Evidence for the functional equivalence of the iron-binding sites of rat transferrin

The role of the two iron-binding sites of rat transferrin in the exchange of iron with cells has been assessed using urea polyacrylamide gel electrophoresis to separate and quantitate the four possible molecular species of transferrin generated during the incubation of ¹²⁵I-labelled transferrin with rat reticulocytes and hepatocytes. Addition of diferric transferrin to reticulocytes led directly to the appearance of apotransferrin together with small and comparable amounts of the two monoferric transferrins. After 2 h 44.8% of the iron had been removed by the cells, and of the iron-depleted transferrin 71.8% was apotransferrin, the remainder being monoferric transferrin, 16.1% with N-terminal iron and 12.1% with C-terminal iron. A similar pattern emerged with hepatocytes, but the rate of iron removal was slower and the proportion of apotransferrin generated was lower. After 4 h 10.9% of the iron had been removed from the transferrin and the distribution of the iron-depleted protein was: apotransferrin 26.9% and monoferric (N-terminal) 39.2%, (C-terminal) 33.9%. The appearance of apotransferrin during each incubation and the generation of both monoferric transferrins suggest that both cell types are able to remove iron from differic transferrin in pairwise fashion and that they do not appreciably distinguish between the two iron-binding sites of the protein. Release of iron from hepatocytes to apotransferrin lead to the appearance of both monferric species and then to increasing amounts of diferric transferrin. The process of iron release did not seem to distinguish between the vacant iron-binding sites of transferrin.     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

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.     

Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine

We have investigated the effect of increasing concentrations of methylamine (5, 10, and 25 mM) on the removal of iron from the two iron-binding sites of transferrin during endocytosis by human erythroleukemia (K562) cells. The molecular forms of transferrin released from the cells were analyzed by polyacrylamide gel electrophoresis in 6 M urea. Endocytosis of diferric transferrin was efficient since greater than 10% of surface-bound protein escaped endocytosis and was released in the diferric form. Although transferrin exocytosed from control cells had been depleted of 80% of its iron and contained 65-70% apotransferrin, iron-bearing species were also released (15% C-terminal monoferric; 10% N-terminal; 10% diferric). The ratio of the two monoferric species (C/N) was 1.32 +/- 0.12 (mean +/- SD; n = 4), suggesting that iron in the N-terminal site was more accessible to cells. In the presence of methylamine there was a concentration-dependent increase in the proportion of diferric transferrin release (less than 80% at 25 mM) and a concomitant decrease in apotransferrin. Small amounts of the iron-depleted species, especially apotransferrin, appeared before diferric transferrin, suggesting that these were preferentially released from the cells. The discrepancy between the proportions of the monoferric transferrin species noted with control cells was enhanced at all concentrations of methylamine, most markedly at 10 mM when the C/N ratio was 2.4. The N-terminal site of transferrin loses its iron at a higher pH than the C-terminal site, and so by progressively perturbing the pH of the endocytic vesicle we have increased the difference between the two sites observed with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites

Iron release to PPi from N- and C-terminal monoferric transferrins and their complexes with transferrin receptor has been studied at pH 7.4 and 5.6 in 0.05 M HEPES or MES/0.1 M NaCl/0.01 M CHAPS at 25 degrees C. The two sites exhibit kinetic heterogeneity in releasing iron. The N-terminal form is slightly less labile than its C-terminal counterpart at pH 7.4, but much more facile in releasing iron at pH 5.6. At pH 7.4, iron removal by 0.05 M pyrophosphate from each form of monoferric transferrin complexed to the receptor is considerably slower than from the corresponding free monoferric transferrin. However, at pH 5.6, complexation of transferrin to its receptor affects the two forms differently. The rate of iron release to 0.005 M pyrophosphate by the N-terminal species is substantially the same whether transferrin is free or bound to the receptor. In contrast, the C-terminal form releases iron much faster when complexed to the receptor than when free. Urea/PAGE analysis of iron removal from free and receptor-complexed diferric transferrin at pH 5.6 reveals that its C-terminal site is also more labile in the complex, but its N-terminal site is more labile in free diferric transferrin. Thus, the newly discovered role of transferrin receptor in modulating iron release from transferrin predominantly involves the C-terminal site. This observation helps explain the prevalence of circulating N-terminal monoferric transferrin in the human circulation.     

Functional heterogeneity and pH-dependent dissociation properties of human transferrin.

Human diferric transferrin was partially labeled with 59Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe3+, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2--5.8) was required to effect dissociation of iron that has remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-donating properties of human transferrin and identifies that the near neutral iron-binding site initially surrenders its iron to these cells.     

Characterization of monoferric fragments obtained by tryptic cleavage of bovine transferrin.

1. The electrophoretically fast (F) and slow (S) fragments obtained by tryptic cleavage of bovine iron-saturated transferrin differed in carbohydrate content and peptide 'maps'. 2. A fragment capable of binding one Fe3+ ion per molecule was isolated after brief tryptic digestion of bovine apotransferrin and shown closely to resemble the S fragment obtained from the iron-saturated protein. 3. Fragments F and S are probably derived from the N- and C-terminal halves of the transferrin molecule respectively. 4. Bovine transferrin could donate iron to rabbit reticulocytes, but the monoferric fragments possessed little iron-donating ability.     

Effects of spermine on transferrin and iron uptake by reticulocytes: II. Changes in intravesicular pH

An increase in extracellular spermine concentration brought about a progressive rise in intralysosomal pH in rabbit reticulocytes. Since intracellular release of iron from transferrin is believed to involve the protonation of the iron-transferrin complex, the rise in intralysomal pH could account for the inhibitory effect of spermine on iron uptake. The inhibition could be reversed if spermine was removed by washing. As a result of spermine treatment, more acid-labile N-terminal monoferric transferrin and less apotransferrin were released from the cell. These results are consistant with the protonation theory of iron release.     

Functional heterogeneity and pH-dependent dissociation properties of human transferrin

Human diferric transferrin was partially labeled with ⁵⁹Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte ⁵⁹ uptake experiments with chemically labeled preparations indicated that iron bound at near neutral ph was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe³⁺, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2–5.8) was required to effect dissociation of iron that had remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-binding properties of human transferrin and identifies that the near neutral iron-donating site initially surrenders its iron to these cells.     

The influence of pH on the equilibrium distribution of iron between the metal-binding sites of human transferrin.

The dependence of the metal-binding properties of transferrin on pH in the pH 6--9 range was investigated by urea/polyacrylamide-gel electrophoresis. Equations are presented for calculating the relative values of the four conditional site constants for the stepwise binding of iron to the two sites of transferrin and for calculating the equilibrium distribution of the protein among the four principal forms, apotransferrin, the C-terminal and N-terminal monoferric transferrins and diferric transferrin. The relative affinity of iron for the two sites and the co-operativity of iron-binding follow characteristic "pH titration' curves. A mathematical model that can account for the former behaviour is presented. In both cases the metal-binding sites are affected by the ionization of functional groups with apparent pKa values near physiological pH approx. 7.4. There is strong positive co-operatively in the release of protons from these groups. The results indicate that pH must be accurately controlled in studies of the differential properties of the two sites of the transferrin molecule.     

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.     

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     

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.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.     

Transferrin and iron uptake by rat reticulocytes

The uptake of transferrin labeled with 3H and 59Fe by rat reticulocytes was studied to clarify the characteristics of the uptake process and intracellular transport. Rat reticulocytes took up transferrin in a saturable, time- and temperature-dependent manner. Scatchard analysis of the binding parameters indicated that transferrin molecules were bound to cell-surface receptors with high affinity. Monodansyl- cadaverine, a potent inhibitor of transglutaminase, reduced the amount of internalized transferrin but has no effect on the total amount of cell-associated transferrin, suggesting that transferrin is taken up by rat reticulocytes via receptor-mediated endocytosis. About 50% of the internalized 3H label was released from the cells after reincubation for 1 h in fresh medium. In contrast, no release of 59Fe label was observed. By immunoprecipitation and subsequent SDS-PAGE the released 3H-labeled product was identified as apotransferrin. Lysosomotropic reagents and a proton ionophore reduced the uptake of 59Fe. These results indicated that iron was removed from transferrin at an intracellular site in an acidic environment. The released iron was found not to associate with any intermediate ligands before it was utilized for heme synthesis in mitochondria.     

Terephthalamide-containing ligands: fast removal of iron from transferrin

The mechanism and effectiveness of iron removal from transferrin by three series of new potential therapeutic iron sequestering agents have been analyzed with regard to the structures of the chelators. All compounds are hexadentate ligands composed of a systematically varied combination of methyl-3,2-hydroxypyridinone (Me-3,2-HOPO) and 2,3-dihydroxyterephthalamide (TAM) binding units linked to a polyamine scaffold through amide linkers; each series is based on a specific backbone: tris(2-aminoethyl)amine, spermidine, or 5-LIO(TAM), where 5-LIO is 2-(2-aminoethoxy)ethylamine. Rates of iron removal from transferrin were determined spectrophotometrically for the ten ligands, which all efficiently acquire ferric ion from diferric transferrin with a hyperbolic dependence on ligand concentration (saturation kinetics). The effect of the two iron-binding subunits Me-3,2-HOPO and TAM and of the scaffold structures on iron removal ability is discussed. At the low concentrations corresponding to therapeutic dose, TAM-containing ligands exhibit the fastest rates of iron removal, which correlates with their high affinity for ferric ion and suggests the insertion of such binding units into future therapeutic chelating agents. In addition, urea polyacrylamide gel electrophoresis was used to measure the individual microscopic rates of iron removal from the three iron-bound transferrin species (diferric transferrin, N-terminal monoferric transferrin, C-terminal monoferric transferrin) by the representative chelators 5-LIO(Me-3,2-HOPO)(2)(TAM) and 5-LIO(TAMmeg)(2)(TAM), where TAMmeg is 2,3-dihydroxy-1-(methoxyethylcarbamoyl)terephthalamide. Both ligands show preferential removal from the C-terminal site of the iron-binding protein. However, cooperative effects between the two binding sites differ with the chelator. Replacement of hydroxypyridinone moieties by terephthalamide groups renders the N-terminal site more accessible to the ligand and may represent an advantage for iron chelation therapy.     

Removal of iron from diferric rabbit serum transferrin by rabbit reticulocytes.

When radioiron-labelled transferrin with 55Fe located predominantly in the N-terminal iron-binding site and 59Fe predominantly in the C-terminal iron-binding site was incubated with rabbit reticulocytes, both radioisotopes of iron were removed at similar rates. Electrophoresis of transferrin samples taken during the course of an incubation, in polyacrylamide gels containing 6 M-urea, showed that iron was removed in a pairwise fashion, giving rise to iron-free transferrin.     

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.