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.     

Comparative aspects of transferrin-reticulocyte interactions: membrane receptors and iron uptake

1. A comparative study was made of transferrin and iron uptake by rabbit, rat and human reticulocytes and chick embryo erythrocytes from rabbit, rat, human, chicken and porcine transferrins, human lactoferrin and chicken conalbumin. 2. Three methods were used, viz. direct and competitive uptake studies of transferrin and iron by the four species of cells, and competitive studies of transferrin binding by solubilized membrane receptors (rabbit reticulocytes only). 3. Methods were devised to analyse the data so as to obtain indices of relatedness or relative affinities of each type of heterologous transferrin in rates of iron uptake found with transferrin and cells from various species are largely due to variation in the affinity of cellular receptors for different transferrins. 5. It is concluded that the procedure used in this investigation allow the assessment of phylogenetic relationships and evolutionary trends obtained by structural studies of proteins.     

Nature of the iron requirement for Chinese hamster V79 cells in tissue culture medium

The Chinese hamster V79 cell line can be grown in medium containing iron instead of lactalbumin hydrolysate and containing defined low molecular weight components instead of peptone. A rather large amount of inorganic iron must be supplied for optimum growth. Dose-response curves done with commercially available transferrins from various species show that this Chinese hamster cell line grows well with human and rabbit transferrins but poorly with porcine, bovine, and chicken egg white (conalbumin) transferrins. An assay of Chinese hamster serum in the presence and absence of iron shows that hamster serum is better at providing the V79 cells with iron than human or rabbit transferrin. Thus, the nature of the iron requirement of V79 cells lies in the requirement for a specific transferrin.     

Receptors for transferrin in pathogenic bacteria are specific for the host's protein

Transferrin receptors detected by a solid-phase binding assay were shown to be specific for the host's transferrin in the representative bacterial pathogens Neisseria meningitidis (human), Pasteurella haemolytica (bovine), and Actinobacillus pleuropneumoniae (porcine). Consistent with the receptor specificity, iron-deficient bacteria were only capable of utilizing transferrin from the host as a source of iron for growth.     

Fhua and HgbA, outer membrane proteins of Actinobacillus pleuropneumoniae: their role as virulence determinants

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (DeltahgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.     

Specificity of hepatic iron uptake from plasma transferrin in the rat.

1. The role of specific interaction between transferrin and its receptors in iron uptake by the liver in vivo was investigated using 59Fe-125I-labelled transferrins from several animal species, and adult and 15-day rats. Transferrin-free hepatic uptake of 59Fe was measured 2 or 0.5 hr after intravenous injection of the transferrins. 2. Rat, rabbit and human transferrins gave high and approximately equal levels of hepatic iron uptake while transferrins from a marsupial (Sentonix brachyurus), lizard, crocodile, toad and fish gave very low uptake values. Chicken ovotransferrin resulted in higher uptake than with any other species of transferrin. 3. Iron uptake by the femurs (as a sample of bone marrow erythroid tissue) and, in another group of 19-day pregnant animals by the placentas and fetuses, was also measured, for comparison with the liver results. The pattern of uptake from the different transferrins was found to be similar to that of iron uptake by the liver except that with femurs, placentas and fetuses ovotransferrin gave low values comparable to those of the other non-mammalian species. 4. It is concluded that iron uptake by the liver from plasma transferrin in vivo is largely or completely dependent on specific transferrin-receptor interaction. The high hepatic uptake of iron from ovotransferrin was probably mediated by the asialoglycoprotein receptors on hepatocytes.     

Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin

Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96-102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropneumoniae during growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.     

The nucleotide sequence of rabbit liver transferrin cDNA

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.     

Production of transferrin receptors by Histophilus ovis: three of five strains require two signals

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.     

Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1.

Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidin-agarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (approximately 64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer-membrane protein. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.     

Headspace solid-phase microextraction with 1-pyrenyldiazomethane on-fibre derivatisation for analysis of fluoroacetic acid in biological samples

Four molecular forms of transferrins with different iron-binding states were separated by HPLC using a pyridinium polymer column. The elution order was monoferric transferrin bound to the C-site, holotransferrin, apotransferrin and monoferric transferrin bound to the N-site. Human sera were also analyzed with the column, and ICP-MS combined with HPLC was used to detect iron in each peak. Transferrin peaks separated by HPLC were also confirmed by an immunological method. The percentages of iron saturation in transferrins obtained by the HPLC method were compared with the values calculated from clinical data.     

Iron-binding properties and amino acid composition of marsupial transferrins: comparison with eutherian mammals and other vertebrates

1. Some physicochemical properties of transferrin from three marsupials, viz a possum (Trachosurus vulpecula), a kangaroo (Macropus fuliginosus) and the quokka (Setonix brachyurus) were studied and compared with those of transferrins from mammalian and non-mammalian vertebrate species. 2. The molecular weight of the marsupial transferrins fell within the range of 76,000-79,000 daltons. 3. The marsupial transferrins were similar to the transferrins of eutherian mammals with respect to optical spectral properties, iron binding capacity and the pH-dependence of iron binding, and iron release mediated by 2,3-DPG. 4. The amino acid compositions of the marsupial transferrins were compared with each other and with the transferrins from the other vertebrate species. The compositions of the marsupial transferrin were closely related to each other, and also showed similarities with transferrins from eutherian mammals and chicken ovotransferrin.     

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.     

Isolation and characterization of the iron-binding properties of a primitive monolobal transferrin from Ciona intestinalis

Transferrins are bilobal glycoproteins responsible for iron binding, transport, and delivery in many higher organisms. The two homologous lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form. In the present study, a 37.7-kDa primitive monolobal transferrin (nicatransferrin, or nicaTf) from the serum of the model ascidian species Ciona intestinalis was isolated by using an immobilized iron-affinity column and characterized by using mass spectrometry and N-terminal sequencing. The protein binds one equivalent of iron(III) and exhibits an electron paramagnetic resonance spectrum that is anion-dependent. The UV/vis spectrum of nicaTf has a shoulder at 330 nm in both the iron-depleted and the iron-replete forms, but does not display the approximately 460 nm tyrosine-to-iron charge transfer band common to vertebrate serum transferrins under the conditions investigated. This result suggests that iron may adopt a different binding mode in nicaTf compared with the more highly evolved transferrin proteins. This difference in binding mode could have implications for the physiological role of the protein in the ascidian. The genome of C. intestinalis has genes for both a monolobal and a bilobal transferrin, and the sequences of both proteins are discussed in light of the known features of vertebrate serum transferrins as well as other transferrin homologs.     

Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center

Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.     

Uptake of sialo and asialo transferrins by isolated rat hepatocytes. Comparison of a heterologous and a homologous system

Incubation of isolated rat hepatocytes with different human sialo transferrins shows that interaction with the specific transferrin receptor is insensitive to differences in the carbohydrate composition of the glycans. Asialo transferrins lead to an increased iron uptake, which is dependent on the amount of exposed galactose. This is explained by the presence of the asialo glycoprotein (AsGP) receptor. Experiments with selective saturation of the two receptor systems show that on incubation with human asialo transferrin (AsHTf) transferrin uptake proceeds increasingly via the AsGP receptor on raising the concentration. Homologous rat asialo transferrin (AsRTf) behaves similarly, but less pronounced. Iron is accumulated via both receptor systems in the heterologous system, but only via the transferrin receptors in the homologous system. The difference in interaction with the AsGP-receptor may be caused by the difference in galactose content of the two asialo transferrins. As an explanation for the differences in intracellular metabolism a hitherto unknown recognition system for species specificity is postulated which protects homologous AsRTf from degradation, but directs foreign AsTf to lysosomes.     

The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study

Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.     

Iron metabolism pathways in the rat hepatocyte

A small to moderate inhibitory effect of iron uptake by isolated rat hepatocytes in short-term studies was seen with oxidative phosphorylation and electron transport inhibitors, and no inhibition by agents affecting pinocytosis. Intracellular transferrin was able to donate iron to the small-molecular weight iron pool, and the latter was able to transfer, by a process not requiring energy or movement of serum transferrin, iron to ferritin. Serum transferrin was not able to lose iron to any cytosol components. Reducing agents were not able to abstract iron from rat serum transferrin to any great extent. It is concluded that iron is taken up by the rat hepatocyte from serum transferrin by a process not requiring energy or movement of serum transferrin into the cell interior; and that intracellular transferrin is involved in acquiring iron from serum transferrin at the cell surface, with iron then being transferred to the small-molecular weight iron pool and hence to ferritin. It is also proposed that intracellular transferrins may have the general function of interacting with serum transferrin at cell surfaces.     

Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae.