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1.  Iron and bismuth bound human serum transferrin reveals a partially-opened conformation in the N-lobe 
Scientific Reports  2012;2:999.
Human serum transferrin (hTF) binds Fe(III) tightly but reversibly, and delivers it to cells via a receptor-mediated endocytosis process. The metal-binding and release result in significant conformational changes of the protein. Here, we report the crystal structures of diferric-hTF (FeNFeC-hTF) and bismuth-bound hTF (BiNFeC-hTF) at 2.8 and 2.4 Å resolutions respectively. Notably, the N-lobes of both structures exhibit unique “partially-opened” conformations between those of the apo-hTF and holo-hTF. Fe(III) and Bi(III) in the N-lobe coordinate to, besides anions, only two (Tyr95 and Tyr188) and one (Tyr188) tyrosine residues, respectively, in contrast to four residues in the holo-hTF. The C-lobe of both structures are fully closed with iron coordinating to four residues and a carbonate. The structures of hTF observed here represent key conformers captured in the dynamic nature of the transferrin family proteins and provide a structural basis for understanding the mechanism of metal uptake and release in transferrin families.
PMCID: PMC3525939  PMID: 23256035
2.  Ionic Residues of Human Serum Transferrin Affect Binding to the Transferrin Receptor and Iron Release 
Biochemistry  2012;51(2):686-694.
Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤ 6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of eleven charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) in TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF/TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367 and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and iron release from the hTF/TFR complex.
PMCID: PMC3267578  PMID: 22191507
3.  Inequivalent Contribution of the Five Tryptophan Residues in the C-lobe of Human Serum Transferrin to the Fluorescence Increase when Iron is Released 
Biochemistry  2009;48(13):2858-2867.
Human serum transferrin (hTF), with two Fe3+ binding lobes transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant timescale (2-3 min), the factors critical to iron release include pH, anions, a chelator and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (± sTFR). Only four of the five recombinant Trp→ Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (FeC hTF) and the four Trp mutants in the FeC hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the FeC hTF control and the four Trp mutants in complex with the TFR: kobsC1 reports conformational change(s) in the C-lobe initiated by the TFR and kobsC2 is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex.
PMCID: PMC2664620  PMID: 19281173
4.  The Unique Kinetics of Iron Release from Transferrin: The Role of Receptor, Lobe-Lobe Interactions and Salt at Endosomal pH 
Journal of molecular biology  2009;396(1):130.
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. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe-lobe interactions and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or of diferric hTF (with iron locked in one lobe), provided distinct kinetic end points allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped flow spectrofluorimetry, in the presence and absence of the TFR, unequivocally establishes the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7- to 11-fold), and slows the rate of iron release from the N-lobe (6- to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe2hTF. Calculated cooperativity factors, in addition to plots of time dependent species distributions in the absence and presence of the TFR clearly illustrate the differences. Accurate rate constants for the pH and salt induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished.
PMCID: PMC2815179  PMID: 19917294
Stopped flow fluorescence; iron release kinetics; salt effect; iron release model
5.  Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of the pH induced changes in intrinsic fluorescence when iron is released 
Biochimica et biophysica acta  2008;1794(3):532-540.
Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome (~pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to FeC hTF alone, the increase in the fluorescent signal from the preformed complex of FeC hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from FeC hTF. Quantum yield determinations of TFR, FeC hTF and the FeC hTF/TFR complex strongly support our interpretation of the kinetic data.
PMCID: PMC2637931  PMID: 19103311
Metalloproteins; protein-receptor interaction; tryptophan analogues; tryptophan fluorescence; stopped-flow kinetics; BHK cells
6.  Identification of a Kinetically Significant Anion Binding (KISAB) Site in the N-Lobe of Human Serum Transferrin† 
Biochemistry  2010;49(19):4200-4207.
Human serum transferrin (hTF) binds two ferric iron ions which are delivered to cells in a transferrin receptor (TFR) mediated process. Critical to the delivery of iron to cells is the binding of hTF to the TFR and the efficient release of iron orchestrated by the interaction. Within the endosome, iron release from hTF is also aided by lower pH, the presence of anions, and a chelator yet to be identified. We have recently shown that three of the four residues comprising a loop in the N-lobe (Pro142, Lys144, and Pro145) are critical to the high-affinity interaction of hTF with the TFR. In contrast, Arg143 in this loop does not participate in the binding isotherm. In the current study, the kinetics of iron release from alanine mutants of each of these four residues (placed into both diferric and monoferric N-lobe backgrounds) have been determined ± the TFR. The R143A mutation greatly retards the rate of iron release from the N-lobe in the absence of the TFR but has considerably less of an effect in its presence. Our data definitively show that Arg143 serves as a kinetically significant anion binding (KISAB) site that is, by definition, sensitive to salt concentration and critical to the conformational change necessary to induce iron release from the N-lobe of hTF (in the absence of the TFR). This is the first identification of an authentic KISAB site in the N-lobe of hTF. The effect of the single R143A mutation on the kinetic profile of iron release provides a dramatic illustration of the dynamic nature of hTF.
PMCID: PMC3197725  PMID: 20397659
7.  Biochemical and Structural Characterization of Recombinant Human Serum Transferrin from Rice (Oryza sativa L.) 
The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications.
PMCID: PMC3483368  PMID: 23010327
human serum transferrin; Optiferrin™; recombinant transferrin; mass spectrometry; iron delivery; kinetics; transferrin receptor
8.  Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced iron release from human serum transferrin 
Biochemistry  2012;51(10):2113-2121.
The recent crystal structure of two monoferric human serum transferrin (FeNhTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details of this binding interaction which dictates iron delivery to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two FeNhTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca2+ binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of iron release at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of a H318A sTFR mutant allows assignment of a small pH dependent initial decrease in the fluorescent signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated iron release from the C-lobe of the Fe2hTF/sTFR Δ757–760 complex. The loss is accounted for by the inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs in order to promote receptor-stimulated iron release from the C-lobe of hTF.
PMCID: PMC3302921  PMID: 22356162
9.  Transferrin-Mediated Cellular Iron Delivery 
Essential to iron homeostasis is the transport of iron by the bilobal protein human serum transferrin (hTF). Each lobe (N- and C-lobe) of hTF forms a deep cleft which binds a single Fe3+. Iron-bearing hTF in the blood binds tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane protein. After undergoing endocytosis, acidification of the endosome initiates the release of Fe3+ from hTF in a TFR-mediated process. Iron-free hTF remains tightly bound to the TFR at acidic pH; following recycling back to the cell surface, it is released to sequester more iron. Efficient delivery of iron is critically dependent on hTF/TFR interactions. Therefore, identification of the pH-specific contacts between hTF and the TFR is crucial. Recombinant protein production has enabled deconvolution of this complex system. The studies reviewed herein support a model in which pH-induced interrelated events control receptor-stimulated iron release from each lobe of hTF.
PMCID: PMC4479283  PMID: 23046645
10.  Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin1 
Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated FeChTF. The new bridge sequence of this construct, designated FeCTEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe.
PMCID: PMC2860049  PMID: 20064616
Tobacco etch virus protease; transferrin; stopped-flow fluorescence; intrinsic tryptophan fluorescence; absorption coefficient determination; cooperativity
11.  Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH 
Biochimica et Biophysica Acta  2011;1820(3):326-333.
Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe3+ and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated.
Scope of Review
We provide an overview of the kinetics of iron release from hTF ± the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided.
General Significance
Delivery of Fe3+ by to actively dividing cells by hTF is essential; too much or too little Fe3+ directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential.
Major Conclusions
Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe3+ release within a physiologically relevant time frame.
PMCID: PMC3253137  PMID: 21699959
transferrin; transferrin receptor; kinetics; fluorescence
12.  A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping 
Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR.
PMCID: PMC4479294  PMID: 19693784
human serum transferrin; isothermal titration calorimetry; binding isotherms; transferrin receptor; epitope; recombinant protein; BHK cells; His-tag
13.  Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.) 
BMC Biotechnology  2012;12:92.
Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications.
Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production.
The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.
PMCID: PMC3521190  PMID: 23194296
Recombinant human serum transferrin; Transferrin receptor; Endocytosis; Cell growth and proliferation; Antibody production
14.  The protective role of transferrin in Müller glial cells after iron-induced toxicity 
Molecular Vision  2008;14:928-941.
Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection.
We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl3-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively.
mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice.
hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to control iron-induced stress.
PMCID: PMC2391081  PMID: 18509548
15.  Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B. 
Journal of Bacteriology  1997;179(20):6400-6407.
Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.
PMCID: PMC179556  PMID: 9335289
16.  Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor 
Iron release from human serum transferrin (hTF) has been studied extensively; however, the molecular details of the mechanism(s) remain incomplete. This is in part due to the complexity of this process, which is influenced by lobe–lobe interactions, the transferrin receptor (TFR), the salt effect, the presence of a chelator, and acidification within the endosome, resulting in iron release. The present work brings together many of the concepts and assertions derived from previous studies in a methodical, uniform, and visual manner. Examination of earlier work reveals some uncertainty due to sample and technical limitations. We have used a combination of steady-state fluorescence and urea gels to evaluate the effect of conformation, pH, time, and the soluble portion of the TFR (sTFR) on iron release from each lobe of hTF. The use of authentic recombinant monoferric and locked species removes any possibility of cross-contamination by acquisition of iron. Elimination of detergent by use of the sTFR provides a further technical advantage. We find that iron release from the N-lobe is very sensitive to the conformation of the C-lobe, but is insensitive to the presence of the sTFR or to changes in pH (between 5.6 and 6.4). Specifically, when the cleft of the C-lobe is locked, the urea gels indicate that only about half of the iron is completely removed from the cleft of the N-lobe. Iron release from the C-lobe is most affected by the presence of the sTFR and changes in pH, but is unaffected by the conformation of the N-lobe. A model for iron release from diferric hTF is provided to delineate our findings.
PMCID: PMC2733522  PMID: 19290554
Cooperativity; Urea gels; Steady-state tryptophan fluorescence; Transferrin/transferrin receptor complex; Iron-release model
17.  Human serum transferrin: Is there a link between autism, high oxalate and iron deficiency anemia? 
Biochemistry  2013;52(46):10.1021/bi401190m.
It has been previously suggested that high amounts of oxalate in plasma could play a role in autism by binding to the bilobal iron transport protein transferrin (hTF) thereby interfering with iron metabolism by inhibiting iron delivery to cells. By examining the effect of the substitution of oxalate for the physiologically utilized synergistic carbonate anion in each lobe of hTF we sought to provide a molecular basis for or against such a role. Our work clearly shows both qualitatively (6 M urea gels) and quantitatively (kinetic analysis by stop flow spectrofluorimetry) that the presence of oxalate in place of carbonate in each binding site of hTF does indeed greatly interfere with iron removal from each lobe (both in the absence and presence of the specific hTF receptor). However, we also clearly demonstrate that once the iron is bound within each lobe of hTF, neither anion can displace the other. Additionally, as verified by urea gels and electrospray mass spectrometry, formation of completely homogeneous hTF-anion complexes requires that all iron must first be removed and hTF then reloaded with iron in the presence of either carbonate or oxalate. Of significance, experiments described herein show that carbonate is the preferred binding partner, i.e., even if an equal amount of each anion is available during the iron loading process the hTF-carbonate complex is formed.
PMCID: PMC3887466  PMID: 24152109
18.  Localization of the meningococcal receptors for human transferrin. 
Infection and Immunity  1993;61(2):751-759.
The interaction between gold-labelled human transferrin (Au-HTF) with live meningococci after growth in vivo or in different in vitro conditions was examined by electron microscopy to localize and quantify the numbers of HTF-binding sites on the cell surface. It was clearly demonstrated that HTF binds to the surface of live meningococci (of different serogroups and serotypes) after growth in either iron-sufficient or iron-restricted cultures, although the degree of labelling was always higher (2- to 35-fold) in the latter case. The commensal Neisseria polysaccharea behaved similarly. Ultrathin sections showed that Au-HTF was localized predominantly on the outer membrane of the cells and vesicles, with hardly any internalization. Au-HTF labelling on meningococci was significantly reduced after incubation with unlabelled HTF or with rabbit antiserum containing antibodies against transferrin-binding proteins (TBPs), demonstrating the specificity of the interaction. These sera also blocked binding between HTF and outer membrane proteins on Western immunoblots. Direct evidence of the expression of the TBPs (Western blots) and localization of the HTF receptor (electron microscopy) on in vivo-grown meningococci was obtained from organisms derived without laboratory culturing from the cerebrospinal fluid of a patient. There was considerable cell-to-cell variation in the amount of labelling present on cells of the same sample (in vitro- or in vivo-grown organisms) and between different strains. The degree of binding varied with time of incubation of the cells with Au-HTF. The gold particles frequently formed discrete circles on the cell surfaces of the in vitro-grown organisms; these circles appear to be associated with outer membrane vesicle formation. The results show that the TBPs, which form part of the active components of the HTF receptor(s), are expressed in vivo and are surface exposed and immunogenic and that antibodies against them can interfere with the HTF binding of the meningococcal cells, which may affect iron utilization. This study further supports the concept of regarding the TBPs as future vaccine candidates.
PMCID: PMC302789  PMID: 8423101
19.  The structural basis of transferrin sequestration by transferrin-binding protein B 
Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin (Tf) during infection via a surface Tf receptor system composed of proteins TbpA and TbpB. Here in we present the crystal structures of TbpB from N. meningitidis, in its apo form and in complex with human Tf (hTf). The structure reveals how TbpB sequesters hTf and initiates iron release from hTf.
PMCID: PMC3981719  PMID: 22343719 CAMSID: cams4091
20.  Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin 
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.
PMCID: PMC4505821  PMID: 20711621
Protein–protein interactions; Human serum transferrin; Strategic mutagenesis; Stopped-flow fluorescence; Kinetics
21.  The Complex Role of Multivalency in Nanoparticles Targeting the Transferrin Receptor for Cancer Therapies 
Journal of the American Chemical Society  2010;132(32):11306-11313.
Transferrin receptor (TfR, CD71) has long been therapeutic target due to its over-expression on many malignant tissues. In this study, PRINT® nanoparticles were conjugated with TfR ligands for targeted drug delivery. Cylindrical poly(ethylene glycol)-based PRINT nanoparticles (diameter [d] = 200 nm, height [h] = 200 nm) labeled with transferrin receptor antibody (NP-OKT9) or human transferrin (NP-hTf), showed highly specific TfR-mediated uptake by all human tumor cell lines tested, relative to negative controls (IgG1 for OKT9 or bovine transferrin (bTf) for hTf). The targeting efficiency was dependent on particle concentration, ligand density, dosing time and cell surface receptor expression level. Interestingly, NP-OKT9 or NP-hTf showed little cytotoxicity on all solid tumor cell lines tested but were very toxic to Ramos B-cell lymphoma, whereas free OKT9 or hTf was not toxic. There was a strong correlation between TfR ligand density on particle surface and cell viability and particle uptake. NP-OKT9 and NP-hTf were internalized into acidic intracellular compartments but were not localized in EEA1 enriched early endosomes or lysosomes. Elevated caspase 3/7 activity indicates activation of apoptosis pathways upon particle treatment. Supplementation of iron suppressed the toxicity of NP-OKT9 but not NP-hTf, suggesting different mechanisms by which NP-hTf and NP-OKT9 exerts cytotoxicity on Ramos cells. Based on such an observation, the complex role of multivalency in nanoparticles is discussed. In addition, our data clearly reveal that one must be careful in making claims of “lack of toxicity” when a targeting molecule is used on nanoparticles and also raise concerns for unanticipated off-target effects when one is designing targeted chemotherapy nano-delivery agents.
PMCID: PMC2923393  PMID: 20698697
22.  Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice 
Molecular Vision  2010;16:2612-2625.
Retinal degeneration has been associated with iron accumulation in age-related macular degeneration (AMD), and in several rodent models that had one or several iron regulating protein impairments. We investigated the iron concentration and the protective role of human transferrin (hTf) in rd10 mice, a model of retinal degeneration.
The proton-induced X-ray emission (PIXE) method was used to quantify iron in rd10 mice 2, 3, and 4 weeks after birth. We generated mice with the β-phosphodiesterase mutation and hTf expression by crossbreeding rd10 mice with TghTf mice (rd10/hTf mice). The photoreceptor loss and apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling in 3-week-old rd10/hTf mice and compared with 3-week-old rd10 mice. The neuroprotective effect of hTf was analyzed in 5-day-old rd10 mice treated by intraperitoneal administration with hTf for up to 25 days. The retinal hTf concentrations and the thickness of the outer nuclear layer were quantified in all treated mice at 25 days postnatally.
PIXE analysis demonstrated an age-dependent iron accumulation in the photoreceptors of rd10 mice. The rd10/hTf mice had the rd10 mutation, expressed high levels of hTf, and showed a significant decrease in photoreceptor death. In addition, rd10 mice intraperitoneally treated with hTf resulted in the retinal presence of hTf and a dose-dependent reduction in photoreceptor degeneration.
Our results suggest that iron accumulation in the retinas of rd10 mutant mice is associated with photoreceptor degeneration. For the first time, the enhanced survival of cones and rods in the retina of this model has been demonstrated through overexpression or systemic administration of hTf. This study highlights the therapeutic potential of Tf to inhibit iron-induced photoreceptor cell death observed in degenerative diseases such as retinitis pigmentosa and age-related macular degeneration.
PMCID: PMC3002967  PMID: 21179240
23.  N-linked oligosaccharides of human transferrin are not required for binding to bacterial transferrin receptors. 
Infection and Immunity  1990;58(9):2972-2976.
Derivatives of human transferrin (hTf) with removed or modified N-linked oligosaccharides were compared with native hTf with respect to their binding to bacterial hTf receptors from Neisseria meningitidis, N. gonorrhoeae, and Haemophilus influenzae. Partially and fully deglycosylated hTf were prepared by enzymatic deglycosylation with glycopeptidase F and isolated by concanavalin A-Sepharose affinity chromatography. Oligosaccharide-modified hTf was prepared via mild periodate oxidation. Competition and direct binding experiments with the hTf derivatives demonstrated that the hTf oligosaccharides are not essential for binding to the bacterial hTf receptors.
PMCID: PMC313597  PMID: 2117577
24.  Structural insight into the lactoferrin receptors from pathogenic Neisseria 
Journal of structural biology  2013;184(1):83-92.
Neisseria are pathogenic bacteria that cause gonorrhea, septicemia, and meningitis. Like other pathogenic bacteria, Neisseria must acquire iron for survival from their local environment within the human host. Instead of secreting siderophores to scavenge iron, Neisseria steal iron from human iron binding proteins such as hemoglobin, transferrin and lactoferrin for survival. Recently we reported the crystal structures of the N. meningitidis transferrin receptors TbpA and TbpB, as well as the structures of apo and holo human transferrin. We also analyzed these proteins using small angle X-ray scattering and electron microscopy to provide the molecular details explaining how Neisseria are able to interact with and extract iron from transferrin. Here, we utilize the structural reports, as well as the recently reported structure of the N-lobe of LbpB from Moraxella bovis, to assemble improved 3D homology models for the neisserial lactoferrin import receptors LbpA and LbpB, both of which are important vaccine targets against N. meningitidis. We then analyzed these models to gain structural insights into the lactoferrin-iron import system and form a mechanistic model fashioned in parallel to the homologous transferrin-iron import system.
PMCID: PMC3716850  PMID: 23462098
Neisseria; meningitidis; gonorrhoeae; lactoferrin; transferrin; iron acquisition; TonB
25.  Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor 
PLoS Biology  2003;1(3):e51.
Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.
Differences in the contact points between the transferrin receptor and the two forms of transferrin (with or without iron) are consistent with pH-dependent conformational changes in transferrin, its receptor, or both
PMCID: PMC300677  PMID: 14691533

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