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Metal binding properties of p97 and the trafficking of a novel iron internalization pathway by GPI-anchored p97 Tiong, Jacqueline Woang Cheing

Abstract

Iron is an essential for cellular metabolism yet excessive iron can lead to deleterious oxidative damages in the cells. Since humans have no physiological pathways for iron excretion, dietary iron absorption in the intestine is tightly regulated. Although the essential nature of iron for normal celular functions is beyond doubt, only in the two decades has its uptake and regulation been relatively wel understood. The transferrin/ transferrin receptor-independent iron transport pathways play a major role in iron overload diseases such as hemochromatosis. However, very little known about the molecular details of these pathways. P97, also known as melanotransferrin, is an example of iron binding proteins that mediates such pathways. P97 is a member of the transferrin family, which includes serum transferrin, lactoferrin and ovotransferrin. Unlike members of the transferrin family, which are all serum proteins, p97, in addition to being a serum protein, also exists as a glycosylphosphatidylinositol-anchored protein. The primary iron-binding site in the Nterminal lobe is highly conserved among the other transferrin members. However, the iron binding site at the C-terminal lobe of p97 is not. This leads to the question whether p97 is a functional iron transport protein. In addition, the iron binding affinity p97 has never been ascertained. Therefore, an aspect of this thesis was to determine binding affinity of iron to p97 and to establish whether the C-lobe of p97 can bind The results in this thesis will demonstrate that the iron binding affinity for the N-lobe p97 is 2.2 x 10¹⁷ M⁻¹ at 25 °C. In addition, the data will show that C-lobe is able iron albeit very weakly. Metals such as iron, copper, aluminum and zinc have been implicated in pathology of several neurological disorders. The expression of p97 in the brain capilary endothelium and the elevated level of serum p97 associated with Alzheimer s disease have led to the hypothesis that p97 may play an important role in disease progression the disease. Therefore, another goal of this study was to establish whether p97 can metals such as copper, aluminum and zinc. The results will show that copper, aluminum and zinc are able to interfere with iron binding to GPI-anchored p97 and soluble able to bind to these metals as shown by urea polyacrylamide gel electrophoresis. Though the internalization pathways of many GPI-anchored proteins have been studied, debate still exists as to whether caveolae or clathrin-coated vesicles are involved in their uptake. Surprisingly, very little is known about the fate of the ligands internalized by GPI-anchored molecules. Also, there is controversy as to whether iron bound to p97 can be donated to ferritin. Therefore, another aspect of this thesis was examine the uptake of GPI-anchored p97 and its ligand, iron. The results from confocal immunofluorescence microscopy and sub-celular fractionation demonstrate that GPIanchored p97 bound with iron is endocytosed into SK-MEL 28 cells via caveolae traffics to early endosomes. In addition, the data also shows that GPI-anchored p97 present in the same vesicles as Nramp2, a metal transporter thought to mediate transport across the vesicular membrane into the cytoplasm. Furthermore, it is shown that iron taken up by p97 becomes bound by the iron storage molecule, ferritin. The establishes that iron bound to GPI-anchored p97 is internalized through caveolae and donated to ferritin for cellular metabolism and supports an improved understanding of both GPI anchor mediated uptake of ligand and the novel transferrin/ transferrin receptorindependent mechanism of iron uptake.

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