It is well-known that the bioavailability of iron from human milk is very high. The investigators have hypothesized that this high bioavailability is the result of a receptor-mediated mechanism in the small intestine that facilitates the absorption of iron from LF, the major iron-binding protein in human milk. Preliminary data demonstrate the presence of a specific LFR in the small intestinal brush border membrane of human infants and piglets. In the proposed project, the investigators intend to isolate and characterize the human LFR with regard to molecular size, isoelectric point, subunit arrangement, amino acid and carbohydrate composition and to study the molecular interaction between LF and the LFR. A polyclonal antibody towards LFR will be developed and the gene will be cloned from a human small intestinal cDNA library in lamda-gt11. The gene will be sequenced and the amino acid sequence deduced from the nucleotide sequence. In parallel experiments, the pig LFR will be characterized, an antibody produced and the LFR gene cloned and sequenced. The piglet will then be used as an animal model to study the ontogeny of the LFR, the localization in the small intestine and the effect of iron status on the concentrations of LFR and its mRNA, using the isolated cDNA as a probe. Using piglet enterocytes in suspension, the potential internalization of Lf and LfR will be studied. In conclusion, the results from this proposal should provide a detailed insight into the molecular nature of the LFR, its interaction with LF and the mechanism of iron uptake into the enterocyte. Information on the ontogeny of the LFR, localization in the gut and the effect of iron status on LFR expression will be obtained from the animal model chosen.
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