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Elucidating the interaction between human serum transferrin and human transferrin receptor using electrospray ionization mass spectrometry (ESI-MS)
Abstract
The primary route most cells utilize to acquire iron is the human serum transferrin (hTf)-human transferrin receptor (TfR) mediated uptake pathway. This pathway has been exploited in the treatment of tumor cells; therefore it is vital to understand the interactions between hTf and TfR at the cell surface (pH 7.4) and in the endosome (pH 5.6). The present study investigates the role of iron in hTf binding to TfR to determine whether iron free (apo) hTf can bind TfR at pH 8.3 and understand the conformational changes (associated with iron release) in hTf-TfR at pH 5.6. Electrospray ionization mass spectrometry (ESI-MS) was used to detect the binding products of competitive, single and displacement assays between different forms of hTf (apo, monoferric, diferric) or lobe fragments and TfR at pH 8.3 and 5.6. All of the hTf-bound TfR in a specific but reversible manner at basic and acidic pH. At pH 8.3, diferric (holo)-hTf was the preferred ligand for TfR followed by monoferric-hTf and then apo-hTf. The iron-free lobe of monoferric-hTf was stabilized by the iron-bound lobe (interlobe communication) whereas the apo-hTf was stabilized by the glycan moieties on the C-terminal lobe (C-lobe). Binding assays performed with the lobe fragments suggest the C-lobe of hTf initially binds TfR, which induces allosteric conformational changes resulting in the binding of the N-lobe. However, the latter is incapable of binding TfR on its own. At pH 5.6, apo-hTf and FeNTf (iron in the N-lobe) were the preferred TfR ligands followed by Fe2Tf and FeCTf (iron in the C-lobe). Therefore, iron status of the C-lobe dictated the binding stability to TfR at pH 5.6. When the C-lobe contains iron it cannot completely open, as required for stable TfR binding at pH 5.6; iron occupancy of the N-lobe had no affect on the interaction due to the lack of binding to TfR when the C-lobe has an open conformation.
Subject Area
Analytical chemistry|Biochemistry
Recommended Citation
Leverence, Rachael C, "Elucidating the interaction between human serum transferrin and human transferrin receptor using electrospray ionization mass spectrometry (ESI-MS)" (2008). Doctoral Dissertations Available from Proquest. AAI3315494.
https://scholarworks.umass.edu/dissertations/AAI3315494