Transformation-dependent alterations is glycoproteins of extracellular matrix of human fibroblasts. Characterization of GP250 and the collagen-like GP140.
The extracellular matrix, prepared by extraction of confluent cultures of human lung WI-38 fibroblasts with a dipolar tonic detergent, contains four major glycoproteins: fibronectin, GP250, GP170, and GP140. All the glycoproteins can be surface-labeled; however, only fibronectin and GP170 can be readily removed by digestion with trypsin (Carter, W. G., and Hakomori, S. (1981) J. Biol. Chem. 256, 6953-6960). Most of the noncovalently bound GP250, GP170, and GP190, an additional minor glycoprotein, can be dissociated from the matrix by extraction with 8 M urea. The remaining insoluble matrix is stabilized by extensive intermolecular disulfide bonds and contains primarily GP140 and fibronectin (Carter, W. G. (1982) J. Biol. Chem. 257, 3249-3257). Affinity-purified, monospecific antibodies were prepared against GP[140 and fibronectin and utilized for detection of GP140 and fibronectin in extracts and conditioned media of WI-38, WI-38 VA13, WI-26, WI-26 VA4, and HT-1080 cells. Additional affinity-purified, polyspecific antibodies that react with GP250, GP190 GP170, and GP140 were also utilized. Fibronectin, GP250, GP190, GP170, and GP140 were all absent from transformed cells. With the exception of GP140, the absence of these glycoproteins from the matrix of transformed cells was paralleled by their accumulation in the conditioned culture media. Incubation of conditioned culture media with collagenase indicated that GP190, GP170, and GP140, as well as other glycoproteins, were digested. Antibodies to GP140 did not react with any other cellular component indicating that it is not a processing product of other matrix glycoproteins. GP140 has characteristics unlike all reported collagen types and appears to be a new collagen-like glycoprotein. In contrast, neither Gp250 nor fibronectin were sensitive to digestion with collagenase. Antibodies that react with GP250 did not react with fibronectin and vice versa, suggesting that GP250 and fibronectin do not share antigenic determinants. The interaction of labeled fibronectin and the labeled, gelatin-binding domain of fibronectin with cells after fractionation on polyacrylamide gels indicated that GP170 is the primary procollagen receptor for fibronectin in the extracellular matrix. GP140 also bound fibronectin but to a lesser degree. Soluble GP170 and GP190 present in the conditioned medium of cultured cells also bound to insolubilized fronectin, confirming the association of GP170 and GP190 with fibronectin. The interaction of the glycoprotein components in the matrix are discussed in relation to their potential cooperative function in cell attachment and their failure to adhere to the surface of transformed cells.
D002460:Cell Line; D002472:Cell Transformation, Viral; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008168:Lung; D008970:Molecular Weight; D013539:Simian virus 40 / Q000235:genetics
D006023:Glycoproteins; D003094:Collagen