The isolation and properties of multiple forms of folate binding protein in cultured KB cells

Luhrs, Carol; Sadasivan, Easwara; Costa, Maria Da; Rothenberg, Sheldon P.

doi:10.1016/0003-9861(86)90705-8

Cited by 28 publications

(23 citation statements)

References 22 publications

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“…The total FBP of the transfected 3T3 cells did not increase when these cells were cultured in FD medium for 4 mo (column 3) (1.36 ng/,ug Triton solubilized protein). In contrast, the total FBP of KB cells did increase when these cells were cultured in FD medium (column 5) (6 ng/Ag Triton solubilized protein) and this has been previously reported by our laboratory and others (3)(4)(5) (Fig. 4) The concentration of total folate in these cells is shown in Table I.…”

Section: Methodssupporting

confidence: 81%

“…It is of interest that although the expression of the GPIlinked FBP in KB cells increases when these cells are cultured in low folate medium (3)(4)(5), no similar increase is observed in the transfected 3T3 cells. Two factors may explain this observation.…”

Section: Discussionmentioning

confidence: 99%

“…The membrane FBP was purified from cells cultured in FD medium using an epoxy-activated Sepharose 6B matrix coupled to folate (24), as previously described (5,25).…”

Section: Methodsmentioning

confidence: 99%

“…The KB cell, a cultured cell line derived from a human epidermoid carcinoma, has a high level ofexpression ofa membrane FBP, a small fraction of which is released as a soluble form into the culture medium (3)(4)(5). We have recently demonstrated that the membrane FBP in KB cells is a member of a unique group ofproteins that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail, rather than by a transmembrane hydrophobic peptide (6).…”

Section: Introductionmentioning

confidence: 99%

“…However, unlike these cell lines, KB cells survive without selection in medium containing a low concentration of folate (1 nM), i.e., 2,000-fold less than contained in normal, folate-replete medium (2.3 ,uM) (3)(4)(5). Although the GPI-anchored FBP has a higher affinity for oxidized than reduced folates, it has been demonstrated that this membrane FBP in KB cells mediates the uptake of the physiologic folate, 5-methyltetrahydrofolate (5-methyl THF) (12,13).…”

Section: Introductionmentioning

confidence: 99%

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

Luhrs¹,

Raskin²,

Durbin³

et al. 1992

J. Clin. Invest.

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KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated 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/,ug protein, respectively. The capacity for binding 13HIfolate to the surface of transfected 3T3 cells cultured in folatedeficient 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 13HI 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. (J. Clin. Invest. 1992. 90:840-847.)

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Section: Methodssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

“…The membrane FBP was purified from cells cultured in FD medium using an epoxy-activated Sepharose 6B matrix coupled to folate (24), as previously described (5,25).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations