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.

The binding of titanium(IV) to human serum transferrin in 50 mM Tris with 20 mM bicarbonate and 10 mM citrate at pH 7.4 was studied by UV/vis kinetics and by isothermal titration calorimetry. Ti(IV) citrate, [Ti(C6H4O7)3]8-, employed in this study was previously characterized and delivers the metal to transferrin rapidly, allowing the quantification of the intrinsic binding constants for Ti(IV) to the C- and N-sites of transferrin. The results after correcting for blood plasma conditions (pH 7.4, [HCO3-] = 27 mM) reveal that Ti(IV) binds with greater affinity (log K = 26.8 and 25.7) than Fe(III) (log K = 22.5 and 21.4) to transferrin, a finding not previously observed for other examined metal ions. The strength of metal binding to transferrin correlates with the Lewis acidity of the metal. Ti(IV) is more Lewis acidic than Fe(III) and is nearly the same size. The study also reveals that Ti(IV) binds more tightly to one site than the other, and this difference is due to both entropic and enthalpic contributions. The study has implications for the role of transferrin in the anticancer activity of Ti(IV) drugs and the serum binding of Ti(IV) ions released from implants or imaging reagents.

The trafficking of titanium(IV) by human serum transferrin (HsTf) has been implicated in the physiology of this hydrolysis-prone metal. The current work broadens to include the further interactions of Ti(IV) in serum that bear on this model. Ti2HsTf (2 equiv) binds the transferrin receptor TfR1 with Kd1 = 6.3 +/- 0.4 nM and Kd2 = 410 +/- 150 nM, values that are the tightest yet measured for a metal other than iron but weaker than the corresponding ones for Fe2HsTf due to both slightly slower on rates and slightly faster off rates. Comparing the affinities of metals for HsTf with the affinities of the resulting M2HsTf species for TfR1, we speculate that the formation of an M2HsTf complex of high affinity may predict a lobe-closed conformation that leads to a favorable interaction with TfR1. Human serum albumin (HSA), an important serum competitor for metal binding, can bind up to 20 equiv of Ti(IV) supplied in several forms. With some ligands, Ti(IV) may bind to the N-terminal metal binding site of albumin, forming a ternary complex. However, the dominant type of HSA binding is via Ti(IV) in complex form, probably at surface sites. Notably, HSA greatly stabilizes the titanocene moiety of the drug candidate Cp2TiCl2 with respect to hydrolysis and precipitation. HSA binds Ti(IV) citrate supplied as a hydrolyzed or unhydrolyzed source, with 1 equiv of citrate remaining bound. Titanium(IV) monocitrate neither competes with the binding of reporter molecules known to dock at canonical drug sites I or II nor binds at the N-terminus. HsTf outcompetes HSA for soluble Ti(IV) in a direct competition, but once bound to albumin, the transfer of Ti(IV) from HSA to HsTf is quite slow. Each of these findings has implications for the metabolism of Ti(IV) in human serum.

In recent years, the biological sciences have seen a surge in the development of methods, including high-throughput global methods, for the quantitative measurement of biomolecule levels (i.e., RNA, proteins, metabolites) from cells and tissues. Just as important as quantitation of biomolecules has been the creation of approaches that uncover the regulatory and signaling connections between biomolecules. Our specific interest is in understanding peptide metabolism in a physiological setting, and this has led us to develop a multidisciplinary approach that integrates genetics, analytical chemistry, synthetic chemistry, biochemistry, and chemical biology to identify the substrates of peptidases in vivo. To accomplish this we utilize a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics platform to measure changes in the peptidome-all peptides in a cell, tissue, or organism-as a function of peptidase activity. Previous analysis of mice lacking the enzyme dipeptidyl peptidase 4 (DPP4 −/− mice), a biomedically relevant peptidase, using this approach identified a handful of novel endogenous DPP4 substrates. Here, we utilize these substrates and tissues from DPP4 −/− mice to improve the coverage of the peptidomics platform by optimizing the key steps in the workflow, and in doing so, discover a total of 70 renal DPP4 substrates (up from 7 at the beginning of our optimization), a 10-fold improvement in our coverage. The sequences of these DPP4 peptide substrates support a broad role for DPP4 in proline-containing peptide catabolism and strengthen a biochemical model that interlinks aminopeptidase and DPP4 activities. Moreover, the improved peptidome coverage also led to the detection of greater numbers of known bioactive peptides (e.g., peptide hormones) during the analysis of gut samples suggesting additional uses for this optimized workflow. Together these results strengthen our ability to identify endogenous peptide substrates through improved peptidome coverage and demonstrate a broader potential of this peptidomics platform.

Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) at different pH values models the two forms of Ti(IV)-loaded transferrin spectrally and structurally. 13C NMR and stopped-flow kinetic experiments reveal that when the metal is delivered to the protein using an unhydrolyzed source, Ti(IV) can coordinate in the typical distorted octahedral environment with a bound synergistic anion. The crystal structure of TiHBED obtained at low pH models this type of coordination. The solution structure of the complex compares favorably with the solid state from pH 3.0 to 4.0, and the complex can be reduced with E1/2 = -641 mV vs NHE. Kinetic and thermodynamic competition studies at pH 3.0 reveal that Ti(citrate)3 reacts with HBED via a dissociative mechanism and that the stability of TiHBED (log beta = 34.024) is weaker than that of the Fe(III) complex. pH stability studies show that Ti(IV) hydrolyzes ligand waters at higher pH but still remains bound to HBED until pH 9.5. Similarly, at a pH greater than 8.0 the synergistic anion that binds Ti(IV) in transferrin is readily displaced by irreversible metal hydrolysis although the metal remains bound to the protein until pH 9.5. Thermal denaturation studies conducted optically and by differential scanning calorimetry reveal that Ti(IV)-bound transferrin experiences only minimal enhanced thermal stability unlike when Fe(III) is bound. The C- and N-lobe transition Tm values shift to a few degrees higher. The stability, competition, and redox studies performed provide insight into the possible mechanism of Ti2-Tf transport in cells.