Isolation of Cell Surface Glycoproteins from Normal and Transformed Cells by Affinity Chromatography on Plant Lectin Columns

Carbohydrate-containing components of the cell surface have been implicated in many important biological functions, including cell-cell interactions, receptors for some hormones and viruses and immunological specificity (Kraemer, 1971). Further, studies on normal cells and their virally-transformed derivatives have shown some alterations in plasma-membrane glycoproteins (Warren et al., 1973) and increased agglutinability of transformed cells by plant lectins (Eckhart et af. , 1971). Isolation of the glycoproteins from purified plasmamembranes would permit a direct comparison of these proteins from normal and transformed cell, and, in addition, might make possible the analysis of their biological functions, as well as their organization in the cell surface membrane. The binding of lectins occurs through their interaction with saccharides, and, although each lectin has its own saccharide specificity, a wide diversity can be obtained from various plants and animals (Sharon & Lis, 1972). Recently a method was described for the isolation of lymphocyte plasma-membrane glycoproteins (Hayman & Crumpton, 1972) and virus glycoproteins (Hayman et al., 1973) by means of affinity chromatography of sodium deoxycholate-solubilized membrane on Lens culinaris lectin covalently attached to Sepharose. These reports suggest that sodium deoxycholate-solubilized membrane glycoproteins may be fractionated by affinity chromatography on immobilized lectins of different specificities. The present paper describes the application of this technique to the isolation and partial characterization of plasma-membrane glycoproteins from normal mouse (3T3), simian virus 40-transformed mouse (SV3T3), normal hamster (BHK) and polyoma virus-transformed hamster (PyBHk) fibroblast cell lines. Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % (v/v) calf serum. To facilitate removal of membrane-bound serum proteins, the medium was replaced with fresh medium minus calf serum 24 h before harvesting. Cells were harvested by scraping, washed with phosphate-buffered saline and plasma membrane prepared by the two-phase polymer procedure of Brunette &Till (1971). In some experiments cells were labelled for a 24h period before harvesting with [14C]leucine (2/1Ci/ml) in medium containing 10% dialysed calf serum and leucine at 10% of its normal concentration. The labelled normal or transformed cells were harvested and then mixed with an excess of unlabelled transformed cells before membrane preparation. The final plasma-membrane pellet was suspended in 2 % sodium deoxycholate and immediately frozen at -70°C. Plant lectins were prepared from L. culinaris by the method of Hayman & Crumpton (1972), and from Ricinus communis by the method of Nicolson et al. (1974). The lectin from L. culinaris (LcH) possesses a specificity for glucoseand mannose-related sugars, whereas the lectin from R . Communis (RCAII) exhibits a specificity for D-galactoserelated sugars. Lectin-Sepharose 4B conjugates were prepared as described by Hayman & Crumpton (1972). The frozen plasma-membrane solution was thawed and diluted with an equal volume of H20 beforecentrifugation at 1OOOOOgfor 1 h.The supernatant, the‘soluble’membrane components, was applied to the desired lectin column equilibrated with 1 % sodium deoxycholate. After application of the solution the column was washed extensively with 1 % sodium deoxycholate and the adsorbed glycoproteins were subsequently eluted with 0.3hi-a-methyl-~-mannose (LcH) or 0.3hi-~-galactose (RCAd in 1 % sodium deoxycholate. The eluted fractions were dialysed against 1 % sodium deoxycholate before freezing and freeze-drying. Sodium deoxycholate was removed from the freeze-dried residues by extraction with ethanol at -20°C. The precipitated proteins were analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate as described by Studier (1972).