Comparative studies of the binding and growth‐supportive ability of mammalian transferrins in human cells

Penhallow, Robert C.; Brown-Mason, Anne; Woodworth, Robert C.

doi:10.1002/jcp.1041280217

Cited by 43 publications

(20 citation statements)

References 26 publications

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“…Substitution of His349 with a lysine is particularly interesting because (1) it allows assessment of the effect of a positive charge at position 349 at all pH values tested and (2) lysine is found in the homologous position in ovo-transferrin (oTF), a TF family member that does not bind to the human TFR [29]. We note that since a tryptophan residue equivalent to Trp641 in the human TFR and a phenylalanine residue equivalent to Phe760 are both conserved in the chicken TFR, differences in the oTF sequence may dictate the binding specificity.…”

Section: Discussionmentioning

confidence: 99%

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

et al. 2010

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His349 in human transferrin (hTF) is a residue critical to transferrin receptor (TFR)-stimulated iron release from the C-lobe. To evaluate the importance of His349 on the TFR interaction, it was replaced by alanine, aspartate, lysine, leucine, tryptophan, and tyrosine in a monoferric C-lobe hTF construct (FeChTF). Using a stop-ped-flow spectrofluorimeter, we determined rate processes assigned to iron release and conformational events (in the presence and in the absence of the TFR). Significantly, all mutant/TFR complexes feature dampened iron release rates. The critical contribution of His349 is most convincingly revealed by analysis of the kinetics as a function of pH (5.6–6.2). The FeChTF/TFR complex titrates with a pKa of approximately 5.9. By contrast, the H349A mutant/TFR complex releases iron at higher pH with a profile that is almost the inverse of that of the control complex. At the putative endosomal pH of 5.6 (in the presence of salt and chelator), iron is released from the H349W mutant/TFR and H349Y mutant/TFR complexes with a single rate constant similar to the iron release rate constant for the control; this suggests that these substitutions bypass the required pH-induced conformational change allowing the C-lobe to directly interact with the TFR to release iron. The H349K mutant proves that although the positive charge is crucial to complete iron release, the geometry at this position is also critical. The H349D mutant shows that a negative charge precludes complete iron release at pH 5.6 both in the presence and in the absence of the TFR. Thus, histidine uniquely drives the pH-induced conformational change in the C-lobe required for TFR interaction, which in turn promotes iron release.

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Section: Discussionmentioning

confidence: 99%

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

et al. 2010

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“…In general, the TF of a given species binds with highest affinity to the TFR of the same species, although some exceptions exist. As an extreme example bovine TF binds very poorly to the human TFR [45, 46]. Thus human cells cultured in fetal calf serum are starved for iron leading to the synthesis of more and more TF receptors; HeLa cells passaged in fetal calf serum typically have 1–2 million TFR/cell [45].…”

Section: Targeting the Htf/tfr For Chemotherapeutic Treatmentsmentioning

confidence: 99%

“…As an extreme example bovine TF binds very poorly to the human TFR [45, 46]. Thus human cells cultured in fetal calf serum are starved for iron leading to the synthesis of more and more TF receptors; HeLa cells passaged in fetal calf serum typically have 1–2 million TFR/cell [45]. Therefore, species compatibility must be considered when attempting to utilize the hTF/TFR system as a drug carrier.…”

Section: Targeting the Htf/tfr For Chemotherapeutic Treatmentsmentioning

confidence: 99%

Structure and dynamics of drug carriers and their interaction with cellular receptors: Focus on serum transferrin

Luck

Mason

2013

Advanced Drug Delivery Reviews

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Highly proliferative cells have a dramatically increased need for iron which results in the expression of an increased number of transferrin receptors (TFR). This insight makes the transferrin receptor on these cells an excellent candidate for targeted therapeutics. In this regard, it is critical to understand at a molecular level exactly how the TFR interacts with its ligand, hTF. Understanding of the hTF/TFR pathway could, in theory, maximize the use of this system for development of more effective small molecules or toxin-conjugates to specifically target cancer cells. Many strategies have been attempted with the objective of utilizing the hTF/TFR system to deliver drugs; these include conjugation of a toxin or drug to hTF or direct targeting of the TFR by antibodies. To date, in spite of all of the effort, there is a conspicuous absence of any successful candidate drugs reaching the clinic. We suggest that a lack of quantitative data to determine the basic biochemical properties of the drug carrier and the effects of drug-conjugation on the hTF-TFR interaction may have contributed to the failure to realize the full potential of this system. This review provides some guidelines for developing a more quantitative approach for evaluation of current and future hTF-drug conjugates.

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“…Tables 5 and 6 show residues in the N-and C-lobes of hTF that have been implicated in TFR binding by a variety of different approaches. Sequence alignments are shown for the regions of TF that bind human TFR with high affinity (human, rabbit, and pig TF), low affinity (bovine TF) (77,78), or not at all (oTF and human LTF) (77). Clearly residues that are critical to the interaction should be conserved in the rabbit and pig TFs and not in the other three sequences.…”

Section: Triad-lysmentioning

confidence: 99%

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

Wally

Halbrooks

Vonrhein

et al. 2006

Journal of Biological Chemistry

229

259

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Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-Å resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4°and 49.5°rotations are required to open the N-and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N-and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.The transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal in the body (1, 2). Human serum transferrin (hTF) 4 is synthesized in the liver and secreted into the plasma; it acquires Fe(III) from the gut and delivers it to iron requiring cells by binding to specific transferrin receptors (TFR) on their surface. The entire hTF⅐TFR complex is taken up by receptor-mediated endocytosis culminating in iron release within the endosome (3). Essential to the re-utilization of hTF, iron-free hTF (apo-hTF) remains bound to the TFR at low pH. When the apo-hTF⅐TFR complex is returned to the cell surface, apo-hTF is released to acquire more iron.Strong homologies exist, both between TF family members, and between the two lobes of any given TF (4, 5). Each N-and C-lobe is divided into two subdomains (designated N1 and N2, and C1 and C2) connected by a hinge that gives rise to a deep cleft containing the iron-binding ligands. Iron is coordinated by four highly conserved amino acid residues: an aspartic acid (the sole ligand from the N1-or C1-subdomain), a tyrosine in the hinge at the edge of the N2-or C2-subdomain, a second tyrosine within the N2-or C2-subdomain, and a histidine at the hinge bordering the N1-or C1-subdomain. In addition, the iron atom is bound by two oxygen atoms from the synergistic anion (carbonate), w...

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Comparative studies of the binding and growth‐supportive ability of mammalian transferrins in human cells

Cited by 43 publications

References 26 publications

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

Structure and dynamics of drug carriers and their interaction with cellular receptors: Focus on serum transferrin

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

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