Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe–lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies

Abstract

Recombinant non-glycosylated human serum transferrin and mutants in which the liganding aspartic acid (D) in one or both lobes was changed to a serine residue (S) were produced in a mammalian cell system and purified from the tissue culture media. Significant downfield shifts of 20, 30, and 45 nm in the absorption maxima were found for the D63S-hTF, D392S-hTF and the double mutant, D63S/D392S-hTF when compared to wild-type hTF. A monoclonal antibody to a sequential epitope in the C-lobe of hTF reported affinity differences between the apo- and iron-forms of each mutant and the control. Cell-binding studies performed under the same buffer conditions used for the antibody work clearly showed that the mutated lobe(s) had an open cleft. It is not clear whether the receptor itself may play a role in promoting the open conformation or whether the iron remains in the cleft.

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

“…The results of earlier cell binding assays suggested that iron removal from hTf results in a dramatic reduction of its receptor affinity (6)(7)(8)(9), while the results of more recent biophysical studies were consistent with a complete loss of the receptorbinding competence for the apo-hTf under conditions mimicking the extra cellular environment (11)(12)(13)(14). ESI MS provides conclusive evidence that aTf has the ability to form relatively stable complexes with TfR at neutral and mildly basic pH.…”

Epitope mapping of anti‐human transferrin monoclonal antibodies: potential uses for transferrin–transferrin receptor interaction studies

“…Both groups used the Mabs as tools to study the interaction between hTf and the hTf receptor (hTf-R). In other work, Mason et al (1998) (www.interscience.wiley.com) DOI:10.1002/jmr.878 describe the use of a Mab named E-8 as a complementary tool for the analysis of conformational changes in the hTf molecule following iron binding by the iron-free apotransferrin isoform (apo-hTf ). However, until now the characterization of the antigenic surface of the hTf molecule and their potential uses in the above-mentioned studies have not been fully addressed.…”

Anion-mediated Iron Release from Transferrins

“…Considerable interest has been focused to delineate the mechanism of iron release from transferrins in vitro, mainly by using small molecular weight chelators (9, 11-14, 16 -20), and, rarely, by using receptors (21)(22)(23). Because the iron release mechanism seems to be very complex with diferric transferrin, studies are now being undertaken using either independent lobes or selectively iron-loaded lobe on the full-length transferrin (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

“…Because the iron release mechanism seems to be very complex with diferric transferrin, studies are now being undertaken using either independent lobes or selectively iron-loaded lobe on the full-length transferrin (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The iron-binding site on each lobe of transferrin involves two tyrosine residues, one each of aspartic acid and histidine residues.…”

“…The remaining two ionic coordinates of iron are shared by a synergistic bicarbonate anion that anchors using an arginine residue of the protein. Hence, recently a number of iron release studies are focused using mutants of these functional sites of transferrin or its isolated lobes to delineate the mechanism (13,17,22,23,25). The concept of iron release mechanism proposed by Bates and co-workers (27,28) involving a "mixed ligand" intermediate between "domain closed" holo form and "domain opened" apo form is widely accepted, where the rate-limiting step is the conformational change because of anion binding followed by the rapid removal of bound iron.…”