Protein sialylation affects the pH-dependent binding of ferric ion to human serum transferrin.

Physiological or pathophysiological changes lead to posttranslational changes in the sialic acid content of human serum transferrin (hTf), an essential mediator of iron transport in the human body, resulting in a significantly increased concentration of desialylated hTf. The intrinsic fluorescence quenching upon binding of iron to hTf was successfully modeled using the binding polynomial for two iron-binding sites, allowing measurements in a high-throughput format. Removal of sialic acid residues resulted in a 3-fold increase in iron binding affinity for both sites of hTf at pH 7.4. The pH-dependence of iron binding showed significant differences in equilibrium constants, resulting in a 10-fold increase in binding affinity for desialylated hTf at pH 5.9. The changes in hTf sialylation apparently result in tuning of the stability of the conformational state, which in turn contributes to the stability of the diferric hTf. The observed differences in the conditional thermodynamic equilibrium constants suggest that the desialylated protein has a higher preference for diferric hTf over monoferric hTf species down to pH 6.5, which may also influence the interaction with transferrin receptors that preferentially bind to diferric hTf. The results suggest a link between changes in hTf glycan structure and alterations in iron binding equilibrium associated with tissue acidosis.