Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium

Abstract

KB cells express a folate-binding protein that is anchored to the plasma membrane by a glocosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/μg protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/106 cells, and this is ∼ 50% of the surface binding capacity of KB cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of [3H] folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions.