Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity

Abstract

Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf’s role as a potential transporter of other metals like titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. Based on in vitro studies it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We find that sTf binds Ti(IV) in an open conformation with both carbonate and citrate as synergistic anions at the metal binding sites, an unprecedented role for citrate. Studies with cell lines suggest that Ti(IV)-sTf is transported into cells and that sTf and citrate regulate the metal’s blood speciation and attenuate its cytotoxic property. Our results provide the first glimpse into the citrate-transferrin synergism in the regulation of Ti(IV) bioactivity and offers insight into the future design of Ti(IV)-based anticancer drugs.

A ubiquitous metal, difficult to track: towards an understanding of the regulation of titanium(iv) in humans

“…Viability studies performed by our own lab have shown that even at 100 μM, this form of Ti(IV) shows no cytotoxicity. 33 Using these concentrations as a benchmark above which soluble Ti(IV) in serum would exhibit cytotoxicity, the amount of Ti(IV) released into the body even by implant sources should not pose a cytotoxic threat.…”

“…A recent x-ray crystal structure we obtained of Ti(IV)-bound sTf, the first ever Ti(IV) protein structure, demonstrated sTf binding of Ti(IV) in a manner that is quite distinct from that of Fe(III). 33 The Ti(IV) is coordinated to only the two tyrosine residues in the metal binding site. The remainder of the coordination sites are fulfilled by carbonate and, in a surprise, citrate (Fig.…”

“…In solution under physiologically relevant conditions, the Ti(IV) coordination remains identical as revealed by a 13 C NMR experiment using isotopically labelled carbonate and citrate to show that they are coordinated to the Ti(IV) at the metal binding site. 33 The citrate coordination results in a metal-bound open conformation for the protein because in citrate blocking the histidine and aspartate from binding the metal, the subdomain containing these residues does not move. A metal uptake study was performed with A549 human lung cancer cells, which have an overexpression of the transferrin receptor, using ICP-optical emission spectroscopy (ICP-OES) to measure the levels of intracellular Ti(IV) following treatment with Ti 2 -sTf.…”

“…67–71 Even at 100 μM concentration Ti 2 Tf does not exhibit any cytotoxic behavior against A549 and MRC (normal) human lung cell lines. 3, 33 …”

“…104, 105 These ligands mimic STf binding of Fe(III) in order to favor Fe(III) binding relative to Ti(IV). 33 The Ti(IV) cTfm complexes are expected to work by the two-prong method of intracellularly binding Fe(III) and simultaneously releasing Ti(IV) resulting in depletion of bioavailable Fe(III) and Ti(IV) delivery to its sites of attack. The FDA approved iron chelator deferasirox serves as an excellent cTfm ligand.…”