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

Sebastien Farnaud, Maryam Amini, Chiara Rapisarda, Richard Cammack, Tam Bui, Alex Drake, Robert W. Evans, Yohan Suryo Rahmanto, Des R. Richardson

Research output: Contribution to journalArticle

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 6 M 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.

Original languageEnglish (US)
Pages (from-to)2739-2745
Number of pages7
JournalInternational Journal of Biochemistry and Cell Biology
Volume40
Issue number12
DOIs
StatePublished - Aug 5 2008
Externally publishedYes

Fingerprint

Transferrins
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Transferrin
Iron
Binding Sites
Circular Dichroism
Metals
Iron-Binding Proteins
Electrophoresis
Metal ions
Conformations
Urea
Blood Proteins
Chickens
Melanoma
Carrier Proteins
Proteins
Homeostasis
Gels

Keywords

  • Circular dichroism (CD)
  • Electron-paramagnetic resonance spectroscopy
  • Iron
  • Melanotransferrin
  • Transferrin

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

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. / Farnaud, Sebastien; Amini, Maryam; Rapisarda, Chiara; Cammack, Richard; Bui, Tam; Drake, Alex; Evans, Robert W.; Suryo Rahmanto, Yohan; Richardson, Des R.

In: International Journal of Biochemistry and Cell Biology, Vol. 40, No. 12, 05.08.2008, p. 2739-2745.

Research output: Contribution to journalArticle

Farnaud, Sebastien ; Amini, Maryam ; Rapisarda, Chiara ; Cammack, Richard ; Bui, Tam ; Drake, Alex ; Evans, Robert W. ; Suryo Rahmanto, Yohan ; Richardson, Des R. / 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. In: International Journal of Biochemistry and Cell Biology. 2008 ; Vol. 40, No. 12. pp. 2739-2745.
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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 6 M 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.",
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T2 - Electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

AU - Farnaud, Sebastien

AU - Amini, Maryam

AU - Rapisarda, Chiara

AU - Cammack, Richard

AU - Bui, Tam

AU - Drake, Alex

AU - Evans, Robert W.

AU - Suryo Rahmanto, Yohan

AU - Richardson, Des R.

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Y1 - 2008/8/5

N2 - 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 6 M 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.

AB - 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 6 M 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.

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