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The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co- receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process., Neisseria comprise a large family of Gram-negative bacteria that colonize humans. Two family members, Neisseria gonorrhoeae and Neisseria meningitidis, are pathogens that invade the urogenital tract and nasopharynx, respectively, causing [...]

The transferrins (TF) are a family of bilobal glycoproteins that tightly bind ferriciron, each of the homologous N- and C-lobes contains a single iron-binding site situated in a deep cleft. The importance of charge and geometry of the amino acids at the positions as a partial explanation for differences in behavior of individual TF family members, human serum transferrin, ovotransferrin and lactoferrin are provided.

An attempt is made to create mutant hTF molecules in which the N-lobe binds iron with high affinity or not all, in order to simplify the assessment of mutations in the C-lobe. The equilibrium binding experiments indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive.

Research has been conducted on the recombinant nonglycosylated human serum transferrins. The N-terminal His-tagged recombinant nonglycosylated human serum transferrins has been produced and the results indicate that the C-terminal His tag slows iron release rate from each lobe and that the N-terminal His tag cannot affect this release rate.

Research has been conducted on the proteins of the transferrin family. The role of these proteins in the iron homeostasis in vertebrates has been investigated and discussed.

Crystal structure analysis of the N-lobe half-molecule of human transferrin, which mediates iron transport, reveals a highly conserved iron binding site and hydrogen bonding interaction. Data point out that the binding and release of iron is influenced by the dilysine interaction.

Research has been conducted on the effect of histidine ligand of human transferin on iron binding. Site-directed mutagenesis, spectroscopy and structural analysis are examined.

Research has been conducted on the iron-binding properties of the N-lobe following ligand mutations. The changes resulting from mutating His 249 are discussed.

The function of the dilysine (Lys206-Lys296) pair in human transferrin N-lobe (hTF/2N) is investigated. Results show that the dilysine pair modulates the release of iron from the protein and is an anion-binding site for chelators. It is also observed that the release of iron is slower compared to the wild-type protein when both lysines are mutated to glutamate. Moreover, findings provide evidence to support the hypothesis that the apo-hTF/2N exists in equilibrium between closed and open conformations.

The expression of the ferric form of the N-lobe of human serum transferrin in the space group P4(sub 1)2(sub 1)2 in Pichia pastoris has been undertaken. X-ray crystallography and mass spectroscopy are then used in studying the crystal structure. Results indicate that the folding pattern is identical with that of serum-derived transferrin. Spectroscopic data show that glycosylation of Ser-32 occurs with a single hexose and is the first instance of the localization of an O-linked glycan in P. pastoris.

Transferrins are serum proteins responsible for the transport of iron in the body. The three-dimensional structure of these proteins were determined in the free and bound form to investigate the mechanics of iron release during transport. Results reveal that conformational changes that open up the channel through the formation of salt bridges involving two iron ligands before iron is released. Further, protonation of the histidine at position 249 has been found to be an important step in the release of iron.

The crystalline structures of the iron-bound, recombinant N-lobe of human serum transferrin has been studied at resolutions 1.6 and 1.8 angstroms. Transferrins are bilobal proteins that bind strongly with ferric iron at two similar binding sites. Both structures have folding patterns that resemble those of previously discovered transferrin and lactoferrin N-lobe structures. Its amino acid sequence has a polypeptide chain that has similar amino- and carboxyl-terminal halves.

The significance of tyrosine ligands in transferrin was investigated through using different techniques including high-resolution electroparamagnetic resonance spectra. Results revealed that inequivalence of the two tyrosine ligands in iron and copper binding with transferrins was opposite rendering the coordination geometry and charge of the metal ion critical for metal interaction with transferrin. Tyrosine 188 ligand mediated the normal binding of the protein with iron.

The effects of mutations of aspartic acid 63 on the metal-binding properties of the recombinant N-lobe of human serum transferrin were evaluated through utilization of recombinant DNA technology. Results have shown that aspartic acid position 63 is an important ligand for tight iron binding in transferrin. Furthermore, mutations from Asp to Ser, Glu, Asn, and Ala weakened the ferric iron binding capacity.

Metal-induced conformational changes in the blood serum protein transferrin are studied by (1H, 13C) nuclear magnetic resonance (NMR) spectroscopy. NMR resonance assignments for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin are shown based on investigations of recombinant N-lobe, nuclear Overhauser effect spectroscopy-relayed (1H, 13C) heteronuclear multiple-quantum coherence spectra and structural considerations.

The lysine residues at positions 296 and 206 in the N-lobe of recombinant human serum transferrin (hTF/2N) are necessary for the binding of divalent anions. A study conducted on the binding of synergistic anions to the wild-type and mutant protein using ultraviolet spectroscopy indicates that the binding is proportional to pH. The binding of anions to hTF/2N occurs with the help of a proton. The mutation has little effect on the binding of synergistic anions such as oxalate and malonate and no effect on the binding of nonsynergistic anions such as phosphate and sulfate.