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Jalaludeen, Mohamed Riyazudeen Ahamed (2012) Studies on the mechanism of the ferroxidase hephaestin in the absorption of dietary iron. Masters thesis, London Metropolitan University.
Iron is one of the most abundant metals in nature and a necessary element for life. Dietary iron enters the intestinal epithelium via the brush border reductase/transporter Dcytb/DMTI or haem pathway and exits through the basolateral membranes. The basolateral transfer of iron requires two components: a membrane transport protei ferroportin and a ceruloplasmin (Cp) homologue known as hephaestin (Hp), which was identified as the defective gene in the sla (sex- linked anaemia) mice and plays a critical role in intestinal iron absorption, ascribed to its multicopper ferroxidase activity. Knowledge of the synthesis, distribution, activity and regulation of hephaestin will help to understand its role in cellular iron efflux. The aims of this investigation are i) to study the , expression and localisation of hephaestin using GFP-tagged constructs; ii) to investigate ferroxidase activity and oxidase activity in the presence of different copper concentrations of recombinant hephaestins tagged with GFP and expressed in COS cells and iii) to determine the optimal pH of the ferroxidase activity.
Four GFP-tagged variants of hephaestin were successfully expressed in COS cells. Subcellular localisation was assessed by fluorescence microscopy and assay of enzyme activity confirmed that the recombinant proteins were all biologically active. Three variants were transmembrane forms of hephaestin with cytoplasmic tails either full length, half-length, or with the di-leucine motif mutated to di-alanine. All variants showed a similar increase in oxidase and ferroxidase activity when extra copper was added to the cell culture medium, suggesting that normal medium is not optimal for expression of copper proteins. The pH dependence of the ferroxidase activity showed, for all variants, a marked decline from pH 5.5 to pH 7.4. These results, and the localisation studies indicating a large presence of hephaestin inside cells, suggest that hephaestin may work optimally as a ferroxidase in intracellular compartments. If so, this model raises questions how transferrin can interact with hephaestin in vesicular compartments, and suggestions how this question can be addressed are discussed.
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