A number of human diseases have been described which are not due to a quantitative deficiency of folic acid but which may respond to large doses of the vitamin. These include hematopoietic disorders, such as some forms of aplastic anemia, a hereditary form of dyserythropoiesis, neurologic disorders such as dementia, some forms of schizophrenia, the recently recognized fragile X chromosome syndrome, and drug induced disorders of folate metabosism. The response to large doses of folate suggests that a common defect may underlie these disorders. The folate binding proteins (FBPs) which have now been identified in the membrane, in the soluble, and most recently in the nuclear fraction of mammalian cells could be the common link. Most of the work on these proteins from mammalian cells have focused on their structural properties rather than on their function and turnover in the cell. In addition, there have been no studies on the molecular genetics of the FBP to characterize the structure, organization and expression of the gene. The primary aims of this project, therefore, are to establish the precise function(s) of the folate binding proteins (FBPs) in the cellular uptake and intracellular metabolism of folate and to characterize fully the molecular genetics of this protein(s). The experiments will be designed to achieve the following objectives: 1) define the kinetics for the synthesis and degradation of the FBP in human KB cells and study the factors that affect these parameters using intrinsic labeling with (35S)methionine 2) map the cellular and topographic distribution of the FBP in the KB cell using electron microscopy immunocytology and study the factors that modify this distribution using monoclonal antibodies 3) clone the FBP gene from KB cells and then characterize its organization and nucleotide sequence, determine its chromosomal location, and examine different human cell lines and tissues for expression and restriction fragment length polymorphism of the gene 4) determine if transfection of the functional FBP gene into cells which express little or no FBP improves the viability of these cells in folate deficient medium. The study could provide definitive evidence that the FBP provides a critical function in cellular metabolism.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA032369-08
Application #
3170324
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1982-05-01
Project End
1992-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
8
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Suny Downstate Medical Center
Department
Type
Schools of Medicine
DUNS #
068552207
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
Sadasivan, E; Cedeno, M M; Rothenberg, S P (1994) Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein. J Biol Chem 269:4725-35
Luhrs, C A; Raskin, C A; Durbin, R et al. (1992) Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium. J Clin Invest 90:840-7
Sadasivan, E; Cedeno, M; Rothenberg, S P (1992) Genomic organization of the gene and a related pseudogene for a human folate binding protein. Biochim Biophys Acta 1131:91-4
Luhrs, C A (1991) The role of glycosylation in the biosynthesis and acquisition of ligand-binding activity of the folate-binding protein in cultured KB cells. Blood 77:1171-80
Sadasivan, E; Rothenberg, S P (1989) The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA. J Biol Chem 264:5806-11
Luhrs, C A; Slomiany, B L (1989) A human membrane-associated folate binding protein is anchored by a glycosyl-phosphatidylinositol tail. J Biol Chem 264:21446-9
da Costa, M; Rothenberg, S P (1988) Characterization of the folate-binding proteins associated with the plasma membrane of rat liver. Biochim Biophys Acta 939:533-41
Sadasivan, E; da Costa, M; Rothenberg, S P et al. (1987) Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells. Biochim Biophys Acta 925:36-47
Luhrs, C A; Pitiranggon, P; da Costa, M et al. (1987) Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation. Proc Natl Acad Sci U S A 84:6546-9
Luhrs, C A; Sadasivan, E; da Costa, M et al. (1986) The isolation and properties of multiple forms of folate binding protein in cultured KB cells. Arch Biochem Biophys 250:94-105