The cooperative role of the transformation-sensitive glycoproteins, GP140 and fibronectin, in cell attachment and spreading

The detergent-insoluble matrix of cultured human fibroblasts contains cytoskeletal and nuclear components, as well as two major, noncollagenous glycoproteins, fibronectin and GP140. These glycoproteins are stabilized by extensive intermolecular disulfide bonding. GP140, in contrast to fibronectin, is resistant to digestion with trypsin and is not cross-reactive with antisera prepared against fibronectin (Carter, W. G., and Hakomori, S. (1981) J. Biol. Chem. 256, 6953-6960). GP140 was partially purified, under nonreducing conditions, by differential extraction of trypsinized cells with sodium trichloroacetate. Alternatively, a higher yield of GP140 could be obtained under reducing conditions by extraction with urea-dithiothreitol followed by molecular sieve chromatography in the presence of sodium dodecyl sulfate and dithiothreitol. The purified GP140 contained mannose, galactose, and N-acetylglucosamine residues, totaling 2.7% of the molecule. In addition, mild periodate oxidation of GP140 followed by reduction with NaB3H4 under conditions designed to label sialic acid also labeled the peptide portion of the molecule. Amino acid analysis of GP140 detected periodate-sensitive hydroxylysine residues, as well as hydroxylproline, accounting for the periodate/NaB3H4-induced label in the peptide. These hydroxylated amino acids are major components of collagens and collagen-like proteins. The GP140 isolated under nonreducing conditions was found to induce stable cell attachment and cell spreading when coated on plastic surfaces. The cell attachment could not be inhibited with affinity purified anti-fibronectin antibodies. However, trypsinization of cells under conditions that removed surface fibronectin reduced the ability of the cells to bind to the GP140-coated surface. Metabolic labeling of cells with radioactive glucosamine during 1-h cell attachment experiments incorporated label into both fibronectin and GP140, as well as four other carbohydrate-containing components as part of a stable detergent-insoluble matrix, indicating that the cells rapidly glycosylate both fibronectin and GP140 and incorporate them into the matrix. Long term labeling and chase experiments indicated that fibronectin and GP140 in the matrix are subject to very slow turnover. Neither fibronectin nor GP140 were detectable in the detergent-insoluble matrix of SV40-transformed human fibroblasts by either metabolic or cell surface labeling. These results are consistent with the conclusion that fibronectin and GP140 may function in a cooperative manner in cell adhesion and spreading.