RUI: Secreted Ferritin: Structure and Cloning

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9731745 Linder The investigators have obtained evidence that ferritin secreted by hepatic cells, and serum ferritin, have a different structure than the well-studied intracellular ferritins. They hypothesize that secreted/serum ferritin has only partial homology to intracellular ferritins and is encoded by different genes. The objective is to clone cDNAs for the subunits of secreted/serum ferritin to test the hypothesis. Ferritin from horse serum and the medium of rat hepatoma cells will be isolated. N-terminal and internal amino acid sequence will be obtained. cDNAs will be cloned by using the amino acid sequence (a) to design probes with which to screen a rat liver library, and (b) to design primers with which to perform RT-PCR on rat liver mRNA. Full sized clones will be sequenced and used to determine the primary structure of the subunits of secreted ferritin for comparison with that of known intracellular ferritins. Clones will be validated by demonstrating (a) that they encode mRNAs transcriptionally responsive to iron, and (b) that amino acid sequences for different portions of the subunits are encoded by nucleotide sequence in appropriate regions of the clones. If needed, clones will also be validated by showing that rat liver mRNA selected by hybridization to the clones encodes a product of the appropriate size and is recognized by antibody against intracellular ferritin. Ferritin is an iron storage molecule found in significant amounts inside all cells. A great deal is known about its structure, function and genetics. Traces of a ferritin are also found in blood serum, but almost nothing is known about its structure, function and genetics. The investigators have evidence that blood serum ferritin differs markedly from that in cells and will test this concept. If they are correct, their findings will greatly change views about what is ferritin, and exactly what is being measured as ferritin in serum will become apparent.