The influence of inorganic anions on the formation and stability of Fe3+-transferrin-anion complexes

Harris (Biochemistry 24 (1985) 7412) reports that inorganic anions bind to human apotransferrin in such a way as to perturb the ultraviolet spectrum. The locus of binding is thought to involve the specific metal/anion-binding sites since no perturbation is observed with Fe3+-transferrin-CO3(2-). Paradoxically, we were unable to demonstrate the formation of Fe3+-transferrin-inorganic anion complexes despite the presence of high concentrations of SO4(2-), H2PO4-, Cl-, ClO4- or NO3-. Similar results were found for human lactoferrin. Electron paramagnetic resonance spectroscopy and visible spectrophotometry were used to monitor the results. An attempt to form the H2PO4- complex by displacement of glycine from Fe3+-transferrin-glycine resulted only in the disruption of the ternary complex. A series of inorganic anions varied in their ability to release iron from Fe3+-transferrin-CO3(2-) at pH 5.5, the approximate pH of endosomes where iron release takes place within cells. The order of effectiveness was H2P2O7(2-) much greater than H2PO4- greater than SO4(2-) greater than NO3- greater than Cl- greater than ClO4-. The rate of iron removal from Fe3+-transferrin-CO3(2-) at pH 5.5 by a 4-fold excess of pyrophosphate was greatly enhanced by physiological NaCl concentration. Iron removal was complete within 10 min, the approximate time for iron release from Fe3+-transferrin-CO3(2-) in developing erythroid cells. Thus, inorganic anions may have a significant effect on the release of iron under physiological conditions despite the fact that such inorganic anions cannot act as synergistic anions. The results are discussed in relation to a special role for the carboxylate group in allowing ternary complex formation.     

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.     

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.     

Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae

The expression of human transferrin and lactoferrin binding activity in Haemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in all H. influenzae isolates tested but not detected in other Haemophilus species or in species of Pasteurella or Actinobacillus. Lactoferrin binding activity was only detected in 1/15 H. influenzae isolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron-loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron-loaded lactoferrin and apolactoferrin.     

Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron.

Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these iron-binding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.     

Factors affecting the adenosine triphosphate induced release of iron from transferrin.

The release of iron from transferrin was investigated by incubating the diferric protein in the presence of potential iron-releasing agents. The effective chemical group appears to be pyrophosphate, which is present in blood cells as nucleoside di- and triphosphates, notably adenosine triphosphate (ATP). An alternative structure with comparable activity is represented by 2,3-diphosphoglycerate. Neither 1 mM adenosine monophosphate (AMP) nor 1 mM orthophosphate released iron from transferrin. The ATP-induced iron-releasing activity was dependent on weak acidic conditions and was sensitive to temperature and sodium chloride concentration. The rate of iron release rapidly increased as transferrin was titrated with HCl from pH 6.8 to 6.1 in the presence of 1 mM ATP and 160 mM NaCl at 20 degrees C. Iron release from transferrin without ATP was observed below pH 5.5. Ascorbate (10(-4) M) reduced Fe(III), but only after iron release from transferrin by a physiological concentration of ATP. A proposal for the mechanism of iron release from transferrin by ATP and the utilization of reduced iron by erythroid cells is described.     

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     

Effects of host iron transport compounds on growth kinetics and outer-membrane protein expression of Bilophila wadsworthia

Since the environmental iron concentration has emerged as an important attribute in the expression of bacterial virulence, the purpose of this study was to determine the effects of transferrin, lactoferrin, heme compounds, and inorganic iron sources (ferric and ferrous sulfate) on the growth of Bilophila wadsworthia and to study its outer membrane composition when grown under these different simulated in vivo conditions. Lactoferrin, transferrin, hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources. Bilophila wadsworthia was also capable of growing in the presence of ferrous and ferric sulfate. Profiles obtained by SDS-PAGE showed two iron-regulated outer membrane proteins (IROMPs) of 190 kDa and 88 kDa. The 190 kDa was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. These two major IROMPs were expressed in iron-restricted media supplemented with iron-bound organic sources and repressed by the addition of inorganic iron sources.     

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.     

Response of Neisseria meningitidis to iron limitation

In the human body, the concentration of free iron is limiting for bacterial growth, since iron is bound to transport and storage proteins such as transferrin and lactoferrin. When grown under iron starvation, Neisseria meningitidis produces receptors for these proteins in the outer membrane. These receptors are presently being characterized at the molecular level. Here, we summarize our current knowledge of these receptors, with special emphasis on the LbpA and FrpB proteins, which are studied in our laboratories. Furthermore, the genetic and antigenic variability of these proteins and their vaccine potential are discussed.     

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.     

Aerobactin-mediated utilization of transferrin iron

Aerobactin and enterobactin, hydroxamate- and catechol-type siderophores, respectively, were found capable of removing iron (III) from transferrin in buffered solution. Although under these conditions aerobactin displaced the iron much more slowly than did enterobactin, the rate for the former could be accelerated by addition of pyrophosphate as mediator. Transfer of iron (III) from transferrin to aerobactin appeared to proceed via a ternary complex. Cells of Escherichia coli BN 3040 NalR iuc containing transport systems for both enterobactin and aerobactin, the genetic determinants for the latter specified on a ColV-type plasmid, took up iron from [55Fe]transferrin in minimal medium. In this case aerobactin was effective at a much lower concentration, although enterobactin still displayed superior ability to transfer the iron. In serum, however, the rate measured with aerobactin exceeded that found with enterobactin. The results indicate that aerobactin, in spite of its relatively unimpressive affinity for iron (III) as a siderophore, is nonetheless equipped with structural features or properties that enhance its ability to remove the metal ion from transferrin, especially when receptor-bearing cells of E. coli are present to act as a thermodynamic sink for the iron. These attributes of the aerobactin system of iron assimilation may account for its status as a virulence determinant in hospital isolates of E. coli.     

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.     

Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin-agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.     

Interaction of lactoferrin and transferrins with the outer membrane of Bordetella pertussis

Bordetella pertussis was able to grow in vitro under conditions where the only iron present was bound to the iron-binding proteins ovotransferrin, transferrin or lactoferrin. Under these conditions the bacteria produced neither hydroxamate nor phenolate-catecholate siderophores to assist in the procurement of iron. Examination of B. pertussis outer-membrane preparations by SDS-PAGE and immunoblotting showed that the iron-binding protein ovotransferrin was bound directly to the bacterial surface. Assays of the binding of radiolabelled transferrin by the bacteria showed that the association was a specific process and that there was turnover of the bound proteins. Competitive binding assays indicated that lactoferrin could be bound in the same way. It is suggested that B. pertussis obtains iron directly from host iron-binding proteins during infection.     

Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions

Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes. The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.     

Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.     

Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Are lactoferrin and transferrin promoters of hydroxyl-radical generation?

Apo-lactoferrin and apo-transferrin protect against iron-ion-dependent hydroxyl-radical (.OH) generation from H2O2 in the presence of superoxide radicals or ascorbic acid at pH 7.4, whether the necessary iron is added as ionic iron or as ferritin. Iron-loaded transferrin and lactoferrin [2 mol of Fe(III)/mol] show no protective ability, but do not themselves accelerate .OH production unless chelating agents are present in the reaction mixture, especially if the proteins are incorrectly loaded with iron. At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation. The physiological significance of these observations is discussed.