Isolation of a Major Cell Surface Glycoprotein from Fibroblasts

Abstract

A cell surface component has been isolated in partially purified form from cultured chick embryo and chick heart fibroblasts. This glycoproteiri is similar to a protein recently reported to be present at the surface of normal cells, and missing after neoplastic transformation. It is a major cell surface glycoprotein that is synthesized by cultured fibroblasts, but is not collagen. It is shown to be markedly trypsin-sensitive, and its recovery from the cell surface is dependent on cell density. It is excluded from Sephadex G-200, but is not rapidly sedimented by ultracentrifugation, and has an apparent molecular weight of 220,000. Recently, there have been several reports that a cell surface glycoprotein of 200,000-250,000 daltons is present on several "normal" cell types, but is markedly diminished or absent from their transformed variants (9-13). We report the extraction and preliminary characterization of such a protein from chick embryo and chick heart fibroblasts. The conditions of isolation should be gentle enough to permit future evaluation of its biological function. MATERIALS AND METHODSCulture Methods. Secondary cultures of 10-day chick embryo fibroblasts were prepared by a modification of the methods of Rein and Rubin (14). After 3-4 days of primary culture, cells were harvested with 0.125% (w/v) trypsin (Difco) plus 10 ug/ml of DNase (Schwarz/Mann), passed to 100-mm plastic dishes at 5 X 101 per 12 ml of medium, and cultured to a necessary final cell density of about 2.5 X 105/cm2. Primary cultures of minced 7-day chick embryo heart ventricles were established in 25-cm2 plastic culture flasks (Falcon). Secondary cultures were prepared at densities of 6 to 8 X 100 per 100-mm dish from the outgrowths after 2 days in culture.Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; NaDodSO4, sodium dodecyl sulfate; CSP, cell surface protein; MW, molecular weight; PAS, periodic acidSchiff. * Isolation of Surface Protein. The following extraction procedure maximized purity and yield of the cell surface protein, and minimized retraction of the cell monolayers. Cultures were rinsed three times with Hanks' balanced salts solution buffered with 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4 (Calbiochem). Cells on 100-mm dishes were then extracted 2. hr at 370 in 5 ml of serum-free Dulbecco's. medium with glutamine on a gyratory shaker (model G-2, New Brunswick Scientific) at 30 rpm.This first extract in serum-free medium was discarded, and the monolayers were washed once with HEPES-buffered Hanks' solution. The cells were then further extracted for 2 hr in serum-free medium to which urea (Schwarz/Mann, ultra-pure) was -added to a final concentration of 0.2 M immediately before use. This second extract was removed, chilled, and centrifuged at 25,000 X g for 15 min.Cell loss after this extraction protocol was minimal: <0.8% of the total was found in the pellets, and no loss was detected when parallel dishes before and after extraction were compared (1.4 X 107...

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