Iron is one of the essential elements for maintaining cell growth, reproduction, and metabolic activities. The vast majority of free iron in the body is transported and delivered by transferrin (Tf). Transferrin is the major β-globulin that binds and transports iron in the plasma, mainly responsible for carrying iron absorbed from the gastrointestinal tract and iron released from the degradation of red blood cells. Besides iron transport, transferrin also possesses many physiological and biochemical functions, such as antimicrobial activity, promotion of cell growth and differentiation, and cell protection, making it a multifunctional protein. Additionally, research has found that transferrin can serve as a novel drug-targeting carrier, with broad prospects for application in targeted therapy for tumors.
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Fig 1. Structure of diferric human serotransferrin (holo-hTf) showing domain organization and highlighting the two ferric ions (orange) (Silva A.M., et al. 2021).
The structure of Tf
Tf contains 679 amino acid residues with a molecular weight of approximately 79 kD. The amino acid sequence of Tf includes 38 cysteine residues, which can form 19 pairs of disulfide bonds, playing an important role in stabilizing the protein structure. Tf is composed of two structurally similar domains, including an N-terminal domain (336aa) and a C-terminal globular domain (343aa), connected by a short spacer sequence between the two domains. Each domain contains a series of α-helices and β-sheets, with hydrophilic metal ion binding sites formed through interactions between the domains. Within the domains, there are four conserved amino acids constituting Fe2+ binding sites, including two tyrosines, one aspartate, and one histidine, arranged in a tetrahedral manner. Additionally, the Fe2+ binding sites require two oxygen molecules provided by carbonate ions to maintain the stability of the iron atom. Near the Tf binding site, Gly-65, Glu-83, Tyr-85, Arg-124, Lys-206, Ser-248, and Lys-296 play crucial roles in the iron release process. Studies have shown that protonation effects of the Lys-206 and Lys-296 base pairs located on the opposite structural domain at the N-terminus can induce Tf to "open" or "close" its conformation. Currently, Tf is primarily categorized into three types: serum Tf, ovotransferrin, and lactotransferrin.
Polymorphism and Distribution of Tf
Tf protein is widely present in eukaryotes, with expression detected in at least 30 different species, showing significant polymorphism. Among them, Tf protein has three major subtypes: B, C, and D. Studies have shown a strong correlation between the polymorphism of Tf and susceptibility to certain diseases, including allergic iron proteinemia, cardiovascular diseases, and Alzheimer's disease.
In the human body, Tf is primarily synthesized by hepatocytes. Additionally, it is expressed in supporting cells, choroid plexus, oligodendrocytes, glioblastoma, metastatic melanoma cell lines, and human breast cancer cell lines. Tf can be detected in plasma, bile, amniotic fluid, cerebrospinal fluid, lymph, and breast milk.
Functions of Tf
Transport Iron
During DNA replication, iron serves as a cofactor for ribonucleotide reductase, directly participating in DNA synthesis and repair. Additionally, iron directly participates in the process of transporting O2 by hemoglobin. Free iron induces lipid peroxidation by converting hydrogen peroxide into reactive peroxides and alkoxides. Therefore, the primary role of Tf is to safely transport iron into cells.
Tf-Tf Receptor System
Iron-loaded Tf (holo-transferrin) binds to transferrin receptors (TfR) on the cell surface, forming a complex called holo-transferrin-transferrin receptor (holo-Tf-TfR), which is transported into endosomes via clathrin-mediated endocytosis. The decrease in pH within the endosome promotes the release of iron. Finally, the iron-free Tf-Tf receptor (apo-transferrin-transferrin receptor) complex is exocytosed to the cell surface via vesicles. Then Tf molecules circulate in the body, mediating the next cycle of iron transport. One Tf molecule can circulate up to 100 times.
Antimicrobial Activity of Apo-Tf
Recent studies indicate that an increase in free iron concentration in the body promotes pathogen growth, while chelating free iron with apo-Tf can reduce the incidence of bacterial infections. The antimicrobial activity of apo-Tf is not limited to simply reducing free iron levels; research has shown that apo-Tf can effectively inhibit the growth of both Gram-positive and Gram-negative bacteria.
Growth, Differentiation, and Cell Protection
Tf is extensively involved in the growth and differentiation of organism cells, including muscle cell growth, embryonic formation, cell proliferation, neurogenesis, chemotaxis, and angiogenesis. Furthermore, the paracrine effects of Tf have been discovered in cancer cell lines, for example, Tf produced by brain cells promotes the proliferation of brain melanoma metastases.
Holo-Tf has been shown to inhibit apoptosis in ovarian cancer cells. Holo-Tf mainly restores intracellular iron levels by upregulating Tf, thereby preventing cell death. Similarly, Tf has been reported to protect lymphocytes and hepatocytes, reducing Fas-mediated cell apoptosis.
Clinical Applications of Tf
Recent research on the Tf-TfR system has provided a basis for the clinical application of targeted drug delivery. Tf and TfR serve as new clinical indicators, offering new insights and approaches for the diagnosis and treatment of diseases such as blood disorders and tumors.
Tf Deficiency Syndrome
Tf deficiency syndrome is a rare autosomal recessive genetic disorder first reported in a 7-year-old girl in 1961. Patients experience anemia due to a lack of serum Tf. Clinical trials have shown that intravenous infusion of apo-Tf effectively alleviates symptoms. Additionally, apo-Tf can be used to treat juvenile transfusion-dependent anemia, reducing growth retardation and other symptoms associated with iron deficiency.
Cardiovascular Diseases
Low levels of Tf (<2 g/L) and protein glycation enhance iron-induced oxidative reactions, contributing to cardiovascular disease risk in diabetes patients. Tf is an important negative acute-phase protein in inflammatory diseases. Therefore, intravenous infusion of apo-Tf in diabetic patients reduces free iron levels, alleviating oxidative damage and reducing the risk of cardiovascular diseases.
Radiotherapy
Clinical data show that Tf levels decrease during radiotherapy, possibly due to increased oxidative stress reactions in the circulatory system. Intravenous injection of apo-Tf during radiotherapy reduces the release of free iron, thereby mitigating oxidative damage. Apo-Tf effectively lowers free iron levels and alleviates oxidative damage through mechanisms such as stimulating bone marrow cells and modifying nucleotides.
Targeted Drug Development with Tf
Studies on Tf-TfR system iron transport and uptake mechanisms have revealed the potential for targeted therapy using various therapeutic metal ions, drugs, proteins, and genes. TfR protein is highly expressed in tumors and lesion tissues, suggesting the potential for precise targeted therapy against tumor cells.
Tf transports not only iron ions but also reversibly binds to over 30 multivalent metal ions, including copper, zinc, and cobalt. Therefore, Tf can be used to transport other metals in the body, particularly gallium (Ga3+) and indium (In3+), for diagnostic imaging and cancer therapy.
Tf can also bind to drug molecules, proteins, or genes. For example, conjugating diphtheria toxin Tf-CRM107 and administering it intravenously to patients with malignant brain tumors has shown positive anti-tumor effects. Anti-cancer compounds used in combination with Tf include doxorubicin, chlorambucil, and paclitaxel.
Cancer Therapy
TfR is universally overexpressed in tumor tissues. Therefore, utilizing the Tf-TfR system to target tumor cells with anti-cancer drugs is a current focus and challenge in cancer research. Tf promotes cytotoxicity and proliferation of lymphocytes and natural killer cells by binding to other cytokines such as insulin-like growth factor 1 (IGF-1) and interleukin-2 (IL-2). Using Tf-containing cell growth medium RDSF stimulates the growth and proliferation of cytotoxic cells.
Artemisinin (ART) decomposes into toxic compounds in the presence of Fe2+. Pre-treatment of ART with Tf has been shown to kill small cell lung cancer cells at nanomolar concentrations. Similarly, combining ART compounds with Tf has strong cytotoxic effects on leukemia cells.
Conclusion
Tf, the primary β-globulin in plasma, binds and transports iron, mediating iron absorption through Tf receptors, maintaining iron and energy balance, and vitalizing vertebrate fluids and cells. Besides iron transport, Tf exhibits antimicrobial properties, supports cell growth and protection, rendering it multifunctional. Furthermore, it acts as a carrier for targeted drug delivery, showing potential in anti-tumor treatments like chemotherapy, biological drugs, and gene therapies. With advancements in protein engineering and nanobiomaterials, Tf holds promising prospects for tumor-targeted therapy.
References
- Silva A.M., et al. Human transferrin: An inorganic biochemistry perspective. Coordination Chemistry Reviews. 2021, 449: 214186.
- Gomme P.T., et al. Transferrin: structure, function and potential therapeutic actions. Drug Discovery Today. 2005, 10(4): 267-73.
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