Folate is a water-soluble vitamin that is essential for nucleic acid and amino acid metabolism. Folate absorption requires a two-stage process of hydrolysis of dietary pteroylpolyglutamates and binding and transport of the pteroylmonoglutamate derivative at the brush-border surface of the intestine. The overall objective of the proposed research is to further understanding of the process of folate absorption by study in the pig of the molecular properties and tissue expression of two intestinal folate-interactive proteins: jejunal brush-border folate hydrolase (BBFH) and folate-binding protein (FBP).
Specific aims i nclude: a) to develop cDNA and antibody probes for both proteins; b) to determine the full-length cDNA for both proteins; and c) to study the tissue expression of both proteins. Methods include use of oligonucleotide probes for BBFH and a gift full-length cDNA for a human FBP to isolate specific clones for BBFH and FBP from a pig jejunal cDNA library. Antibodies will be developed from purified proteins, fusion proteins, and synthetic peptides, and will be used to confirm oligonucleotide screens and for tissue expression studies. Sequencing of full-length cDNAs for each protein will be followed by homology comparisons. Tissue studies will utilize Northern blots and in-situ hybridization for transcripts, and immunoprecipitation, rocket immunoelectrophoresis, immunoblots, and immunohistochemistry to determine molecular sizes, amounts, and cellular distribution of each protein in the intestine and other tissues. Data from These studies will be used to develop a subsequent proposal to study the regulation of protein synthesis in animal models, thus providing a framework for study of the regulation of folate absorption in human tissue from patients with different disorders of folate absorption and deficiency.
