Transformation-specific cell killing by a cancer-associated galactosyltransferase acceptor and cellular binding

Cancer-associated galactosyltransferase acceptor (CAGA glycoprotein), a small glycoprotein purified from human malignant effusion that selectively kills transformed cells, was tritiated by reductive methylation in the presence of NaB(3)H(4). CAGA-glycoprotein-sensitive cells (baby-hamster kidney cells transformed by polyoma virus and chick-embryo fibroblasts infected with Ts68 temperature-sensitive mutant of Rous sarcoma virus grown at 37 degrees C, the permissive temperature) bound 3-5-fold more (3)H-labelled CAGA glycoprotein than did their CAGA-glycoprotein-resistant non-transformed counterparts. The Rous-sarcoma-virus-infected chick-embryo fibroblasts grown at non-permissive temperature (41 degrees C) bound an intermediate amount of (3)H-labelled CAGA glycoprotein; however, this intermediate amount appeared to be sufficient to induce inhibition of cell growth when the infected chick-embryo fibroblasts treated at 41 degrees C were switched to 37 degrees C. Binding of (3)H-labelled CAGA glycoprotein was time- and temperature-dependent and was not inhibited by monosaccharide. Binding was completely inhibited by the oligosaccharide liberated by endoglucosaminidase H treatment or by exhaustive Pronase digestion of intact CAGA glycoprotein. However, the isolated oligosaccharide failed to demonstrate the growth-inhibition characteristics of the intact glycopeptide. Binding of (3)H-labelled CAGA glycoprotein was unaffected by co-incubation with the peptide core released by endoglucosaminidase H treatment. (3)H-labelled CAGA glycoprotein bound to intact cells could be removed by trypsin treatment up to 4h after addition of the glycoprotein but not thereafter. This time course paralleled the decreasing reversibility of growth inhibition. However, all (3)H-labelled CAGA glycoprotein was found in the supernatant when cells were first disrupted by sonication followed by trypsin treatment for up to 12h. (3)H-labelled CAGA glycoprotein linked to Sepharose 4B failed to cause growth inhibition in CAGA-glycoprotein-sensitive cells. These findings suggest that binding of CAGA glycoprotein occurs via its oligosaccharide moiety. Binding appears to be a necessary but not sufficient condition to induce cell killing. Growth inhibition appears to depend on internalization of the glycoprotein and the presence of a transformation-specific cell process.