Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor

Evelyn M. Teh, Jeff Hewitt, Karen C. Ung, Tanya A.M. Griffiths, Vinh Nguyen, Sara K. Briggs, Anne B. Mason, Ross T.A. MacGillivray

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Abstract

The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N- and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.

Original languageEnglish (US)
Pages (from-to)6344-6353
Number of pages10
JournalFEBS Journal
Volume272
Issue number24
DOIs
StatePublished - Dec 1 2005

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Keywords

  • Epitope mapping
  • Monoclonal antibody
  • Transferrin C-lobe
  • Transferrin-transferrin receptor interaction

Cite this

Teh, E. M., Hewitt, J., Ung, K. C., Griffiths, T. A. M., Nguyen, V., Briggs, S. K., Mason, A. B., & MacGillivray, R. T. A. (2005). Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor. FEBS Journal, 272(24), 6344-6353. https://doi.org/10.1111/j.1742-4658.2005.05028.x