Site-specific rate constants for iron acquisition from transferrin by the Aspergillus fumigatus siderophores N′,N′′,N′′′-triacetylfusarinine C and ferricrocin

Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N',N'-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system

Intact cells are known to reduce external, impermeable electron acceptors. We now show that cells can reduce the iron in diferric transferrin at the cell surface and that this reduction reaction depends on the transferrin receptor as well as the transmembrane electron transport system. Reduction of external diferric transferrin is accompanied by oxidation of internal NADH which indicates that the transmembrane enzyme is an NADH diferric transferrin reductase. Highly purified liver plasma membranes have NADH diferric transferrin reductase activity which shows properties similar to the diferric transferrin reductases activity of intact cells. Cell growth stimulation by diferric transferrin and other impermeable oxidants which can react with the diferric transferrin reductase can be based on electron transport through he plasma membrane.     

Iron uptake and metabolism by hepatocytes

The hepatocytes form part of the iron storage system of the body. In serving this function they exchange iron bidirectionally with the plasma iron transport protein transferrin (Tf). Iron uptake involves binding of the iron-Tf complex to cell membrane receptors and endocytosis into low-density vesicles, where the iron is released from its carrier protein before the Tf is returned undegraded to the extracellular medium. Two components of the iron uptake process can be distinguished, one saturable at low concentrations of diferric Tf and the other not saturable by increasing the Tf concentration. Both result in net uptake of iron by the cells and both appear to depend on specific binding to the cell membrane and endocytosis. Hepatocytes also obtain some iron from haptoglobin-hemoglobin, heme-hemopexin, and ferritin (Fn), in each case by interaction with membrane receptors and endocytosis. Within the cell iron from all sources enters one or more transit pools, where it is available for exchange with the iron storage protein Fn, and for release from the cell to plasma Tf or to iron chelators administered therapeutically or experimentally. Chelator-mediated iron release occurs to the plasma and/or to the bile, depending on the nature of the chelator and the source of the iron.     

Effects of alterations in cellular iron on biosynthesis of the transferrin receptor in K562 cells.

Treatment of K562 cells, a human erythroleukemia cell line, with desferrioxamine raised the levels of the receptor for transferrin (Tf) two- to threefold over that of the control cells. The levels of receptor were reduced by at least 50 and 35% of that of the control in cells treated with diferric Tf and ferric ammonium citrate, respectively. These changes were of total cellular receptors with no alteration in the proportion of receptors found on the cell surface. The half-lives of the receptor were identical in cells treated with desferrioxamine, diferric Tf, or ferric ammonium citrate. Cells metabolically labeled with [35S]methionine showed a 2.5-fold increase in the rate of receptor synthesis when treated with desferrioxamine and a 35 and 65% decrease when treated with ferric ammonium citrate and diferric Tf, respectively. In vitro translations of polyadenylated mRNA isolated from cells incubated with desferrioxamine showed a 2.5-fold increase in translatable mRNA for the receptor, whereas treatment of cells with ferric ammonium citrate and diferric Tf resulted in a 25 and 50% reduction, respectively, in translatable mRNA for this receptor.     

Manganese and iron transport across pulmonary epithelium

Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with (54)Mn or (59)Fe. Unlike (59)Fe, interactions between (54)Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled (54)Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of (54)Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca(2+) channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.     

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

Structure of the human transferrin receptor-transferrin complex

Iron, insoluble as free Fe(3+) and toxic as free Fe(2+), is distributed through the body as Fe(3+) bound to transferrin (Tf) for delivery to cells by endocytosis of its complex with transferrin receptor (TfR). Although much is understood of the transferrin endocytotic cycle, little has been uncovered of the molecular details underlying the formation of the receptor-transferrin complex. Using cryo-electron microscopy, we have produced a density map of the TfR-Tf complex at subnanometer resolution. An atomic model, obtained by fitting crystal structures of diferric Tf and the receptor ectodomain into the map, shows that the Tf N-lobe is sandwiched between the membrane and the TfR ectodomain and that the C-lobe abuts the receptor helical domain. When Tf binds receptor, its N-lobe moves by about 9 A with respect to its C-lobe. The structure of TfR-Tf complex helps account for known differences in the iron-release properties of free and receptor bound Tf.     

Diferric transferrin reduction by K562 cells. A critical study

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.     

Single particle reconstruction of the human apo-transferrin-transferrin receptor complex

Most organisms depend on iron as a co-factor for proteins catalyzing redox reactions. Iron is, however, a difficult element for cells to deal with, as it is insoluble in its ferric (Fe3+) form and potentially toxic in its ferrous (Fe2+) form. Thus, in vertebrates iron is transported through the circulation bound to transferrin (Tf) and delivered to cells through an endocytotic cycle involving the transferrin receptor (TfR). We have previously presented a model for the Tf-TfR complex in its iron-bearing form, the diferric transferrin (dTf)-TfR complex [Cheng, Y., Zak, O., Aisen, P., Harrison, S.C., Walz, T., 2004. Structure of the human transferrin receptor-transferrin complex. Cell 116, 565-576]. We have now calculated a single particle reconstruction for the complex in its iron-free form, the apo-transferrin (apoTf)-TfR complex. The same density map was obtained by aligning raw particle images or class averages of the vitrified apoTf-TfR complex to reference models derived from the structures of the dTf-TfR or apoTf-TfR complex. We were unable to improve the resolution of the apoTf-TfR density map beyond 16A, most likely because of significant structural variability of Tf in its iron-free state. The density map does, however, support the model for the apoTf-TfR we previously proposed based on the dTf-TfR complex structure, and it suggests that receptor-bound apoTf prefers to adopt an open conformation.     

The interaction of transferin with isolated hepatocytes

Freshly isolated rat heptocytes display about 36 700 high-affinity sites to which deferric transferrin may bind with an apparent association constant of 1.62·10⁷ 1·mol^?1.Uptake of iron from diferric transferrin by hepatocytes is linear with time and is accelerated at increased differric transferrin concentrations.Apotransferrin is able to decrease net iron uptake by hepatocytes from diferric transferrin by a process not dependent on the apotransferrin concentrations used or on the rate at which the cells take up iron. Immunoprecipitation of the apotransferrin during these incubations indicates that iron is being released from the cells to apotransferrin at the same time as iron is being taken up from diferric transferrin. The simultaneous uptake and release of iron, and the insensitivity to apotransferrin concentration, suggest that the processes of iron uptake and release occur via separate mechanisms. The effect of apotransferrin on net retention of iron may be one way in which the in vivo distribution of iron between sites of storage and utilization is controlled.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

Amonabactin-mediated iron acquisition from transferrin and lactoferrin by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> : direct measurement of individual microscopic rate constants

 The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe. The ability of A. hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments. The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis. The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored. Addition of transferrin or lactoferrin promoted bacterial growth. Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing. Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube. Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin. The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access. Taken together, these results support an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin. The implications of these findings for an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed. Received: 8 August 1999 / Accepted: 22 October 1999     

Diferric transferrin reductase in the plasma membrane is inhibited by adriamycin

Diferric transferrin which is often necessary for growth of cells is reduced by the transplasma membrane electron transport system of HeLa cells with release of ferrous iron outside the cell. Reduction of external diferric transferrin is reflected in oxidation of internal NADH. Adriamycin, an antitumor drug, inhibits diferric transferrin reduction by the HeLa cells and inhibits concomittant oxidation of cytosolic NADH at concentrations, 10(-8)-10(-6)M, which inhibit cell growth. Isolated liver plasma membranes have an NADH diferric transferrin reductase activity which is inhibited by similar adriamycin concentrations. We propose that inhibition of cell growth by adriamycin can be based on inhibition of transplasmalemma diferric transferrin reductase.     

The role of cellular iron deficiency in controlling iron export

BackgroundIron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover.MethodsWe ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency.Conclusions/General significanceWe show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.     

Iron release from transferrin induced by mixed ligand complexes of copper(II)

Summary Copper(II) complexes CuL₁L₂ with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25°C. Half-times of the reaction with Cu(PG)(en)^– were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS) ₂ ^4– and virtually none with Cu(serine)(en) nor Cu(PS)(2,2-bipyridyl)^–, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.Abbreviations PG phosphoglycerate - PS phosphoserine - en ethylenediamine - Fe2Tf diferric transferrin - Fe_cTf and Fe_NTf transferrin with iron bound to the lobe containing the C- or N-terminus, respectively - apoTf apotransferrin - K-3 all-cis-1,3,5-tris(trimethylammonio)-2,4,6-cyclo-hexanetriol - NTA nitrilotriacetic acid; bipy, 2,2-bipyridine; mal, malonate