title: Neither human hephaestin nor ceruloplasmin forms a stable complex with transferrin.
abstract: Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Cell toxicity is caused by the generation of hydroxyl-free radicals that result from redox reactions involving Fe(II). Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of these harmful by-products. Ceruloplasmin (Cp) is the major multicopper ferroxidase in blood; however, hephaestin (Hp), a membrane-bound Cp homolog, was recently discovered and has been implicated in the export of iron from duodenal enterocytes into blood. In the intracellular milieu, it is likely that iron exists as reduced Fe(II), yet transferrin (Tf), the plasma iron transporter, is only capable of binding oxidized Fe(III). Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between a ferroxidase and the iron transporter. As such, it has been suggested that as a means of preventing the release of unbound Fe(III), a direct protein-protein interaction may occur between Tf and Hp during intestinal iron export. In the present study, the putative interaction between Tf and both Cp and a soluble form of recombinant human Hp was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance (SPR), a stable interaction between the two proteins was not detected. We conclude that a stable complex between these ferroxidases and Tf does not occur under the experimental conditions used. We suggest alternative models for loading Tf with Fe(III) during intestinal iron export.
authors.affiliation: Centre for Blood Research and Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: David M
authors.initials: DM
authors.lastname: Hudson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Krisinger
authors.forename: Tanya A M
authors.initials: TA
authors.lastname: Griffiths
authors.forename: Ross T A
authors.initials: RT
authors.lastname: Macgillivray
mesh_terms: D002570:Ceruloplasmin / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D014168:Transferrin / Q000737:chemistry*
chemical_list: C492164:HEPH protein, human; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron; D002570:Ceruloplasmin