Studies on members of the transferrin family of proteins in humans and mice

Iron is required by all cells. Its chemical properties have been exploited by living organisms to drive a variety of life sustaining metabolic reactions. The demand for iron coupled with its highly reactive nature has forced organisms to evolve specific mechanisms for the adsorption, transport, and storage of this element. The iron binding transferrin family of proteins that includes transferrin (Tf), lactoferrin (LTf), and melanotransferrin (MTf) has been the most well studied class of iron transporters. However, much of the molecular physiology of this family, particularly MTf, is still unclear. There is growing evidence that iron imbalances associated with abnormalities in Tf and MTf expression, localization, and function may be involved in the pathologies of a number of diseases such as cancer and Alzheimer's disease (AD). Mouse as a model system to study Tf and MTf has been poorly characterized. To date, no full length cDNA of mouse Tf has been characterized and no mouse homologue to human MTf has been identified. Possession of full length cDNAs of mouse Tf and MTf will allow us to create a variety of recombinant constructs that will allow us to make transgenic and knockout mice. The characterization of these mice may allow us to determine the importance of Tf and MTf in normal iron transport as well as the role of these molecules in diseases such as AD. In developing the mouse as a small animal model, this thesis describes (1) the cloning of a mouse Tf cDNA from a mouse liver cDNA library, (2) Southern analysis of the genomic organization of human MTf, (3) the identification of a mouse homologue of MTf through Southern analysis, (4) Northern analysis of the distribution of Tf and MTf mRNA expression in normal tissues in humans and mice and (5) identification of a mouse melanoma cell line expressing mouse MTf mRNA. A putative 2.2kb mouse Tf cDNA complete with translation start site, an endoplasmic reticulum (ER) signal peptide, 159 bp of 3' untranslated (UTR) sequence, and a poly adenylation signal was cloned and sequenced. The human MTf gene is approximately 49kb long as compared to the putative mouse MTf gene which spans 38kb. The restriction sites of Bam HI, Eco Rl, and Hind III in the human and mouse MTf genes were mapped. The mRNA expression of Tf and MTf appear to be conserved in both humans and mice. Liver tissue appears to express high amounts of Tf mRNA in relation to other tissues. No detectable MTf mRNA expression was found in normal adult human and mouse tissues. In contrast, MTf mRNA was detected in the human melanoma cell line SKMEL-28, the mouse melanoma cell line JB/MS, and mouse fetal and placental tissues. Finally, cell lines and tissues positive for MTf mRNA expression appear to express multiple forms of the mRNA. In SKMEL-28 and JB/MS cells MTf transcripts of the sizes 3.9kb and 2.6kb were detected. In normal mouse liver tissues a 3.6kb MTf transcript was detected. These results establish the feasibility of using mice as a model to study Tf and MTf and have allowed us to go on to the cDNA cloning of mouse MTf and the construction of a variety of fusion proteins and deletion constructs.Science, Faculty ofZoology, Department ofGraduat