GlyMech

Introduction and Background

Human serum transferrin (hTf) is the principal iron-transport protein in blood plasma. It is a bilobal glycoprotein capable of reversibly binding two ferric ions (Fe³⁺) in cooperation with a synergistic anion, typically carbonate, forming stable ternary complexes. A key structural feature of hTf is its extensive N-glycosylation, with two canonical glycosylation sites at Asn413 and Asn611, both located in the C-lobe of the protein. The glycan structures are typically capped with sialic acid residues, which confer negative charge and influence the physicochemical and biological properties of the protein.


The central objective of this research series is to elucidate how the terminal glycosylation pattern—particularly the presence or absence of sialic acids—modulates the thermodynamics and efficiency of iron binding in hTf. This is investigated through a combination of glycoproteomics, isothermal titration calorimetry (ITC), UV/Vis spectroscopy, and chromatographic techniques.

Method Development and Optimization

Initial work focused on developing reliable methods to produce and characterize desialylated transferrin (Tf–s) from its native sialylated form (Tf+s). A preparative protocol based on enzymatic desialylation using either GlycoCleave® or SialEXO® immobilized sialidases was optimized to increase yield, reproducibility, and cost-efficiency (Friganović et al., Maced. Pharm. Bull., 2022). The optimized procedures allowed for large-scale generation of desialylated hTf with >90% removal of sialic acids, significantly improving upon commercial protocols. Chromatographic separation using low-pressure pH-gradient anion exchange columns was then used to isolate and purify the resulting sialoforms, as described in a previous study (Friganović et al., Heliyon, 2021). Detailed N-glycan analysis via UPLC-MS validated the completeness of desialylation and confirmed the structural integrity of the protein.

In parallel, a separate technical study addressed the preparation and rigorous standardization of iron(III)-nitrilotriacetate (FeNTA), the iron donor used in binding studies. The FeNTA complex serves as a model iron source due to its well-defined stoichiometry and ability to fully saturate hTf in a controlled manner. The paper provided molar absorptivity data across relevant pH and ionic strength conditions, allowing precise quantification of FeNTA solutions for use in ITC and other quantitative assays (Borko et al., Anal. Methods, 2023). The concentrations of all species involved in the equilibria between Fe and NTA were determined in the pH range 2-12 using the Jenkins–Traub algorithm to solve the 5th-order polynomial in Microsoft Excel.

Thermodynamic Analysis of Iron Binding

The thermodynamics of iron binding to native and desialylated hTf was systematically studied by isothermal titration calorimetry (Borko et al., J. Inorg. Biochem., 2023). Measurements were conducted under physiological pH (7.4) in the presence of two different synergistic anions: carbonate (the physiological cofactor) and oxalate (a potent competitor under pathological conditions such as hyperoxalemia).
The data revealed that desialylated hTf binds Fe³⁺ more tightly than its native counterpart, with both enthalpic and entropic contributions varying between the two lobes. Specifically, binding to the C-lobe was enthalpy-driven, while N-lobe binding was predominantly entropy-driven. Desialylation significantly increased the exothermicity of the reactions, especially in the presence of carbonate, and also influenced the heat flow kinetics differently for the two lobes. These findings suggest that terminal sialic acids modulate the conformational dynamics and ligand coordination environment in a site-specific manner.
A follow-up study extended this work to lower pH values (pH 5.9), mimicking endosomal conditions where iron release typically occurs (Friganović et al., Dalton Trans., 2024). At this pH, desialylated hTf showed up to a 10-fold increase in binding affinity compared to native hTf, particularly at the N-lobe, which plays a dominant role in iron sequestration in vivo. These results imply that desialylated hTf may be less efficient at releasing iron in acidic environments, potentially altering iron homeostasis under pathological conditions.

Conclusions and Relevance

Collectively, these studies demonstrate that sialylation significantly influences the iron-binding thermodynamics of human serum transferrin. Removal of sialic acids enhances iron affinity, alters site-specific thermodynamic signatures, and potentially affects downstream processes such as receptor recognition and iron release. The findings suggest that glycosylation microheterogeneity - particularly the degree of sialylation - is not a passive structural feature, but a dynamic regulatory element in transferrin function.
From a biomedical perspective, these insights are highly relevant for understanding the pathophysiology of iron overload disorders, inflammatory conditions, and diseases involving altered glycosylation such as cancer, sepsis, and chronic alcohol use. They also support the emerging role of transferrin in ferroptosis and iron-driven oxidative stress. Furthermore, the robust methods developed for transferrin sialoform preparation, purification, and characterization lay the groundwork for future mechanistic and clinical studies into glycoform-specific iron transport pathways.