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PubMed1988

Transferrin endocytosis and iron uptake in developing myogenic cells in culture: effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents.

The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non-dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.

2,4-Dinitrophenol↗
PubMed1991

Correlation between the proliferative response to granulocyte colony-stimulating factor and the positivity of transferrin receptor in acute myeloblastic leukemia cells.

The use of granulocyte colony-stimulating factor (G-CSF) after chemotherapy for acute myeloblastic leukemia (AML) has been reported. However, there is a drawback in that G-CSF may stimulate the proliferation of AML progenitors. To determine the parameter(s) indicative of responsiveness of AML blasts to G-CSF, various surface phenotypes of blasts were examined in relation to the blast colony formation stimulated by G-CSF in 39 AML patients. A correlation was found only with transferrin receptor positivity among the various phenotypes studied. The population mean of percentages of transferrin receptor-positive blasts in the group responding to G-CSF in vitro was significantly higher than that of blasts in the group not responding to G-CSF. A further correlation was found between transferrin receptor positivity and the number of G-CSF receptors on the blasts; that is, blasts expressing more G-CSF receptors have greater transferrin receptor positivity. In our previous study, we observed that blasts with a large number of G-CSF receptors produce more colonies in response to G-CSF. These results indicated that blasts expressing more transferrin receptors have a larger number of G-CSF receptors and may show more active proliferation in response to G-CSF. Therefore, the proliferative response of blasts to G-CSF can be predicted by examining transferrin receptor positivity. The clinical use of G-CSF in AML patients may be recommended when the patient's blasts have a low level of transferrin receptor expression. The measurement of transferrin receptors on blasts, instead of the rather complicated G-CSF receptor determination, would be a useful indicator for the safer application of G-CSF in AML patients.

Antigens, CD↗
PubMed1989

Monoclonal antibody detects embryonic epitope specific for nerve-derived transferrin.

Monoclonal antibodies were generated against transferrin purified from chick embryo extract by fusing spleen cells from BALB/c mice immunized against embryonic transferrin, with myeloma cells. Antibodies produced by the selected hybridoma clones were all type IgG. Twelve clones were selected for secretion of antibodies to the embryo extract-derived transferrin, and three clones were studied extensively. Immunoblotting was used to demonstrate antibody binding to several avian transferrin proteins derived from adult chicken serum, adult chicken peripheral nerves, and ovotransferrin. Screening and detailed epitope analysis were accomplished by solid-phase immunoassay. The results indicated that two clones, 2G9.1 and 2B11.1, recognized the embryonic and egg antigens in preference to the adult proteins. However, a third clone, 6H2.1, recognized the nerve-derived transferrin preferentially to both the embryonic and adult serum antigens. None of the clones recognized the serum-derived transferrin in preference to the other antigens. These results indicate that embryonic epitope(s) are conserved in the nerve- but not the serum-derived transferrin. They also show that the neural antigen has site(s) distinct from the embryonic proteins. No changes in displacement curves were observed after these proteins were digested with neuraminidase, indicating that the epitope differences discovered are not intimately related to sialic acid residues on the various transferrins.

Animals↗
PubMed1993

Transferrin in the central nervous system of the shiverer mouse myelin mutant.

Transferrin, the iron mobilization protein, and its mRNA are normally present in oligodendrocytes. Previous reports using myelin mutants have shown both a decrease in transferrin protein and mRNA when the oligodendrocyte population is compromised. In this study the shiverer mouse mutant in which the oligodendrocyte population is numerically normal, but has both quantitatively diminished and qualitatively abnormal myelin was used. This animal model was chosen to address the question whether expression of the transferrin message and/or protein correlated more closely to the number of oligodendrocytes (normal) or the amount of myelin (abnormally low). A 1/2 to 2/3 decrease in transferrin protein occurred in all brain regions examined except for the spinal cord in the shiverer group compared to both heterozygous littermates and wild type controls. Levels of transferrin transcripts in the brain are not affected by the shiverer mutation. These results taken with previous reports from this laboratory indicate that the presence of oligodendrocytes is a requirement for normal expression of transferrin mRNA in brain but is not sufficient for normal values of the protein. The level of Tf protein correlates more closely with the amount of myelin present than it does with the numbers of oligodendrocytes present. These data are consistent with previous reports from our laboratory that transferrin accumulation by oligodendrocytes is associated with myelin production by these cells. These data further suggest transferrin mRNA may be constitutively expressed by oligodendrocytes and that the protein expression is regulated at the level of translation.

Animals↗
PubMed1995

The 5'-untranslated region of human transferrin mRNA, which contains a putative iron-regulatory element, is bound by purified iron-regulatory protein in a sequence-specific manner.

Human transferrin mRNA contains a 5'-untranslated region that (1) has homology to an iron responsive element and (2) is implicated in translational iron regulation of human transferrin transgenes in transgenic mice. Ferritin mRNA contains a 5'-untranslated region iron-responsive element, but iron regulation of ferritin differs from that of human transferrin transgenes in both magnitude and direction. Structural differences between the ferritin iron-responsive element and the human transferrin putative iron-responsive element may influence their iron-regulatory protein interactions and direct the differing translational responses. This study examines human transferrin RNA nucleotide sequence requirements for binding of cytoplasmic proteins and purified iron-regulatory protein. Mutations of the putative transferrin iron-responsive element similarly affected binding of purified iron-regulatory protein and liver cytoplasmic proteins, providing evidence that the IRP is one of the liver cytoplasmic proteins that binds the human transferrin iron-regulatory element and suggesting that it may be involved in iron-regulation of transferrin.

Animals↗
PubMed1991

Transferrin receptor expression and the regulation of placental iron uptake.

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport. Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors. Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.

Cell Differentiation↗
PubMed1988

Species specificity of transferrin binding, endocytosis and iron internalization by cultured chick myogenic cells.

The ability of unlabelled heterologous transferrin to interact with transferrin receptors on developing chick myogenic cells was investigated by measuring their capacity to inhibit the surface-binding and internalization of 125I- and 59Fe-labelled ovotransferrin. Transferrins from rat, rabbit, human, and a species of kangaroo (Macropus fuliginosus) were unable to inhibit either surface-binding or internalization of labelled ovotransferrin even at concentrations ten times the molar concentration of the ovotransferrin. Transferrins isolated from the serum of a toad (Bufo marinus) and a lizard (Teliqua rugosa), when added at high concentrations, were found to reduce surface-binding of 125I-Tf by 20-25% but did not inhibit internalization of either 125I-Tf or 59Fe. This suggests that the effects of toad and lizard transferrins are due to non-specific binding to the myogenic cells. In contrast, inhibition of both surface-binding and internalization of labelled ovotransferrin was found when myogenic cells were incubated in the presence of the homologous transferrin (ovotransferrin). The species-specificity of transferrin binding, endocytosis and iron internalization did not vary with the state of proliferation or differentiation of the myogenic cells. However, the intracellular iron utilization was found to differ between differentiating presumptive and terminally differentiated myotubes. Internalized 59Fe was fractioned by gel filtration. In dividing and non-dividing presumptive myoblasts 59Fe was found to elute in three peaks, two with elution volumes corresponding to ferritin and transferrin and one at greater elution volume than that of myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Mössbauer studies of electrophoretically purified monoferric and diferric human transferrin.

Electrophoretically purified 57Fe-enriched monoferric and diferric human transferrins and selectively labeled complexes ([C-56Fe,N-57Fe]transferrin and [C-57Fe,N-56Fe]transferrin) were studied by Mössbauer spectroscopy. The data were recorded at 4.2 K over a wide range of applied magnetic fields (0.05-6 T) and were analyzed by a spin-Hamiltonian formalism. Characteristic hyperfine parameters were found and the obtained zero-field splitting parameters (D = 0.25 +/- 0.05 cm-1 and E/D = 0.30 +/- 0.02) agree with previous electron paramagnetic resonance (EPR) findings. The weak-field spectra of the [N-57Fe]transferrin are slightly broader than those of the [C-57Fe]transferrin, indicating that the N-terminal iron site may be more heterogeneous. However, the absorption line positions and the relative intensities of the subspectra originating from the three Kramers doublets of each Fe3+ site are identical. Thus the electronic structures of the two iron sites can be described by the same set of spin-Hamiltonian parameters, indicating that the ligand environments for the two sites are the same, as suggested by the recent X-ray crystallographic studies. This suggestion is further supported by the observation that the strong-field spectra of the two monoferric transferrins are indistinguishable. The selectively labeled mixed-isotope transferrins exhibit spectra that are identical to those of the corresponding monoferric 57Fe-enriched transferrins, implying that the occupation of one iron site has little or no effect on the immediate environment of the other site, a finding that is not surprising since the two sites are separated by approximately 4.2 nm.

Binding Sites↗
PubMed1995

Transferrin is not involved in initial uptake process of iron in rat duodenal mucosa. Ultrastructural study by x-ray energy spectrometry.

The role of transferrin in iron absorption by the duodenal mucosa in rats with iron deficiency and controls was evaluated immunohistochemically. Ferric iron was administered to each rat using a metallic gastric tube. Transferrin was stained by an immunoperoxidase method and iron with Prussian blue in the same duodenal sections and observed by light microscopy. The localization of transferrin differed from that of ferric iron both in rats with iron deficiency and in controls. In iron-deficient rats, transferrin was weakly stained after iron administration but was strongly stained after saline administration. In contrast, in controls, transferrin was weakly stained after saline administration but was strongly stained after iron administration. By electron microscopy, x-ray energy spectrometric analysis of the transferrin-positive areas showed no iron peak. In iron-deficient rats, accumulation of electron-dense transferrin-negative microgranules was observed in some of the duodenal columnar epithelium. X-ray energy spectrometric analysis of this area revealed iron, indicating iron absorption. These results suggest that mucosal transferrin does not act as a shuttle protein in iron absorption via the rat duodenal columnar epithelium, and the function of this protein may be to inform the absorptive cells of the iron status of the body as observed in other organs.

Animals↗
PubMed1999

Transferrin is an essential factor for myelination.

It has been established that oligodendrocytes, the myelin forming cells, participate in iron homeostasis through the synthesis and secretion of transferrin. Here we investigated whether a correlation exists between myelination, the commonly studied function of oligodendrocytes, and that of transferrin synthesis and secretion. We used a proteolipid protein mutant, the myelin deficient rat, whose condition is characterized by severe hypomyelination. We compared the ontogenic profile for transferrin gene expression in mutants with that of unaffected rat pups through northern blot analysis and in situ hybridization. Surprisingly, transferrin synthesis was null in mutant oligodendrocytes. Next, we demonstrated that a single apo-transferrin intraparenchymal injection administered to P5 rat pups enabled mutant oligodendrocytes to synthesize myelin basic protein and to myelinate axons, indicating that transferrin effects mutant oligodendrocyte maturation regardless of its source. Thus, transferrin availability is essential for oligodendrocyte maturation and function, and oligodendrocytes are most vulnerable to transferrin deficiency during the premyelinating stage.

Animals↗
PubMed1995

Transport of [125I]transferrin through the rat blood-brain barrier.

Transferrin receptors are present in the plasma membrane of brain endothelial cells but it is unclear if these receptors mediate transport of transferrin across the blood-brain barrier (BBB). In the present study, the transport of rat holo-transferrin (rTf) across the BBB in vivo was evaluated in ketamine anesthetized rats (250-300 g) both by in situ brain perfusion coupled with capillary depletion analysis and by thaw-mount autoradiography. [125I]rTf was infused through the right internal carotid artery at a rate of 3.65 ml/min for 2.5-5 min. After a 5 min perfusion, the volume of distribution (VD) of [125I]rTf in the brain homogenate, the postvascular supernatant, or the vascular pellet was 55.8 +/- 4.5, 43.5 +/- 4.8, and 8.7 +/- 2.3 microliters/g, respectively. Co-infusion of [125I]rTf with unlabeled rTf or with a high dose of OX26 monoclonal antibody to the rat transferrin receptor significantly reduced the [125I]rTf transport, and in the presence of 10% rat serum [125I]rTf transport was nearly entirely abolished. The transport of [125I]rTf across the BBB in vivo was demonstrated by thaw-mount autoradiography, which showed silver grains well within brain parenchyma following a 5 min internal carotid artery perfusion. These studies are consistent with the following conclusions: (a) in the absence of competing plasma transferrin, [125I]holo-transferrin is transported through the BBB at a rate comparable to the OX26 monoclonal antibody; and (b) the ability to detect measurable transport of perfused [125I]transferrin is greatly inhibited by a small contamination of the perfusate by rat serum, which contains high concentrations of competing transferrin.

Animals↗
PubMed1993

Semi-automatic method for determination of different isoforms of carbohydrate-deficient transferrin.

Carbohydrate deficient transferrin (CDT) has been reported to be one of the best biochemical markers of alcohol abuse. However, a need still exists for a simple and practical method for widespread laboratory use. A semi-automatic (SA) isoelectric focusing (IEF) assay for CDT (SA-IEF-CDT) by a Phast System is introduced here. Different isoforms of transferrin were separated by IEF on polyacrylamide gels (pI 4.0-6.5) and located by immunofixation with an anti-transferrin serum. The precipitation bands were stained with Coomassie Brilliant Blue and quantitated densitometrically. The present method gave a picture of the relative amounts of 10 different transferrin isoforms. The percentage of CDT with pI > or = 5.7 (representing di-, mono- and asialotransferrin) was calculated. For comparisons transferrin bands with pI > or = 5.6 (tri-, di-, mono-, and asialotransferrin), pI > or = 5.8 (mono- and asialotransferrin) and pI > or = 5.9 (asialotransferrin) as well as GGT, ASAT and ALAT were calculated. The method showed good linearity and it identified different isoforms in concentrations of < 10 mg/l of transferrin. The correlation of the present method with a commercially available method employing anion exchange followed by double antibody RIA (AE-RIA-CDT) was good (n = 38, r = 0.924). In 19/20 (95%) of healthy controls, the CDT value was below 4.4% (mean + 2 S.D.) of total transferrin, while higher values were observed in all 20 (100%) alcoholics. In conclusion, the developed semi-automatic method is a practical and reliable alternative for determination of different transferrin isoforms.

Adult↗
PubMed1993

Unfolding of human serum transferrin in urea studied by high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was used to monitor the progress of the unfolding of human serum transferrin in urea. Denaturation curves of the transferrin forms were constructed plotting the migration times corrected for the viscosity vs. the concentration of urea in the buffer. The practical advantage of capillary zone electrophoresis is the short analysis time, 5-15 min, as compared with slab-gel experiments, which require overnight runs for similar purposes. The resolution increased with the urea concentration, and hence high concentrations are beneficial for quantitative and qualitative analysis of mixtures of transferrin forms. Unfolding intermediates of the isoforms, which interconvert to the unfolded state slowly compared with the time scale of the electrophoretic separation, and also the completely unfolded isoforms were resolved and detected simultaneously when iron-free transferrin was subjected to denaturation by urea at concentrations between 3 and 6 M. However, no unfolding intermediates were observed with transferrin isoforms containing two iron atoms (i.e. diferric transferrin molecules), which accordingly are strongly resistant to urea denaturation. The unfolding of the transferrin isoforms depends on the iron content of the complexes, but not the carbohydrate content. HPCE in the presence of urea in this mode has the potential to become an analytical tool for diagnosis of diseases in which the transferrin patterns change.

Chromatography, High Pressure Liquid↗
PubMed1994

Transport of transferrin across the blood-thymus barrier in young rats.

The interaction of transferrin-peroxidase (Tf-HRP) with the capillary endothelium of the rat thymus was analyzed by diaminobenzidine (DAB) cytochemistry and electron microscopy. The thoracic aortas of young rats were cannulated and the upper bodies perfused with the Tf-HRP conjugate. In the thymus, plasmalemmal vesicles of the endothelium mediated the endocytosis and transport of Tf-HRP. Transcytosis of Tf-HRP appeared to occur by micropinocytosis, without morphological evidence of involvement by endothelial endosomes. DAB reaction product was commonly present in the subendothelial interstitial space and, in addition, was often localized in clathrin coated pits and vesicles in epithelial reticular cells that surround the thymic capillaries. In perfusions done at 4 degrees C, no binding of Tf-HRP to the lumenal membranes of capillaries was detected. The transport of Tf-HRP across the capillary endothelium in the thymus was not inhibited by competition with excess native transferrin; however, the uptake of Tf-HRP by epithelial reticular cells was completely inhibited by excess transferrin. Transferrin receptors were localized in the thymus by indirect immunocytochemistry using OX-26, a mouse anti-rat transferrin receptor monoclonal antibody. No transferrin receptors were detected on the capillary endothelium but diffuse reaction product was localized on the subjacent epithelial reticular cells. These results indicate that transport of Tf-HRP across the thymus capillary wall is independent of transferrin receptors at the level of the endothelial cells but that subsequent uptake of Tf-HRP by epithelial reticular cells is a transferrin receptor dependent process.

Age Factors↗
PubMed1991

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.

Apoproteins↗
PubMed1987

Transferrin in chick retina: distribution and location during development.

Chick retinas from embryonic day 6 (E6) to 3 weeks post-hatching were examined for the presence and location of endogenous transferrin. Immunocytochemistry revealed that transferrin was differentially distributed in retinal layers. Furthermore, the pattern of transferrin distribution changed with developmental age. At day E6, transferrin was found in 2 distinct bands which were located in the area of the Müller cell end-feet. By day E9, additional regions of transferrin immunoreactivity could be found in the inner and outer plexiform layers (IPL, OPL) and the nerve fiber layer (NFL). These latter 3 bands (IPL, OPL and NFL) became more prominent from E9 until E17 as the synaptic layers and nerve fiber layer increased in density and maturation. Perikarya in the nuclear layers size, density and maturation. Perikarya in the nuclear layers were negative. At day E17 and later, the newly forming outer segments of photoreceptor cells were strongly reactive for transferrin while the somas of the photoreceptor cells, in the ONL, were negative. Retinas from chicks 1 day to 3 weeks post-hatching retained strong immunoreactivity for transferrin in the photoreceptor cell outer segments and OPL, lessened immunoreactivity in the IPL and loss of immunoreactivity in the NFL. Iron distribution in the retina for all ages examined showed only 2 bands that locally corresponded to the Müller cell end-feet. Iron stores were not found in the synaptic layers or photoreceptor cell outer segments. These studies suggest an iron storage function for retinal glia and a role for transferrin in neuronal development and differentiation.

Animals↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1996

Copper binding selectivity of N- and C-sites in serum (human)- and ovo-transferrin.

Copper binding selectivity of the N- and C-sites in serum (human)- and ovo-transferrin was investigated through copper binding constants, copper dissociation rate constants, and EPR spectra. At pH 7.4, stepwise copper binding constants of serum (human)-transferrin were K1 = 1.8 (+/- 0.6) x 10(12) M-1 and K2 = 1.2 (+/- 0.5) x 10(11) M-1, and those of ovo-transferrin were K1 = 1.9 (+/- 0.5) x 10(11) M-1 and K2 = 2.1 (+/- 0.4) x 10(11) M-1. Absorbance changes resulting from copper binding to the C- or N-site at various ratios of Cu2+/apo-transferrin were separated by a kinetic method. It was clearly indicated that, in serum (human)-transferrin, the copper binding affinity for the C-site was much larger than that for the N-site, whereas in ovo-transferrin, the C- and N-sites have almost the same affinity for copper ions. In the presence of anions (0.1 M KCl or 0.1 M NaClO4), the stepwise copper binding constants of serum (human)-transferrin were almost 10-times smaller than those in the absence of the anions. The selectivity in binding the copper ions to both sites of serum (human)-transferrin in the presence of 0.1 M NaClO4 is much smaller than that in the presence of 0.1 M KCl or in the absence of the anions (0.1 M KCl and 0.1 M NaClO4). EPR spectra of the copper ions of the N-site in dicupric serum-transferrin are dramatically changed respectively by the addition of 0.1 M KCl, 0.1 M NaCl, and 0.1 M NaClO4. This suggests that the change in the coordination geometry of the copper ions occurs at the N-site.

Anions↗