Changes in fucosyl-glycopeptides during early ... - Semantic Scholar

/ . Embryo!, exp. Morph. Vol. 57, pp. 25-36, 1980 Printed in Great Britain © Company of Biologists Limited 1980

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Changes in fucosyl-glycopeptides during early post-implantation embryogenesis in the mouse By TAKASHI MURAMATSU, HUBERT CONDAMINE, GABRIEL GACHELIN 1 AND FRANQOIS JACOB From the Service de Genetique Cellulaire du College de France and the Institut Pasteur, Paris

SUMMARY Six-day to 12-day mouse embryos were dissected and radiolabelled by culture in the presence of [3H]fucose. The radiolabelled embryos were extensively digested with Pronase. The resulting glycopeptides were analysed by Sephadex G-50 column chromatography. Glycopeptides from 6-day-old embryos were separated into two main peaks: one eluted near the excluded volume, the other in a well-retarded position. This elution profile was similar to that observed with glycopeptides prepared from embryonal carcinoma cells. The relative amount of the high-molecular-weight glycopeptides decreased during embryonic development and particularly around day 9. Glycopeptide elution profiles from 10-day embryos, or isolated organs of 12-day embryos, were indistinguishable from those obtained from differentiated teratocarcinoma-derived or adult cells. At least 30% of the large molecular weight glycopeptides appear to be located at the cell surface. INTRODUCTION

The involvement of specific cell-cell interactions in differentiation sequences has often been postulated (Bennett, Boyse & Old, 1971). The evidence, however, for the existence of cell surface components whose function would be to mediate such effects is rather scarce. This problem has been approached in our laboratory by looking for cell surface antigens from undifferentiated embryonal carcinoma (EC) cells, which would disappear, or be altered, as differentiation of these cells proceeds, and thus be likely candidates to play a role in differentiation (reviewed in Jacob, 1977, and Gachelin, 1978). More recently, we have studied membranebound carbohydrates of EC cells, and we have reported the presence in these of large amounts of unusual fucosyl-glycopeptides, characterized by their apparent molecular weight exceeding 7000 daltons, as estimated by gel filtration of the fucose-labelled glycopeptides (Muramats.i et al. 1978). Further chemical analysis has shown that: (a) they are neither glycolipids, nor mucopolysaccharides, nor mucine-type-glycopeptides with short oligo-saccharide chains, nor products of incomplete digestion (Muramatsu et al. 1979 #); (b) they all 1

Author's address: Institut Pasteur, 25 rue du Dr. Roux 75015 Paris, France.

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T. MURAMATSU AND OTHERS

have the 'Glue NAQ? >Gal sequence' as internal core structure (Muramatsu et al. 19796, c); (c) they derive in part from cell surface components (Muramatsu et al. 1919 b, c; Prujansky et al. 1979). Fucosyl-glycopeptides of high molecular weight were not detected in the various differentiated cell types studied, and they were found to disappear from EC cells as these differentiated in vitro (Muramatsu et al. 1978). Since EC cells are currently used as a model for the study of normal embryonic cell differentiation (Graham, 1977), it was important to determine whether glycopeptides of high molecular weight are present in normal embryos: it has been reported that the elution profile of glycopeptides prepared from [3H]fucose-labelled preimplantation embryos was similar to that of EC cells (Muramatsu et al. 1978). In the present paper, we report that elution profiles of fucosyl-glycopeptides obtained from 6- to 10-day embryos are progressively altered. By day 10 the ratio of fucose incorporated into high and low molecular weight glycopeptides reaches a value characteristic of normal adult cells. In addition, we present evidence that at least part of the fucosyl-glycopeptides of early post-implantation cells are located on the surface of (or between) embryonic cells. MATERIALS AND METHODS

(1) Embryo dissection and culture C57B1/6 or ¥x (C57Bl/6xCBA) females were penned with Fx (C57B1/6 x CBA) males and checked daily for the presence of a vaginal plug. The day when a vaginal plug was detected was taken as day 0 of pregnancy. Eight-day and 9-day embryos were dissected as described elsewhere (Buc-Caron, Condamine & Jacob, 1978). Six-day and 7-day embryos were dissected free from the decidua and trophoblast, but retained most of their parietal yolk sac and extraembryonic ectoderm. Whole 6- to 8-day embryos and crudely minced older embryos were cultured for 6 h at 37 °C under an atmosphere of 12 % CO2 in air, in 1-2 ml of Dulbecco's modified Eagle's medium containing 15 % fetal calf serum (Gibco) and 20 /tCi/ml of L-[3H]fucose (10-20 Ci/niM, CEA, France). (2) Preparation and analysis of embryonic cell extracts The labelled embryos were washed three times with Hanks balanced salt solution and processed. Pronase digestion of labelled embryos was carried out as described previously (Muramatsu et al. 1978): the incubation volume was 1 ml for 6- and 7-day embryos, and 2 ml in the other cases. The digest was applied on top of a column of Sephadex G-50 superfine (1-7 x 60 cm) equilibrated and eluted with a pH 6-0, 0-05 M ammonia-acetic acid buffer; 2-3 ml fractions were collected. Radioactivity in the fractions was determined by liquid scintillation counting. Standard glycopeptides for column calibration were as described previously (Muramatsu, Ogata & Koide, 1976). Results are expressed as elution profiles, i.e. as radioactivity counts (in cpm)

Fucosyl-glycopeptides in mouse embryogenesis

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recovered in each fraction eluted from the column, plotted against fraction number. The curves thus obtained generally presented two main peaks corresponding to glycopeptides of relatively high and low molecular weight. In order to characterize these elution profiles more quantitatively, the ratio of counts recovered in low-molecular-weight peak to counts recovered in the highmolecular-weight peak, was used. This ratio should not be considered, however, as reflecting genuine quantification, since little is known of the structure or of turn over of molecules under study (see Discussion). Identification of radioactively labelled saccharides was carried out by paper chromatography of acid-hydrolysis products, as described previously (Muramatsu, Atkinson, Nathenson & Ceccarini, 1973). (3) Release offucosyl-glycopeptides by mild trypsinization Eight- to 10-day embryos were labelled as usual, washed carefully by three successive passages in the pre-warmed Hanks medium and placed in a saline trypsin solution (NaCl 8 g/1, KC1 0-4 g/1, glucose 1 g/1, NaHCO 3 0-5 g/1, trypsin 0-5 g/1, EDTA 0-2 g/1) at 37 °C. After about 10 min the embryos were gently and repeatedly pipetted through a capillary pipette. Trypsin action was allowed for 3 more minutes, then stopped by adding an equal volume of proteinrich medium (Eagles medium+15 % foetal calf serum). The now-dissociated cells were centrifuged gently in a Beckman microfuge (450 #, 4 min). The supernatant was collected, cleared of debris by centrifugation in a Beckman Airfuge (120000 #, 10 min) and digested with pronase. The pellet of cells was resuspended in Eagles medium+15% foetal calf serum. Viable cells were estimated on an aliquot using the Trypan blue exclusion test (Artzt et al. 1973). The remaining cells were digested with Pronase. RESULTS

(1) Fucosyl-glycopeptides from total embryos Six-day embryos were dissected free from their decidua and trophoblast, but retained most of their parietal yolk sac and extra-embryonic ectoderm. After 6 h of culture in the presence of [3H]fucose, the mean incorporation of radioactive fucose into water-soluble glycopeptides was about 400 cpm per 6-day embryo, with a rather large variability probably due to the variability of developmental state and cell number of embryos. Pools of embryos (5-10) were analysed. The soluble glycopeptides of 6-day embryos were separated by Sephadex G-50 column chromatography into two main classes: one class is made of glycopeptides of large molecular weight eluted near the excluded volume, whose apparent molecular weight is higher than 7000 daltons; the other class comprises glycopeptides eluted in well-retarded position, with a molecular weight around 2500 daltons, a value based on the calibration of the column with standard glycopeptides (see legend to Fig. 1). This elution profile (Fig. 1A) appears to be

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T. MURAMATSU AND OTHERS 1

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very similar to the one obtained with both [ H]fucose-labelled EC cells and preimplantation embryos (Muramatsu et al. 1978). Unlike 6- or 7-day embryos, 8-day and older embryos were dissected out of their parietal yolk sac. Seven- and 8-day embryos were incubated for 6 h in culture medium plus [3H]fucose, as above, without further treatment. In contrast, older embryos were cut into five to eight pieces, in order to facilitate the diffusion of metabolites through the embryonic tissues. Again, pooled embryos were used. The mean incorporation of [3H]fucose per embryo into soluble glycopeptides rapidly increased with embryonic age (from 6000 cpm per 7-day embryo, to 80 000 cpm per 10-day embryo). When fucosyl-glycopeptides prepared from 7- or 8-day embryos were analysed by Sephadex G-50 column chromatography, a biphasic elution profile was again observed. However, a slight decrease in the relative amount of high-molecular-weight glycopeptides was noticeable (Fig. IB, C). The decline in the relative amount of high molecular weight material was clearly detectable in 9-day embryos (Fig. ID), and even more evident in 10-day embryos: at that stage, the elution profile was essentially similar to that from fully differentiated cell types (Fig. IF) (Muramatsu et al. 1978). In another set of experiments, pooled 9-day-old embryos were incubated in the culture medium containing [3H]fucose for 24 h instead of the standard 6 h period. Under these conditions, no morphogenesis occurred but cells proliferated and differentiated. The elution profile (Fig. IE) of glycopeptides recovered in that case was now similar to that obtained from 10-day embryos. In previous experiments, such a longer incubation time was found not to alter the glycopeptide elution profile from preimplantation embryos (Muramatsu et al. 1978), and also from EC cells (Gachelin, unpublished result). These results thus show that although the total amount of radioactive fucose incorporated in 6 h into high-molecular-weight fucosyl-glycopeptides per

FIGURE 1

Sephadex column chromatography of [3H]fucose-labelled glycopeptides synthesized by mouse embryos at various stages. The procedure is described in Materials and Methods. The totality of pronase solubilized material was applied on top of the column. Positions of standard substances were as follows: blue dextran (fractions 22-24); [14C]acetylated fetuin glycopeptides (M.W. 3500, fractions 27-39); [14C]acetylated IgG glycopeptides (M.W. 2000, fractions 45-47); fucose (M.W. 164, fractions 60-63). Results are expressed as radioactivity recovered in each fraction versus fraction numbers. Scales have been normalized so as to give the low molecular weight peak the same height. (A) radioactivity recovered from 6-day embryos (6 embryos) (B) radioactivity recovered from 7-day embryos (21 embryos) (C) radioactivity recovered from 8-day embryos (16 embryos) (D) radioactivity recovered from 9-day embryos (5 embryos) (E) radioactivity recovered from 9-day embryos cultured for 24 hours (7 embryos) (F) radioactivity recovered from 10-day embryo? (3 embryos). 57

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Fig. 2. Sephadex G-50 column chromatography (30 x 1 cm) of [3H]fucose-labelled glycopeptides synthesized by parietal yolk-sac cells from 8-day embryos. Blue dextran was eluted in fractions 32-34, IgG glycopeptides in fractions 65-67, and fucose in fractions 77-78.

embryo greatly increases between days 6 and 10 of embryogenesis (from 120 cpm per 6-day embryo to c. 2000 per 10-day embryo), there is at the same time a 10fold decrease in the ratio of fucose incorporated into high- and low-molecularweight glycopeptides respectively, this decrease being particularly striking between day 9 and day 10. The behaviour of these two classes of glycopeptides during post-implantation embryogenesis is thus very reminiscent of what has been previously observed during in vitro differentiation of EC cells (Muramatsu et al. 1978). (2) Fucosyl-glycopeptides from isolated embryonic or extra-embryonic organs As has been noted above, 8-day and older embryos were incubated free from their parietal yolk sac, unlike 6- and 7-day embryos. Since after day 8 of embryogenesis, a steady decrease in the relative incorporation of radioactive fucose into high-molecular-weight glycopeptides is observed, one could wonder whether incorporation was taking place in yolk-sac cells of 8-day and older

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Fig. 3. Sephadex G-50 column chromatography of [3H]fucose-labelled glycopeptides synthesized by dissected embryonic organs. Same column as in Fig. 1. (A) Radioactivity recovered from 10-day embryo head part (10 embryos). (B) Radioactivity recovered from 12-day embryo head part (6 embryos). (C) Radioactivity recovered from 12-day embryo liver (4 embryos).

embryos at a high rate. Therefore, parietal yolk sacs from 8-day embryos were dissected apart and incubated under standard conditions, digested with Pronase extensively, and the resulting soluble glycopeptides were analysed by Sephadex G-50 column chromatography. Yolk-sac cells were found to incorporate substantial amounts of radioactive fucose (c. 104 cpm incorporated per 8-day yolk sac). The fucosyl-glycopeptides obtained from them gave an elution profile essentially similar to that from differentiated cells (Fig. 2), with about ten times more radioactive fucose recovered into glycopeptides of low than high molecular weight. It seems therefore unlikely that the relative decrease of fucose recovery into high-molecular-weight glycopeptides observed in 8-day and older embryos is due to these embryos being incubated free from their yolk sac. 3-2

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Similarly, a few organs from 10- and 12-day embryos were incubated apart to check whether cell populations might remain in some embryonic compartments which would preferentially incorporate fucose in high-molecular-weight glycopeptides at a time when the bulk of the embryo incorporates fucose in low-molecular-weight material preferentially. Head parts from 10-day embryos, brain and liver from 12-day embryos were thus allowed to incorporate radioactive fucose, Pronase digested and their fucosyl glycopeptides analysed in the usual way. The elution pattern obtained in all three cases (Fig. 3 A, B, C) was again found to be very similar to the one yielded by teratocarcinoma-derived differentiated cells, with an amount of radioactivity incorporated into lowmolecular-weight material clearly much higher than in the high-molecularweight peak. (3) Release of fucosyl-glycopeptides from embryonic cells following mild trypsin digestion In order to determine whether the fucosyl-glycopeptides synthesized by early embryonic cells are at least partly located on the outer part of cell membranes, embryos which had incorporated radioactive fucose were submitted to mild tryptic digestion under conditions in which most of the cells were shown to be viable. Eight-day embryos were chosen for these experiments because small batches of embryos of this age have suitable levels of radioactive incorporation and the relative amount of labelling into the high-molecular-weight material is still rather high. Eight-day embryos submitted to trypsinization for about 10 min (see Materials and Methods) yielded well-dissociated cell suspensions with a few aggregates of less than ten cells. After the treatment about 10% of the cells were scored dead in the trypan blue exclusion test. In contrast, 30-40 % of total radioactivity incorporated into non-dialysable material was recovered in the trypsinized cell supernatant. When this soluble material, as well as the radioactive material remaining associated with the cells, were digested by Pronase, and then analysed by Sephadex G-50 column chromatography, both preparations essentially gave the same elution profiles. No enrichment in either lowor high-molecular-weight material was detected in the trypsin-released fraction (Table 1). These results are very similar to those obtained with both murine EC cells (Gachelin, unpublished) and human EC cells (Muramatsu et al. 1979 a). Taken together, these results suggest that at least one third of all fucosyl glycopeptides synthesized by 8-day embryos are present on the surface of embryonic cells, or between embryonic cells. (4) Recovery of ^H]fucose from 8-day embryo fucosyl-glycopeptides Fucose is known to yield few transformation products following its uptake by adult cells (Kaufman & Ginsburg, 1968; Buck, Glick & Warren, 1970; van Beek, Smets & Emmelot, 1973), or by embryonal carcinoma cells (Muramatsu et al. 1978). In order to check whether this was also the case with normal early


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Table 1. Release offucosyl-glycopeptides from 8-day embryonic cells following mild trypsinization Experiment 1 (8 embryos)

Experiment 2 (15 embryos)

Radioactivity recovered in: Trypsinized cell supernatant Total glycopeptides 77,300 181,000 High-molecular-weight glycopeptides 11,400 32,000 Low-molecular-weight glycopeptides 43,000 121,000 Trypsinized cell pellet Total glycopeptides 102,000 390,000 High-molecular-weight glycopeptides 12,400 55,000 49,600 226,000 Low-molecular-weight glycopeptides Dead/live cells after trypsinization 8/219(3%) 24/190(11 %) 3 Eight-day embryos were dissected, incubated with [ H]fucose and trypsinized as indicated in Materials and Methods. Trypsinized cell supernatant and pellet were dialysed and then extensively digested with pronase; the resulting digest was counted (total GP) and then submitted to Sephadex G-50 column chromatography analysis. Radioactivity is expressed in cpm. High- (or low-) molecular-weight fucosyl-glycopeptide radioactivity is the sum of counts found in the high- (or low-) molecular-weight peak fractions (see legend to Table 2 for the definition of these fractions).

embryonic cells, [3H]fucose-labelled 8-day embryos were hydrolysed by HC1 in the presence of unlabelled sugar. Products from hydrolysis were separated by paper chromatography, and the monosaccharides localized. The chromatogram was cut into pieces and the radioactivity associated with each section counted. All the radioactivity recovered in monosaccharides was found to co-migrate with fucose. DISCUSSION

It has been shown previously that embryonal carcinoma cells and preimplantation embryos both share distinctive glycopeptides of unusual physicochemical properties (Muramatsu et al. 1978, 1979a, b, c). When embryonal carcinoma cells are allowed to differentiate in vitro, one observes a progressive decrease in the contribution of these high-molecular-weight glycopeptides and a correlative increase in a heretofore minor glycopeptide family, defined by a lower molecular weight: when the differentiation process has been completed, more than 90 % of the [3H]fucose was recovered into this low-molecular-weight component. The results reported in this paper indicate that a similar shift in the molecular 'landscape' of embryonic cells occurs after day 8 of embryogenesis, which seems to be the normal counterpart of the phenomenon observed with embryonal carcinoma cells first. These results are summarized in Table 2, where the ratios of radioactive fucose incorporated into low-molecular-weight glyco-

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Table 2. Relative [sH]fucose incorporation in low- and high-molecular-weight glycopeptides in various cell lines and embryos at various developmental age Cell types F9 PCC3/A/1 PCC4 aza PCC3/A/1-28 Pys-2 Fibroblasts 129 Lymphocytes 129

Description and references Nullipotent EC (Bernstine et al. 1973) Multipotent EC (Jakob et al. 1973) Multipotent EC resistant to azaguanine (Jakob et al. 1973) Fully differentiated PCC3/A/1 (Nicolas et al. 1975) Endodermal carcinoma (parietal yolk-sac carcinoma) (Lehman et al. 1974) Newborn 129/Sv fibroblasts Normal adult lymph node lymphocytes

Low/high ratio 1 -7 1-7 1-7 26-7 15-0 22 25

Embryos Total: 3-day 6-day 7-day 8-day 9-day 10-day Isolated organs: 8-day 10-day 12-day 12-day

1 7 1-9 2-4 3-2 5-1 22 Parietal yolk sac Head part Head part Liver

11-2 18 26 18

The low/high ratio represents the ratio between the counts recovered in fraction 35-50 ('low', Figs. 1 and 2) and the counts recovered in fraction 21-28 ('high', Figs. 1 and 2) or their equivalent in other columns. Some of the data are taken from results already published as elution profiles (Muramatsu et al. 1978).

peptides towards radioactive fucose incorporated into high-molecular-weight glycopeptides at various embryonic ages are presented. From day 6 to day 10 there is a 10-fold increase in this ratio, so that, by this time the low-molecularweight glycopeptides are obviously largely predominant as they will remain afterwards. The causal relation, if any, which these changes may have with differentiation processes occurring at this time, remains to be found. In looking for it, two facts should be kept in mind. First, some evidence is presented here that the molecular species under study are largely located on the surface of, or between, 8-day embryonic cells. Secondly, the changes in the relative abundance of glycopeptides of high molecular weight are readily apparent at a time of embryogenesis when the early embryonic cell surface marker F9 antigen ceases to be detected on embryonic cell surfaces (Buc-Caron et al. 1978). We have recently shown that high-molecular-weight glycopeptides are associated with the various


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components of the F9 antigen isolated from EC cells (Muramatsu et al. 1979 b), and early embryonic cells (Gachelin, unpublished data): the results represented here point further to a similarity in the evolution of these two classes of EC cell characteristic molecules. It is therefore tempting to interpret these data as meaning that dramatic changes in polysaccharide metabolism occur during days 7-9 of mouse embryogenesis. However, other interpretations are compatible with the data presented here. As an example, the subcellular location as well as the turnover rate of the highand low-molecular-weight glycopeptides could be different: low-molecularweight glycopeptides, such as those associated to the LETS protein (Carter & Hakomori, 1979), could accumulate in extracellular spaces, whereas highmolecular-weight glycopeptides would be permanently turned over. One would thus observe a decrease in the relative contribution of high-molecular-weight species. A more trivial explanation, where the reduction of high-molecularweight glycopeptides in 9- and 10-day embryos would be due to radioactive fucose having no free access to some cellular compartments, seems unlikely, because autoradiographs performed on 10-day-embryo sections did not, in our hands, reveal any limitation to the diffusion of fucose among the various embryonic tissues (Condamine & Gaillard, unpublished result). A detailed structural analysis of these glycopeptides, which would allow the study of specific transferases involved in their synthesis, seems to be the only way leading to a more definitive interpretation of the phenomenon reported above. We thank Francoise Kelly for a critical reading of the manuscript. T.M. has been on leave of absence from Kobe University School of Medicine, and was supported by the Franco-Japanese exchange programme between l'lnstitut National de la Sante et de la Recherche Medicale and the Japan Society for the Promotion of Science. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Delegation Generate a la Recherche Scientifique et Technique, the National Institute of Health, and the Andre Meyer Foundation.

REFERENCES ARTZT, K., DUBOIS, P., BENNETT, D., CONDAMINE, H., BABINET, C. & JACOB, F. (1973). Surface antigens common to mouse cleavage embryos and primitive teratocarcinoma. Proc. natn. Acad. Sci., U.S.A. 70, 2988-2992. VAN BEEK, W. P., SMETS, L. A. & EMMELOT, P. (1973). Increased sialic acid density in surface glycoprotein of transformed and malignant cells. A general phenomenon? Cancer Res. 33, 2913-2922. BENNETT, D., BOYSE, E. A. & OLD, L. J. (1971). Cell surface immunogenetics in the study of morphogenesis. In Cell Interactions, pp. 247-263. Third Lepetit Colloquium. Amsterdam: North Holland. BERNSTINE, E. G., HOPPER, M. L., GRANDCHAMP, S. & EPHRUSSI, B. (1973). Alkaline phosphatase activity in mouse teratoma. Proc. natn. Acad. Sci., U.S.A. 70, 3899-3903. BUC-CARON, M. H., CoNDAMrNE, H. & JACOB, F. (1978). The presence of F9 antigen on the surface of mouse embryonic cells until day 8 of embryogenesis. /. Embryol. exp. Morph. 47, 149-160.

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C. A., GLICK, M. C. & WARREN, L. (1970). A comparative study of glycoproteins from the surface of control and Rous Sarcoma Virus transformed Hamster cells. Biochemistry 9, 4567-4576. CARTER, W. G. & HAKOMORI, S. (1979). Isolation of galactoprotein from hamster embryo fibroblasts and characterization of the carbohydrate unit. Biochemistry 18, 730-738. GACHELIN, G. (1978). The cell surface antigens of mouse embryonal carcinoma cells. Biochim. biophys. Acta 516, 27-60. GRAHAM, C. F. (1977). Teratocarcinoma cells and normal mouse embryogenesis. In Concepts in Mammalian Embyrogenesis (ed. M. I. Sherman), pp. 315-394. Cambridge, Mass: M.I.T. Press. JACOB, H., BOON, T., GAILLARD, J., NICOLAS, J. F. & JACOB, F. (1973). Teratocarcinome de la souris. lsolement, culture et proprietes de cellules in potentialites multiples. Annls Microbiol. Inst. Pasteur, 124B, 269-282. JACOB, F. (1977). Mouse teratocarcinoma and embryonic antigens. Immunological Rev. 33, 3-32. KAUFMAN, R. L. & GINSBURG, V. (1968). The metabolism of L-fucose by HeLa cells. Expl Cell Res. 50, 127-132. LEHMAN, J. M., SPEERS, N. C, SWARTZENDRUBER, D. E. & PIERCE, G. (1974). Neoplastic differentiation. Characteristics of cell lines derived from a murine teratocarcinoma. /. Cell. Physiol. 84, 13-28. MURAMATSU, T., ATKINSON, P. H., NATHENSON, S. G. & CECCARINI, C. (1973). Cell surface glycopeptides. Growth dependent changes in the carbohydrate peptide linkage region. /. molec Biol. 80, 781-799. MURAMATSU, T., AVNER, P., FELLOUS, M., GACHELIN, G. & JACOB, F. (1979O). Distinctive properties of fucosyl-glycopeptides in human teratoma cells. Somat. Cell. Genetics (in the Press). MURAMATSU, T., GACHELIN, G. & JACOB, F. (19796). Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells. Biochim. biophys. Acta 587, 392^06. MURAMATSU, T., GACHELIN, G., DAMONNEVILLE, M., DELARBRE, C. & JACOB, F. (1979C). 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. MURAMATSU, T., GACHELIN, G., NICOLAS, J. F., CONDAMINE, H., JAKOB, H. & JACOB, F. (1978). Carbohydrate structure and cell differentiation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells. Proc. natn. Acad. Sci., U.S.A. 75, 2315-2319. MURAMATSU, T., OGATA, M. & KOIDE, N. (1976). Characterization of fucosyl glycopeptides from cell surface and cellular material of rat fibroblasts. Biochim. biophys. Acta 444, 53-68. NICOLAS, J. F., DUBOIS, P., JAKOB, H., GAILLARD, J. & JACOB, F. (1975). Teratocarcinome de la souris: differentiation en culture de cellules primitives a potentialites multiples. Annls Microbiol. Inst. Pasteur, 126A, 1-20. PRUJANSKY, A., GACHELIN, G., MURAMATSU, T., SHARON, N. & JACOB, F. (1979). External labelling of cell surface galactosyl-glycopeptides of embryonal carcinoma cells. Biochem. biophys. Res. Comm. 89, 448-455. BUCK,

(Received 15 May 1979, revised 20 December 1979)