title: Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins.
abstract: Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.
authors.affiliation: School of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Farnaud
authors.forename: Maryam
authors.initials: M
authors.lastname: Amini
authors.forename: Chiara
authors.initials: C
authors.lastname: Rapisarda
authors.forename: Richard
authors.initials: R
authors.lastname: Cammack
authors.forename: Tam
authors.initials: T
authors.lastname: Bui
authors.forename: Alex
authors.initials: A
authors.lastname: Drake
authors.forename: Robert W
authors.initials: RW
authors.lastname: Evans
authors.forename: Yohan
authors.initials: Y
authors.lastname: Suryo Rahmanto
authors.forename: Des R
authors.initials: DR
authors.lastname: Richardson
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008545:Melanoma / Q000276:immunology*; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011990:Receptors, Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron