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 such as 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. On the basis of 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.

Figure 1

Metal coordinating ligands in Fe (III) bound and Ti(IV) serotransferrin crystal structures, (a) Metal binding site in the C-lobe of serotransferrin (3QYT) showing the iron binding residues (yellow) and the synergistic anion carbonate (blue). (b) N-site structures showing other anions (SO₄²⁻ and NTA³⁻) maintaining metal bound sTf in a semi-open conformation (3QYT and 4H0W). (c) The C-site of Ti-bound serotransferrin. In contrast to A, in Ti-sTf in place of the His-585 and Asp-392 a bidentate synergistic citrate anion provides the necessary contacts to fulfill the coordination of Ti(IV). The Ti(IV) is in an open conformation.

Figure 2

Citrate and carbonate serve as synergistic anions for sTf binding of Ti(IV). The proton-decoupled ¹³C NMR spectra of 1 mM transferrin in the presence of two equivalents of Ti(IV) and ¹³C isotopically labeled bicarbonate (5 mM) and citrate (2 mM). The 165.2 and 165.5 ppm signals are due to bound carbonate at the metal binding site and the 187.5 ppm signal is due to the bound citrate.

Figure 3

STf binds metals in both open and closed conformations mediated by anions. (a) Overlapping ribbon diagram of the apo-sTf (PDB Code: 2-HAU, red) and Ti-sTf (blue) crystal structures. The two structures closely resemble in their conformation (RSMD = 1.217). (b) C (left) and N (right) binding sites of the Ti-sTf protein crystals. (c) CO₃²⁻ polar contacts as seen in the C-site of the sTf-Fe(III) bound form (3QYT).

Figure 4

Transferrin delivers Ti(IV) into cells and controls its cytotoxicity with the aid of citrate. (a) A549 cells exhibit an elevated Ti(IV) content when treated with Ti₂-sTf versus treatment with apo-sTf, Fe₂-sTf, and media alone (control) (N=5). (b) The viability of the cell lines A549 and MRC when treated with [TiOHBED]⁻ (0.1 mM) alone versus with [TiOHBED]⁻ and citrate (0.01–1 mM), apo-sTf (0.03 mM), or citrate (1 mM) and apo-sTf (0.03 mM) combined. The results demonstrate that the combination of apo-sTf and citrate attenuate the cytotoxicity of [TiOHBED]⁻. [Fe(Citrate)₂⁵⁻] = 0.1 mM. Student’s t-test, **, p-value <0.01,*, p-value <0.05.

Figure 5

The proposed mechanism of sTf mediated cellular delivery of Ti from Ti-implants and its LMW compounds as regulated by citrate. Citrate binds Ti(IV) released from implants or from LMW compounds that enter the bloodstream and then delivers the Ti(IV) to sTf. Ti(IV) bound sTf is recognized by the transferrin receptor (TfR), which transports the metal into the cell via endocytosis. Ti(IV) saturation of sTf may not be a requirement. A chelator (L, unknown) then removes Ti(IV) from the protein complex and the metal is released into the cytoplasm by a transporter (unknown).