.A., J. Pennington, K. Uemura, P. Scudder, P.N. Goodfellow, M.J. Evans and T. Feizi: High-molecular- weight glycoproteins are the major carriers of the carbo- hydrate differentiation antigens I, i and SSEA-1 of mouse teratocarcinoma cells. Biochem, J. 215,491-503 (1983).

Dejter-Juszynski, M., N. Harpaz, H.M. Flowers and N. Sharon: Blood group ABH-specific macroglycolipids of human erythrocytes: Isolation in high yield from a crude mem- brane glycoprotein fraction. Eur. J. Biochem. 83, 363-373 (1978).

Dulbecco, R. and M. Vogt: Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182 (1954).

Fairbanks, G., T.L. Steck and D.F.H. Wallach: Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617 (1971).

Fox, N., I. Damjanov, A. Martinez-Hernandez, B.B. Knowles

and D. Solter: Immunohistochemical localization of the early embryonic antigen (SSEA-1) in postimplantation mouse embryos and fetal and adult tissues. Dev. Biol. 83, 391-398 (1981).

Gooi, H.C., T. Feizi, A. Kapadia, B.B. Knowles, D. Solter and M.J. Evans: Stage-specific embryonic antigen involves al ---, 3 fucosylated type 2 blood group chains. Nature 292, 156-158 (1981).

Grover, A,, R.G. Oshima and E.D. Adamson: Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells. J. Cell Biol. 96, 1690-1696 (1983).

Iwakura, Y., P. McCormic, K. Artzt and D. Bennett: A class of large polysaccharides contains the antigenic determinants for the cytotoxic antibodies in a conventional syngeneic anti-F9 serum as well as a monoclonal antibody prepared against F9 cells. Cell Differ. 13, 41-48 (1983).

Kannagi, R., E. Nedelman, S.B. Levery and S. Hakomori: A series of human erythrocyte glycosphingolipids reacting to the monoclonal antibody directed to a developmentally regulated antigen, SSEA-1. J. Biol. Chem. 257, 14865-14874 (1982).

Kasai, M., T. Takashi, T. Takahashi and T. Tokunaga: A new differentiation antigen (FT-1) shared with fetal thymocytes and leukemic cells in the mouse. J, Exp. Med. 159, 971-980 (1984).

Minden, P. and R.S. Farr: The ammonium sulfate method to measure antigen-binding capacity. In: Handbook of Experi- mental Immunology, ed. D.M. Weir, 2nd edn, (Blackwell Scientific Publications, Oxford) pp. 15.1-15.21 (1973).

Muramatsu, H., H. Ishihara, T. Miyauchi, G. Gachelin, T. Fujisaki, S. Tejima and T. Muramatsu: Glycoprotein-bound large carbohydrates of early embryonic cells: Structural characteristic of the glycan isolated from F9 embryonal carcinoma cells. J. Biochem. 94, 799-810 (1983).

Muramatsu, T., G. Gachelin, J.F. Nicholas, H. Condamine, H. Jakob and F. Jacob: Carbohydrate structure and cell differ-

173

entiation: Unique properties of fucosyl-glycopeptides iso- lated from embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA 75, 2315-2319 (1978).

Muramatsu, T., G. Gachelin, M. Damonneville, C. Delarbre and F. Jacob: Cell surface carbohydrates of embryonal carcinoma cells: Polysaccharidic side chains of F9 antigens and of receptors to two lectins, FBP and PNA. Cell 18, 183-191 (1979a).

Muramatsu, T., G. Gachelin and F. Jacob: Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells. Biochim. Biophys. Acta 587, 392-406 (1979b).

Muramatsu, T., H. Muramatsu and M. Ozawa: Receptors for Dolichos biflorus agglutinin on embryonal carcinoma cells. J. Biochem. 89, 473-481 (1981).

Muramatsu, T., H. Muramatsu, M. Ozawa, H. Hamada, G. Gachelin, and S. Tejima: High-molecular-weight carbohydrates in cell surface glycoproteins from teratocar- cinomas and early embryos. In: Teratocarcinoma Stem Cells. Cold Spring Harbor Conferences on Cell Prolifera- tion 10, 173-184 (1983).

Ogata, S. and K.O. Lloyd: Mild alkaline borohydride treatment of glycoproteins - A method for liberating both N- and O-linked carbohydrate chains. Anal. Biochem. 119, 351-359 (1982).

Ozawa, M., S. Yonezawa, T. Miyauchi, E. Sato and T. Mura- matsu: New carbohydrate antigen found in large glyco- peptides of teratocarcinoma cells. Biochem. Biophys. Res. Commun. 105, 495-501 (1982).

Solter, D. and B.B. Knowles: Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Natl. Acad. Sci. USA 75, 5565-5569 (1978).

Solter, D., L. Shevinsky, B.B. Knowles and S. Strickland: The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid. Dev. Biol. 70, 515-521 (1979).