Identification of noncollagenous basement membrane glycopolypeptides synthesized by mouse parietal entoderm and an entodermal cell line

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(1980)

BRIEF Identification Glycopolypeptides

NOTES

of Noncollagenous Basement Synthesized by Mouse Parietal Entodermal Cell Line CHIN

Membrane Entoderm

and an

C. HOWE AND DAVOR SOLTER

The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia,

Pennsylvania

19104

Received June 15, 1979; accepted in revised form December 18, 1979 Using two-dimensional gel electrophoresis, we have identified two noncollagenous basement membrane (BM) glycopolypeptides which are synthesized by the mouse teratocarcinoma-derived parietal yolk sac (PYS) cell line. These glycopolypeptides have molecular weights of about 200,900 and isoelectric points of about 5.6. Polypeptides with identical parameters are synthesized by the parietal entodermal cells of mouse embryos and are found in Reichert’s membrane. Pluripotent embryonal carcinoma cells (ECC) synthesize considerable amounts of the two polypeptides, whereas the yield from nullipotent ECC is negligible. The treatment of nullipotent F9 cells with retinoic acid, which induces entodermal differentiation, activates the synthesis of these polypeptides. These results indicate that the two pol.vpeptides can be used as markers of parietal entoderm -_

differentiation.

INTRODUCTION

It has been demonstrated in previous studies by different techniques that the mouse parietal yolk sac (PYS) embryonic cells and PYS carcinoma cells produce a particular kind of basement membrane (BM) (Jetten et al., 1979; Martinez-Hernandez et al., 1974; Pierce et al., 1962). Parietal entoderm is probably the first differentiated cell type observed during in vitro differentiation of mouse blastocysts and inner cell masses (ICM) (Hogan and Tilly, 1978; Wiley et al., 1978; Solter and Knowles, 1975; Solter et al., 1974). It is also the first observed during in vitro differentiation of embryonal carcinoma cells (ECC) (Martin and Evans, 1975; Martin et al., 1977; Strickland and Mahdavi, 1978). A biochemical marker for parietal entoderm would be very useful in analysis of this event. In one of our recent studies, we examined the in vitro differentiation of murine ECC and ICM to embryoid bodies (EB). Using two-dimensional gel electrophoresis of

[35S]methionine-labeled proteins, we detected two high molecular weight acidic polypeptides in ICM-EB and ECC-EB. These polypeptides were also found in teratocarcinoma-derived PYS entodermal cells but not in teratocarcinoma-derived fibroblasts (Howe et al., 1980). It was therefore of interest to see whether these polypeptides were components of the BM. It is generally recognized that the BM consists of collagenous and one or more noncollagenous glycoprotein components (Kefalides, 1971; Kefalides and Denduchis, 1969; Lee et al., 1969). The amino acid composition and molecular weight of one noncollagenous glycoprotein have been determined (Chung et al., 1979; Johnson and Starcher, 1972), but its isoelectric point is as yet unknown. The high molecular weight acidic polypeptides, which we identified as noncollagenous glycoprotein components of the BM, are synthesized by teratocarcinoma-derived PYS cells and by PYS embryonic cells, and are present in Reichert’s 480

0012.1606/80/080480-08$02.60/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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membrane. We determined both the molec- Nuclear, specific activity, 20 Ci/mmole). ular weights and isoelectric points of these Lactoperoxidase-catalyzed iodination of polypeptides as such parameters are impor- cell surface proteins was performed as detant in the detection of parietal entoderm scribed previously (Hynes, 1973). differentiation. The technique used here enabled us to detect minute quantities of Preparation of NP-40-Insoluble Proteins and Electrophoresis of Proteins these BM components, in particular, a small number of BM-producing parietal enA large proportion of the two high motodermal cells in pluripotent ECC (PSA lecular weight acidic polypeptides syntheand PCC4) cultures. sized in the PYS cell line is apparently NP40 insoluble. Much of it sediments with the MATERIALS AND METHODS nucleus after NP-40 solubilization of the Preparation of Parietal Entodermal Cells, cells (Fig. la) (Howe et al., 1980). The NPVisceral Yolk Sac, and Reichert’s Mem- 40-insoluble proteins were prepared and brane from Mouse Embryos electrophoresed as described previously Parietal entoderm was prepared by dis- (Howe and Solter, 1979; Howe et al., 1980). secting Reichert’s membrane and its asso- Briefly, radiolabeled cells were scraped ciated cells from the trophoblast of lo-day- from the substratum, lysed in 1% NP-40 old mouse embryos (Strickland and Mah- in 0.01 M NaCl, 0.01 M Tris-HCl, pH 7.4, davi, 1978). Yolk sac, composed of the vis- and 2 mM phenylmethylsulfonylfluoride ceral entoderm and mesoderm, was isolated (PMSF, a protease inhibitor) at 4°C for 1 from the same embryos. The cells were hr with occasional vortexing. The lysates labeled with [35S]methionine as described were then centrifuged through a cushion of below. 0.32 M sucrose, 3 mM MgCL, and 0.01 M Acellular Reichert’s membrane was dis- Tris-HCl, pH 7.5, at 2000 rpm for 20 min in sected from lo-day-old mouse embryos that an International centrifuge. The pellets, were frozen at -70°C for 24 hr, the mem- containing NP-40-insoluble proteins and branes were then washed in distilled water nuclear proteins, were prepared for two-di(Jensen et al., 1975). The absence of con- mensional gel electrophoresis as described taminating cells was confirmed histologi- by Peterson and McConkey (1976), and cally. electrophoresed by the method of O’Farrell (1975). Precautions were taken to avoid Cell Cultures and Radiolabeling of Cells degradation or artifactual modification of All cells were cultured in Dulbecco’s the proteins (O’Farrell, 1975). modified Eagle’s medium (DMEM), supImmunoprecipitation. Radiolabeled proplemented with 15% fetal bovine serum teins in the culture medium were immuno(FBS) as described previously (Howe et al., precipitated by the addition of 10 ~1 rabbit 1979). Cells were treated with retinoic acid anti-noncollagenous BM serum, kindly proas described by Strickland and Mahdavi vided by Dr. G. B. Pierce (Johnson and (1978). The cells were then washed and Starcher, 1972)) by normal rabbit serum, or labeled at 37’C for 16 hr in Eagle’s mini- by rabbit anti-LETS (large, external, transmum essential medium (MEM) containing formation-sensitive) glycoprotein serum, half the normal concentration of methio- kindly provided by Dr. R. Hynes, to 2 ml nine, 10% dialyzed FBS, and 20 pCi/ml culture medium. After incubation for 1 hr [35S]methionine (New England Nuclear, at 4”C, 50 ~1 goat (IgG) anti-rabbit IgG specific activity, >400 Ci/mmole), or in (heavy- and light-chain-specific, Cappel MEM containing 10% dialyzed FBS and 20 Laboratories, Cochranville, Pa.), was added @%nl [3H]glucosamine (New England and the mixture incubated overnight. The

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FIG. 1. (a-d) Autoradiograms of two-dimensional gels of NP-40-insoluble proteins of PYS cells: (a) [?!S]methionine-labeled cells, (b) [3H]glucosamine-labeled cells, (c) ‘%surface-iodinated cells, and (d) r3H]glucosamine-labeled cells treated with 0.25% trypsin, 0.1 M EDTA, in phosphate-buffered saline to detach them from the substratum (about 10 min). (e) [3H]glucosamine-labeled proteins secreted into the medium by PYS cells. Culture medium from (b) was dialyzed against 0.2 M triethylammonium bicarbonate buffer, pH 7.8, and 1% mercaptoethanol, and then lyophilized. The proteins were prepared for two-dimensional gel electrophoresis as described under Materials and Methods. Approximate pH range is given at the top and approximate molecular weight on the far left of (a). The molecular weight scale was based on the positions of the following marker proteins: myosin heavy chain (200,060),phosphorylase A (94,000), bovine serum albumin (SS,OOO), actin (45,000),and carbonic anhydrase (29,000). In (a), actin (three forms) is indicated by the letter A and tubulins (55,000and 53,000daltons) by the letter T. The actin and tubulins were identified by electrophoresing purified mouse actin and tubulin. The trichloroacetic acid-precipitable cpm applied to the first dimension of the gels and the duration of X-ray film exposures, respectively, were as follows: (a) 300,000 cpm, 2 weeks; (b) 24,006 cpm, 5 days; (c) 240,000 cpm, 3 days; (d) 160,000 cpm, 1 week; (e) 14,000 cpm, 5 days. Only those parts of the gels corresponding to the area in the block of (a) are shown in (b-e).

were centrifuged, immunoprecipitates washed, and electrophoresed two-dimensionally as described above. Immunofluorescence. PYS cells were grown on glass coverslips and fixed by exposure (10 min) to acetone at -20°C. The coverslips were exposed to rabbit anti-noncollagenous BM serum or normal rabbit serum diluted 1:12. This was followed by the addition of 1:lO diluted goat (IgG) antirabbit IgG (heavy- and light-chain specific), tagged with fluorescein isothiocyanate (GARIG-FITC) (Cappel Laboratories). RESULTS AND DISCUSSION

The two high molecular weight acidic polypeptides (represented as spots a and b, Fig. la), synthesized in the mouse terato-

carcinoma-derived PYS cell line, have molecular weights of about 200,000 and isoelectric points of about 5.6. They were identified as cell surface glycopolypeptides by labeling with [3H]glucosamine (Fig. lb), and by lactoperoxidase-catalyzed lz51-surface iodination (Fig. lc). Both polypeptides also showed sensitivity to mild trypsinization, which lowered their molecular weights (Fig. Id). The variation in the intensity of spots a and b observed in the autoradiograms may have resulted from labeling different components of the glycopolypeptide molecule. It should be noted that the 1251-surface-labeledpolypeptides, a and b, were essentially NP-40 insoluble. But an appreciable amount of NP-40-soluble POSYpeptides was also present in PYS cells as

BRIEF NOTES

revealed by metabolic labeling of what was presumably intracellular material. The same two polypeptides present on the cell surface of PYS cells were also found to be secreted into the medium (Fig. le) and into the extracellular membrane (Fig. 2). PYS embryonic cells synthesize these two poly-

483

peptides (compare Figs. la and 3a) though in proportions different from teratocarcinoma-derived PYS cells (as judged by spot intensities). These polypeptides have the same characteristics (molecular weight and isoelectric points) as the polypeptides in Reichert’s membrane (Fig. 3b). However,

FIG. 2. Staining of PYS cells with (a) normal rabbit serum and (b) rabbit anti-noncollagenous BM glycoprotein serum, followed with GARIG-FITC. The deposits of extracellular material were specifically stained with this antibody. Slides were scanned with a Leitz-Orthoplan microscope with Phloem optics. Violet blue BP390490 was the exciting filter and 515, the suppression filter. Bar = 10 Wm.

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embryo& carcinoma-derived cell line (Chung et al., 1979), and in EHS sarcoma cells (Timpl et al., 1979). At present, it is not clear whether these two polypeptides, seen occasionally as more than two spots, represent charge isomers of a protein resulting from glycosylation or other posttranscriptional modifications. Glycopolypeptides a and b were shown to be noncollagenous components of the BM since they were insensitive to collagenase (results not shown) and immunoprecipitable (Figs. 4a and b) by rabbit anti-noncollagenous BM serum (Johnson and Starcher, 1972). These polypeptides were not LETS protein as shown by their failure to immunoprecipitate with rabbit antiLETS serum (results not shown). Chung et al. (1979), in their work with an embryonal carcinoma-derived cell line, also observed a noncollagenous BM component that was

FIG. 3. Two-dimensional electrophoretjc patterns of NP-40-insoluble proteins of PYS and visceral yolk sac cells, and of Reichert’s membrane dissected from lo-day-old embryos. (a) PYS entodermal cells attached to Reichert’s membrane were labeled with [?S]methionine. Approximately 300,000 cpm was applied to the first dimension of the gel, and the duration of X-ray film exposure was about 2 days. Other details are as described in the legend to Fig. 1. (b) Proteins from acellular Reichert’s membrane displayed on Coomassie blue-stained gel. (c) [%]Methionine-labeled visceral yolk sac. Approximately 300,000cpm was applied to the fist dimension of the gel and the duration of X-ray film exposure was about 3 days.

they are not synthesized by the viscerai yolk sac (Fig. 3~). These results show that these two polypeptides are components of the BM. Using different techniques, other researchers have detected proteins probably identical to ours in PYS embryonic cells (Pierce et al., 1962; Martinez-Hernandez et al., 1974; Jetten et al., 1979), in PYS carcinoma cells (Pierce et al., 1962; Johnson et uZ., 1971; Pierce and Johnson, 1971), in an

FIG. 4. Autoradiograms of two-dimensional gels of immunoprecipitated, [?SJmethionine-labeled proteins from PYS cell line culture medium with (a) normal rabbit serum and (b) rabbit anti-noncollagenous BM serum. The cpm applied to the fast dimension of the gels and the duration of X-ray film exposures, respectively, were: (a) 2000 cpm, 10 days; (b) 8000 cpm, 10 days. Other details are as described in the legend to Fig. 1.

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distinct from LETS protein with a molecular weight similar to what we found. In addition to the 200,000 MW polypeptides, cells of the PYS cell line also synthesize two other noncollagenous BM polypeptides. Their molecular weights are 330,000 and greater than 330,000 (the former in small amounts) as shown by coimmunoprecipitation of these polypeptides with rabbit anti-noncollagenous BM serum (Fig. 5). Whether all these polypeptides belong to subunits of one or more glycoproteins is not as yet known. Strickland and Mahdavi (1978) showed that retinoic acid induces the differentiation of F9 cells into entoderma1 cells, and Solter et al. (1979) showed that these entodermal cells then synthesized BM. The retinoic acid-treated F9 cultures and a clone derived from the resulting

differentiated cells (FS-Ac cl. 9) synthesize the same two polypeptides with molecular weights of 200,000 and greater than 330,000 (Fig. 5). These polypeptides are probably similar or identical to the subunits of laminin, a noncollagenous BM glycoprotein isolated from the transplantable EHS mouse sarcoma (Timpl et al., 1979). The two BM glycopolypeptides (200,000 daltons, isoelectric points 5.6) described in the present study are highly sensitive biochemical markers for the detection of parietal entodermal cells in cell cultures. Pluripotent ECC (PSA or NG2) contain the largest amount of glycopolypeptides a and b, nullipotent F9, the smallest, and oligopotent PCC4, an intermediate amount (Fig. 6). These results suggest that the number of BM-producing PYS-like cells in the ECC cultures can be correlated to their potential to differentiate in vitro (Bernstine et al., 1973; Dewey et al., 1977; Jakob et al., 1973;

I

FIG. 5. Immunoprecipitated [%S]methionine-labeled secreted proteins with rabbit anti-noncollagenous BM serum from (a) PYS culture medium; (b) culture medium from a cloned differentiated cell line (FS-Ac cl. 9) derived from a retinoic acid-treated F9 culture; (c and d) F9 culture medium after 4 and 6 days, respectively, of retinoic acid treatment; (e) F9 control culture medium. The immunoprecipitates were analyzed in a 3-15s linear acrylamide gradient SDS slab gel (Laemmli, 1970). Normal rabbit serum failed to precipitate specific polypeptides (results not shown). Thyroglobulin was used as the molecular weight marker for MW 330,000.

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p&l 4.5

FIG. 6. Autoradiogram of [%]methionine-labeled NP-40-insoluble proteins from various ECC lines: (a) PSA, (b) PCC4, and (c) F9. Approximately 300,000 cpm was applied to the fist dimension of each gel, and the duration of X-ray film exposure was about 2 weeks. Only those parts of the gels showing subunits of noncollagenous BM glycopolypeptides are shown in this figure. Other details are as described in the legend to Fig. 1.

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Martin and Evans, 1975; Martin et al., 1977; Sherman and Miller, 1978), and that the two high molecular weight acidic polypeptides can be used as reference markers for the differentiation of the parietal entoderm in the mouse.

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JOHNSON,L. D., and STARCHER,B. C. (1972). Epitheha1 basement membranes: The isolation and identification of a soluble component. Biochim. Biophys. Acta 290,158-167. JOHNSON,L. D., STARCHER,B. C., and PIERCE, G. B. (1971). Epithelial basement membrane: Characterization of a soluble glycoprotein synthesized in uitro. Amer. J. Pathol. 62, 75a. KEFALIDES,N. A. (1971). Chemical properties of baseThis work was supported by U.S. Public Health ment membranes. Int. Rev. Exp. Pathol. 10, l-39. Service Research Grants CA-10815, CA-17546, and KEFALIDES, N. A., and DENDUCHIS,B. (1969). StrucCA-21069 from the National Cancer Institute, HDtural components of epithelial and endothelial base12487 from NICHHD, by PCM 78-16177 from the ment membranes. Biochemistry 8,4613-4621. National Science Foundation, and by l-695 from the LAEMMLI, U. K. (1970). Cleavage of structural proteins National Foundation March of Dimes. during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. REFERENCES LEE, P. A., BLASEY, K., GOLDSTEIN,I. J., and PIERCE, BERNSTINE, E. G., HOOPER,M. L., GRANDCHAMP,S., G. B. (1969). Basement membrane: Carbohydrates and EPHRUSSI,B. (1973). Alkaline phosphatase acand X-ray diffraction. Exp. Mol. Pathol. 10, 323tivity in mouse teratoma. Proc. Nat. Acad. Sci. 330. MARTIN, G. R., and EVANS, M. J. (1975). DifferentiaUSA 70,3899-3903. CHUNG, A. E., JAFFE, R., FREEMAN, I. L., VERGNES, tion of clonal lines of teratocarcinoma cells: ForJ. P., BRAGINSKI, J. E., and CARLIN, B. (1979). mation of embryoid bodies in uitro. Proc. Nat. Properties of a basement membrane-related glycoAcad. Sci. USA 72,1441-1445. protein synthesized in culture by a mouse embryonal MARTIN, G. R., WILEY, L. M., and DAMJANOV, I. carcinoma-derived cell line. Cdl 16, 277-287. (1977). The development of cystic embryoid bodies DEWEY, M. J., MARTIN, D. W., JR., MARTIN, G. R., in vitro from clonal teratocarcinoma stem cells. and MINTZ, B. (1977). Mosaic mice with teratocarDevelop. Biol. 61,230-244. cinema-derived mutant cells deficient in hypoxanMARTINEZ-HERNANDEZ, A., NAKANE, P. K., and thine phosphoribosyltransferase. Proc. Nat. Acad. PIERCE, G. B. (1974). Intracellular localization of Sci. USA 74.5564-5568. basement membrane antigen in parietal yolk sac HOGAN,B., and TILLY, B. (1978).In vitro development cells. Amer. J. Pathol. 76,549-560. of inner cell masses isolated immunosurgically from O’FARRELL,P. H. (1975). High resolution two-dimenmouse blastocysta. I. Inner cell masses from 3.5-day sional electrophoresis of proteins. J. Biol. Chem. 260,4007-4021. p.c. blastocysts incubated for 24 hr before immunoPETERSON,J. L., and MCCONKEY, E. H. (1976). Nonsurgery. J. Embryo!. Exp. Morphol. 45,93-105. histone chromosomal proteins from HeLa cells: A HOWE,C. C., and SOLTER,D. (1979). Cytoplasmic and survey by high resolution, two-dimensional electronuclear protein synthesis in preimplantation mouse phoresis. J. Biol. Chem. 261,548-554. embryos. J. Embryol. Exp. Morphol. 62,209-225. PIERCE,G. B., and JOHNSON,L. D. (1971). DifferentiaHOWE, C. C., GM~R, R., and SOLTER, D. (1980). Cytion and cancer. In vitro 7, 140-145. toplasmic and nuclear protein synthesis during in PIERCE, G. B., JR., MIDGLEY, A. R., JR., SRIRAM, J., vitro differentiation of murine ICM and embryonal and FELDMAN,J. D. (1962). Parietal yolk sac carcicarcinoma cells. Develop. Biol. 74,351-363. noma: Clue to the histogenesis of Reichert’s memHYNES, R. (1973). Alteration of cell surface proteins brane of the mouse embryo. Amer. J. Pathol. 41, by viral transformation and by proteolysis. Proc. 549-566. Nat. Acad. Sci. USA 70, 3170-3174. JAKOB, H., BOON, T., GAILLARD, J., NICOLAS, J. F:, SHERMAN, M. I., and MILLER, R. A. (1978). F9 embryonal carcinoma cells can differentiate into entoand JACOB,F. (1973). Teratocarcinome de la souris: derm-like cells. Develop. Biol. 63,27-37. isolement, culture et proprfetes de cellules a potenSOLTER,D., and KNOWLES,B. B. (1975). Immunosurtialites multiples. Ann. Microbial. (Inst. Pasteur) gery of mouse blastocyst. Proc. Nat. Acad. Sci. USA 124B, 269-282. 72, 5099-5102. JENSEN, M., KOSZALKA, T. R., and BRENT, R. L. (1975). Production of congenital malformations us- SOLTER,D., BICZYSKO,W., PIENKOWSKI,M., and KoPROWSKI,H. (1974). Ultrastructure of mouse egg ing tissue antisera. Develop. Biol. 42, 1-12. cylinders developed in vitro. Anat. Rec. 180, 263JETTEN, A. M., JETTEN, M. E. R., and SHERMAN, M. 280. I. (1979). Analyses of cell surface and secreted proSOLTER, D., SHEVINSKY, L., KNOWLES, B. B., and teins of primary cultures of mouse extraembryonic STRICKLAND,S. (1979). The induction of antigenic membranes. Develop. Biol. 70.89-104.

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changes in a teratocarcinoma stem cell line (F9) by retinoic acid. Develop. Biol. 70, 515-521. STRICKLAND, S., and MAHDAVI, V. (1978). The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15, 393-403. TIMPL, R., ROHDE, H., ROBEY, P. G., RENNARD, S. I.,

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FOIDART, J. -M., and MARTIN, G. H. (1979). Laminin-a glycoprotein from basement membranes. J. Biol. Chem. 254,9933-9937. WILEY, L. M., SPINDLE, A. I., and PEDERSEN, R. A. (1978). Morphology of isolated mouse inner cell masses developing in vitro. Develop. Bid. 63, I-10.