UBC Theses and Dissertations
Recombinant expression and functional characterization of human hephaestin : a multicopper oxidase with ferroxidase activity Griffiths, Tanya A. M.
Human hephaestin is a transmembrane protein that has been implicated in duodenal iron export. Mutations in the murine hephaestin gene produce microcytic, hypochromic anemia that is refractory to oral iron therapy. Hephaestin shares -50% sequence identity with the plasma multicopper ferroxidase ceruloplasmin including conservation of residues involved in disulfide bond formation and metal coordination. Based on this similarity to ceruloplasmin, human hephaestin may also bind copper and possess ferroxidase activity. To test this hypothesis, human hephaestin cDNA has been cloned by reverse transcription of human duodenal mRNA. Following in vitro mutagenesis to engineer the encoded polypeptide so that it was suitable for expression and purification, the cDNA was cloned into the expression vector pNUT and introduced into baby hamster kidney cells. After selection with methotrexate, the baby hamster kidney cells secreted the recombinant human hephaestin into the medium at a level of -2 mg/L. Purification was achieved by a single immunoaffinity chromatography step. As judged by SDS-PAGE, N-terminal sequence analysis, and matrix assisted laser desorption ionization time-of-flight mass spectrometry, the purified hephaestin was homogeneous with a mass of 129,600 Da suggesting a carbohydrate content of -7.7%. Inductively coupled plasma mass spectrometry revealed that recombinant hephaestin contained an average of 3.13 atoms of copper per protein molecule. A visible absorption maximum was observed at 607 nm that is consistent with the presence of a Type 1 copper site. By using ferrous ammonium sulfate as a substrate, recombinant hephaestin was shown to have ferroxidase activity with a Km of 2.1 fiM for Fe(II). Lastly, urea PAGE showed that hephaestin was able to oxidize Fe(II) to Fe(III) that was acceptable by apo transferrin to form diferric transferrin.
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