Calorimetric Studies of Melanotransferrin (p97) and Its Interaction with Iron

A Louise Creagh,Jacqueline W.C Tiong,Mei Mei Tian,Charles A Haynes,Wilfred A Jefferies

doi:10.1074/jbc.m414650200

Abstract

The mammalian molecule melanotransferrin (mTf), also called p97, is a member of the transferrin family of molecules. It exists in both secreted and glycosylphosphatidylinositol-anchored forms and is thought to play a role in angiogenesis and in transporting iron across the blood brain barrier. The binding affinity of iron to this molecule has not been formally established. Here, the binding of ferric ion (chelated with a 2-fold molar ratio of nitrilotriacetate) to mTf has been studied using isothermal titration calorimetry and differential scanning calorimetry. One iron-binding site was determined for mTf with similar binding characteristics to other transferrins. In the absence of bicarbonate, binding occurs quickly with an apparent association constant of 2.6 x 10(7) M(-1) at 25 degrees C. The presence of bicarbonate introduces kinetic effects that prevent direct determination of the apparent binding constant by isothermal titration calorimetry. Differential scanning calorimetry thermograms of mTf unfolding in the presence and absence of iron were therefore used to determine the apparent binding constant in the bicarbonate-containing system; at pH 7.5 and 25 degrees C, iron binding occurs in a 1:1 ratio with a K(app) of 4.4 x 10(17) M(-1). This affinity is intermediate between the high and low affinity lobes of transferrin and suggests that mTf is likely to play a significant role in iron transport where the high affinity lobe of transferrin is occupied or where transferrin is in proportionally low concentrations.

Highlights

Iron is an essential element for the vast majority of organisms on the planet [1, 2]
Isothermal Titration Calorimetry Studies of Bicarbonate-free System—In all isothermal titrations reported, an apo-mTf-containing sample cell was titrated with ferric ions present in solution in the form of an equilibrium set of Fex1⁄7NTAy1⁄7Hz complexes, where x, y, and z represent the stoichiometric coefficients for each complex present
Many factors can contribute to ⌬Cp,b, negative values observed for binding reactions in aqueous solvent environments are generally associated with the dehydration of apolar surfaces, such as hydrophobic amino acids [29]

Summary

The abbreviations used are

Tf/TfR, transferrin/transferrin receptor; MTf, melanotransferrin; GPI, glycosylphosphatidylinositol; ITC, isothermal titration calorimetry; DSC, differential scanning calorimeclathrin-mediated endocytosis pathway. Unlike the other members of the Tf family, mTf exists in two different isoforms as a result of alternate splicing of the mRNA [11] It can be found as a membrane protein attached to the cell surface via a glycosylphosphatidylinositol (GPI) anchor or as a soluble form in serum [12, 13]. Little is known about the thermodynamics of ferric ion binding to mTf. Spectroscopic studies of mTf [22], as well as iron uptake rates into mammalian cells [7], suggest that the protein binds only one ferric ion, in agreement with the putative binding stoichiometry originally proposed by Baker et al [16] based on sequence alignment with hTf and oTf. In this report, we examine the binding characteristics of Fe1⁄7NTA to soluble mTf using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) both in the presence and absence of bicarbonate. These experiments establish mTf as a mammalian iron binding molecule capable of delivering iron under physiological conditions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION