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

Abstract

The primary route of iron acquisition in vertebrates is the transferrin receptor (TfR) mediated endocytotic pathway, which provides cellular entry to the metal transporter serum transferrin (Tf). Despite extensive research efforts, complete understanding of Tf-TfR interaction mechanism is still lacking owing to the complexity of this system. Electrospray ionization mass spectrometry (ESI MS) is used in this study to monitor the protein/receptor interaction and demonstrate the ability of metal-free Tf to associate with TfR at neutral pH. A set of Tf variants is used in a series of competition and displacement experiments to bracket TfR affinity of apo-Tf at neutral pH (0.2-0.6 microM). Consistent with current models of endosomal iron release from Tf, acidification of the protein solution results in a dramatic change of binding preferences, with apo-Tf becoming a preferred receptor binder. Contrary to the current models implying that the apo-Tf/TfR complex dissociates almost immediately upon exposure to the neutral environment at the cell surface, our data indicate that this complex remains intact. Iron-loaded Tf displaces apo-Tf from TfR, making it available for the next cycle of iron binding, transport and delivery to tissues. However, apo-Tf may still interfere with the cellular uptake of engineered Tf molecules whose TfR affinity is affected by various modifications (e.g., conjugation to cytotoxic molecules). This work also highlights the great potential of ESI MS as a tool capable of providing precise details of complex protein-receptor interactions under conditions that closely mimic the environment in which these encounters occur in physiological systems.

Fig. 1.

ESI mass spectra of 6 μM TfR solutions in 100 mM NH₄HCO₃ containing 10 μM Fe₂Tf (A), aTf (B), Fe_NTf (C), and HSA (D) at pH 8.1. Gray traces represent mass spectra of TfR in the absence of binding partners. Asterisks represent hTf dimers.

Fig. 2.

Competitive binding of aTf and Fe₂Tf to TfR at pH 8.1. The numbers on the right-hand side indicate Fe₂Tf/aTf/TfR molar ratios used in each measurement. The inset shows signals of unbound aTf and Fe₂Tf ions in the absence and presence of the receptor in solution.

Fig. 3.

TfR (6 μM) binding to Fe_CTf (10 μM) in the presence of 10 μM Fe₂Tf (A), Fe_NTf (B), and aTf (C).

Fig. 4.

Acid-induced reversal of TfR binding preferences: ESI MS of aTf, Fe₂Tf (10 μM each) and TfR (6 μM) at pH 8.1 (A) and the same mixture acidified to pH 5.6 (B). The blue and red traces in each spectrum represent ionic signals of (aTf)₂·TfR and (Fe₂Tf)₂·TfR recorded under the same conditions in the absence of a competing transferrin.

Fig. 5.

Nano-ESI MS monitoring of aTf displacement from the receptor by Fe₂Tf at pH 8.1. Concentration of aTf (24 μM) and TfR (13 μM) remained constant, while concentration of Fe₂Tf was increased incrementally as indicated by the numbers shown on the right-hand side of A. B shows full-range mass spectra prior to Fe₂Tf addition (gray) and following addition of Fe₂Tf raising its concentration in solution to 34 μM (black). Relative abundances of ionic species representing all three transferrin/receptor complexes, (aTf)₂·TfR, aTf·Fe₂Tf·TfR and (Fe₂Tf)₂TfR, as a function of the amount of Fe₂Tf added to the solution are shown in C. Relative abundance for each species was calculated by fitting the raw data (A) with Gaussian curves and summing up their heights for all observed charge states.

Fig. 6.

A: nano-ESI MS analysis of complexes generated by incubating bTf and oTf in 13.5 µM TfR at pH 8.1 (total transferrin concentration 22 μM, oTf∶bTf ratio ca. 2.5∶1). B: same mixture following addition of 22 μM aTf. Peaks corresponding to 1∶1 complexes are labeled with single triangles, and 2∶1 complexes are labeled with double triangles (color indicates the bound transferrin(s): bTf, black; oTf, gray; aTf, white).