Utilization of transferrin-bound iron by Listeria monocytogenes

Abstract It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes . This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.     

Transferrin protein and iron uptake by cultured hepatocytes

The binding and uptake of ⁵⁹Fe-loaded ³H-labelled rat transferrin by cultured rat hepatocytes was investigated. At 4°C, there is no evidence for a specific binding of transferrin which could be related to the association of neo-synthesized transferrin with plasma membrane receptors. At 37°C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.     

Utilization of transferrin-bound iron by Haemophilus species of human and porcine origins

Haemophilus influenzae and H. haemolyticus acquired iron bound to human transferrin but not to human lactoferrin, ovo- or porcine transferrins. Conversely the swine pathogens H. pleuropneumoniae and H. parasuis used iron bound only to porcine transferrin. Growth under conditions of iron deprivation induced the production of siderophores and iron-repressible outer membrane proteins in H. parainfluenzae, H. paraphrophilus and H. parasuis but not in H. influenzae, H. haemolyticus or H. pleuropneumoniae. The latter 3 Haemophilus species appear to sequester transferrin bound iron via a siderophore-independent mechanism. However, the ability to produce iron chelating compounds did not enable H. parainfluenzae or H. paraphrophilus to utilize transferrin bound iron.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.     

Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth

The ability of Staphylococcus epidermidis strains to grow in the presence of human transferrin and varying amounts of ferric iron was studied. At initial bacterial densities up to 10(4) cfu ml(-1), none of the three strains grew when transferrin iron saturation was below the full saturation point, whereas the bacteria grew consistently when transferrin was fully iron-saturated and there was non-transferrin-bound iron in the medium. Precultivation of the bacteria under iron-restricted conditions to induce siderophore production did not abolish the growth dependence on non-transferrin-bound iron. At initial bacterial densities of 10(6) cfu ml(-1), the bacteria proliferated consistently also in the presence of partially saturated transferrin. The results indicate that at low bacterial densities, S. epidermidis cannot utilise transferrin-bound iron for growth and that its proliferation is dependent on non-transferrin-bound iron.     

The rate limiting step in the reticulocyte uptake of transferin and transferrin iron Effects of some incubation variables

Reticulocytes incubated in an isotonic NaCl saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticuloctes failed to remove completely the iron from the transferrin which they incorporated.It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unspecific attachment of transferrin.At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.     

Chelator-mediated iron efflux from reticulocytes

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.     

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.     

Large cooperativity in the removal of iron from transferrin at physiological temperature and chloride ion concentration

Iron removal from serum transferrin by various chelators has been studied by gel electrophoresis, which allows direct quantitation of all four forms of transferrin (diferric, C-monoferric, N-monoferric, and apotransferrin). Large cooperativity between the two lobes of serum transferrin is found for iron removal by several different chelators near physiological conditions (pH 7.4, 37 °C, 150 mM NaCl, 20 mM NaHCO₃). This cooperativity is manifested in a dramatic decrease in the rate of iron removal from the N-monoferric transferrin as compared with iron removal from the other forms of ferric transferrin. Cooperativity is diminished as the pH is decreased; it is also very sensitive to changes in chloride ion concentration, with a maximum cooperativity at 150 mM NaCl. A mechanism is proposed that requires closure of the C-lobe before iron removal from the N-lobe can be effected; the open conformation of the C-lobe blocks a kinetically significant anion-binding site of the N-lobe, preventing its opening. Physiological implications of this cooperativity are discussed.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

Critical examination for the presence of a low molecular weight fraction in serum iron

Native human pool serum and individual sera were ultrafiltered by Pellicon ultrafilters (Millipore) and the ultrafiltrates were extracted by an ammonium pyrrolidinedithicarbamate/methylisobutylketone system after addition of different internal iron standards to three of four identical ultrafiltrates. The extracts were examined for iron content by atomic absorption spectrometry. During ultrafiltration pH 7.4 was miantained by a constant atmosphere of a CO₂/air mixture.Low molecular weigth iron in native human sera from normal, normal orally iron substituted and siderotic individuals was found to be less than 0.05 μg/100 ml. Elevating serum citrate to 3-fold normal had no effect on this result.More iron became ultrafiltrable if the serum pH were lowered by citric acid as compared with hydrochloric acid.     

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     

Role of transferrin in iron uptake by the brain: a comparative study

Summary The role of specific transferrin (Tf) and Tf receptor interaction on brain capillary endothelial cells in iron transport from the plasma to the brain was investigated by using Tf from several species of animals labeled with ⁵⁹Fe and ¹²⁵I, and 15-day and adult rats. The rate of iron transfer was much greater in the 15-day rats. It was greatest with Tf from the mammals, rat, rabbit and human, but much lower with chicken ovotransferrin and quokka (a marsupial), toad, lizard, crocodile, and fish Tf. The uptake of Tf by the brain showed a similar pattern, except for a very high uptake of ovotransferrin (ovo Tf). Iron uptake by the femurs (a source of bone marrow) was also high with Tf from the mammalian species and low with the other types of Tf, but showed little change with aging of the animals. It is concluded that iron transport into the brain is dependent on the function of Tf receptors, probably on capillary endothelial cells, and that these receptors show the same type of species specificity as the receptors on immature erythroid cells. Also, the decrease in iron uptake by the brain as rats age from 15 days to adulthood is specific for the brain and is not a general effect of the aging process.Abbreviations Tf transferrin - ovo Tf ovotransferrin     

Long-range charge transport through double-stranded DNA mediated by manganese or iron porphyrins

Guanine oxidation by electron transfer results in the formation of a guanine radical cation, which is at the origin of long-range charge transport through double-stranded DNA. It is possible to observe guanine lesions at a long distance from the oxidative reagent covalently bound to DNA owing to the migration of the positive hole in the DNA pi-stacks. This phenomenon of long-range hole transport is classically studied in the literature with photosensitizers used as one-electron oxidants. It is shown in the present work that the process of long-range charge transport and the concomitant formation of guanine lesions at a long distance can be observed also in the case of two-electron oxidants. This is the signature of the formation of a transient guanine radical cation in the course of the two-electron abstraction process and consequently evidence of the separated one plus one electron abstraction steps. Long-range charge transport is likely to be a universal mechanism for any two-electron oxidant acting by electron abstraction provided that the second electron abstraction is slower than hole transfer.     

Cellular iron transport

Iron has a split personality as an essential nutrient that also has the potential to generate reactive oxygen species. We discuss how different cell types within specific tissues manage this schizophrenia. The emphasis in enterocytes is on regulating the body's supply of iron by regulating transport into the blood stream. In developing red blood cells, adaptations in transport manage the body's highest flux of iron. Hepatocytes buffer the body's stock of iron. Macrophage recycle the iron from effete red cells among other iron management tasks. Pneumocytes provide a barrier to prevent illicit entry that, when at risk of breaching, leads to a need to handle the dangers in a fashion essentially shared with macrophage. We also discuss or introduce cell types including renal cells, neurons, other brain cells, and more where our ignorance, currently still vast, needs to be removed by future research.     

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

Coupled biogeochemical cycling of iron and manganese as mediated by microbial siderophores

Siderophores, biogenic chelating agents that facilitate Fe(III) uptake through the formation of strong complexes, also form strong complexes with Mn(III) and exhibit high reactivity with Mn (hydr)oxides, suggesting a pathway by which Mn may disrupt Fe uptake. In this review, we evaluate the major biogeochemical mechanisms by which Fe and Mn may interact through reactions with microbial siderophores: competition for a limited pool of siderophores, sorption of siderophores and metal–siderophore complexes to mineral surfaces, and competitive metal-siderophore complex formation through parallel mineral dissolution pathways. This rich interweaving of chemical processes gives rise to an intricate tapestry of interactions, particularly in respect to the biogeochemical cycling of Fe and Mn in marine ecosystems.     

Non-transferrin-bound iron in plasma following administration of oral iron drugs

Non-transferrin-bound iron (NTBI) was detected in serum samples from volunteers with normal iron stores or from patients with iron deficiency anaemia after oral application of pharmaceutical iron preparations. Following a 100 mg ferrous iron dosage, NTBI values up to 9 μM were found within the time period of 1–4 h after administration whereas transferrin saturation was clearly below 100%. Smaller iron dosages (10 and 30 mg) gave lower but still measurable NTBI values. The physiological relevance of this finding for patients under iron medication has to be elucidated.