Localization of transferrin and transferrin receptors in rat testes

One of the major proteins secreted by rat Sertoli cells in culture is a transferrin-like protein (Skinner and Griswold, 1980). The purpose of this study was to quantitate the amount of testicular transferrin in fluids isolated from the testis by the use of a radioimmunoassay and to determine the location of transferrin and transferrin receptors in the testis by indirect immunofluorescence. Seminiferous tubule fluid, rete testis fluid, and testicular lymph were collected from rat testes and were found to contain 141 micrograms, 47 micrograms and 3.7 mg transferrin per ml of fluid, respectively. Serum was found to contain 3.7 mg/ml transferrin. Paraffin sections of rat testis were incubated with rabbit anti-rat transferrin, biotinylated goat anti-rabbit and fluorescein-conjugated avidin. Immunoreactive transferrin was thus localized on the proacrosome and nuclear cap of developing spermatids. Late spermatids showed transferrin over the entire region of the head but mature testicular spermatozoa exhibited little fluorescence. The interstitial tissue between seminiferous tubules fluoresced brightly, indicating a large amount of transferrin in this area. By pretreating sections with rat transferrin, the receptor for the protein was localized on and in spermatocytes and early round spermatids. Dividing germ cells were brightly fluorescent.     

脑内的铁,转铁蛋白及转铁蛋白受体

脑铁异常增高可能参与脑神经变性疾病的发生发展。这一发现使得脑铁代谢成为近年广为关注和研究较为广泛的领域。本文综述了这一领域某些方面的目前认识。包括：（１）脑铁分布及功能;（２）铁转铁蛋白及转铁蛋白受体在脑内的合成与分布;（３）脑铁摄取和运输。此外,对铁与某些金属离子,转的蛋白和转铁蛋白受体与脑神经变性疾病的关系,以及转铁蛋白受体内吞在生物大分子跨血脑屏障运输中的作用也作了简要讨论。     

Concentration of transferrin iron and transferrin saturation in blood

Transferrin iron, transferrin protein concentrations, and transferrin saturation have been determined for the first time in the whole blood. Microsamples were taken from healthy adults and patients with occupational secondary haemochromatosis using quantitative electron spin resonance technique. At elevated transferrin saturation, transferrin saturation values determined in the plasma and serum samples were shown to be less than respective values determined in the whole blood of the same patients. At increased transferrin iron concentration the difference between experimental and reference data sets determined in the blood and plasma was statistically significant in contrast to data sets determined in serum. Therefore, the analysis of the blood microsamples ensured an adequate estimation of transferrin iron concentration, especially at high transferrin saturation. A new index--transferrin iron concentration in the formed blood elements--was introduced. The values of the index were determined in the groups of healthy adults, patients with secondary occupational hemochromatosis and healthy newborns.     

Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin.

To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.     

The transferrin receptor

The isolation and analysis of the transferrin receptor has been greatly aided by the use of monoclonal antibodies. The receptor is a disulphide-linked homo-dimer which spans the membrane and binds two molecules of transferrin. Controlling genes for this receptor in humans have been mapped to chromosome 3 using cell hybrids. The expression of transferrin receptors is related to the obligatory and ubiquitous iron requirements associated with cell proliferation or the special iron demand of haemoglobin synthesizing cells and trophoblasts. However, transferrin receptors may also be involved in cell interactions regulating cell growth.     

Human transferrin polymorphism

The application of isoelectric focussing (IEF) has revealed a large amount of heterogeneity in the human transferrin (TF) system and has enhanced its potential value in anthropological and genetic studies. The average heterozygosity has been elevated from 0.05, observed by conventional methods of electrophoresis, to 0.29 detected by IEF. So far approximately 30,000 individuals from 122 population groups have been analyzed for TF subtypes to evaluate the magnitude of genetic variation at the TF locus. Possible environmental and biological factors, which may be operating to maintain the TF polymorphism, are discussed.     

Fibrillation of transferrin

BackgroundThe nature of fibrillar deposits from aqueous solutions of human serum and recombinant human transferrin on mica and carbon-coated formvar surfaces has been investigated.Methods and ResultsAtomic force microscopy showed that the deposition of recombinant transferrin onto the hydrophilic surface of mica resulted in the formation of a monolayer-thick film composed of conformationally-strained flattened protein molecules. Elongated fibres developed on top of this layer and appeared to be composed of single proteins or small clusters thereof. Monomeric and dimeric transferrins were separated by gel permeation chromatography and their states of aggregation confirmed by mass spectrometry and dynamic light scattering. Transmission electron-microscopy showed that dimeric transferrin, but not monomeric transferrin, deposited on carbon-coated formvar grids forms rounded (circular) structures ca. 250 nm in diameter. Small transferrin fibrils ca. 250 nm long appeared to be composed of smaller rounded sub-units. Synchrotron radiation-circular dichroism and, Congo red and thioflavin-T dye-binding experiments suggested that transferrin aggregation in solution does not involve major structural changes to the protein or formation of classical β-sheet amyloid structures. Collisional cross sections determined via ion mobility–mass spectrometry showed little difference between the overall protein shapes of apo- and holo-transferrin in the gas phase.General significanceThe possibility that transferrin deformation and aggregation are involved in neurological disorders such as Parkinson's and Alzheimer's disease is discussed. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.     

Characterization of transferrin binding and specificity of the placental transferrin receptor

This study systematically examined the characteristics of specific binding of adult diferric transferrin to its receptor using a Triton X-100 solubilized preparation from human placentas as the receptor source. The following information was obtained. The ionic strength for maximal binding is in the range of 0.1-0.3 M NaCl. The pH optimum for specific binding extends over the range, from pH 6.0-10.0. Specific binding of diferric transferrin is not affected by 2.5 approximately 50 mM CaCl2 or by 10 mM EDTA. Triton X-100 in the concentration range of 0.02-3.0% does not affect specific binding. Specific binding is saturated within 10 min at 25 or 37 degrees C in the presence of excess amounts of diferric transferrin. The binding is reversible and the dissociation of diferric transferrin from the transferrin receptor is complete within 40 min at 25 degrees C. Apotransferrin, both adult and fetal, showed less binding than the holotransferrin species by competitive binding assay in the presence of 10 mM EDTA independent of up to 20 mM CaCl2. A 1500-fold molar excess of adult and fetal apotransferrin is required to give 40% inhibition for 125I-labeled diferric transferrin binding. Since calcium ion is not a factor, and since apotransferrin has such high binding affinity for iron (Ka = 1 X 10(24], this experiment suggests that the EDTA was necessary to prevent conversion of apotransferrin to holotransferrin from available iron in the reaction system. The specificity of the transferrin receptor for transferrin was examined by competitive binding studies in which 125I-diferric transferrin binding was measured in the presence of a series of other proteins. The proteins tested in the competitive binding studies were classified into three groups; in the first group were human serum albumin and ovalbumin; in the second group were proteins containing iron ions, such as hemoglobin, hemoglobin-haptoglobin complex, heme-hemopexin complex, ferritin, and diferric lactoferrin; in the third group were the metal-binding serum proteins, ceruloplasmin and metallothionein. None of these proteins except ferritin showed inhibition of diferric transferrin binding to the receptor. The effect of ferritin was small since a 700- to 1500-fold molar excess of ferritin is required for 50% inhibition of binding of diferric transferrin to the receptor.     

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells

The mechanisms by which the duodenal mucosa absorbs iron are unknown. Insorption into absorptive cells of luminal iron bound to transferrin via receptor-mediated endocytosis has been hypothesized, but transferrin and transferrin receptor are absent in apical microvillous brush borders of small bowel biopsies taken from fasted patients and normal volunteers. We hypothesized that a normal iron-containing diet might induce the transient appearance of transferrin and transferrin receptor in apical brush borders of small intestinal absorptive cells in a normal mouse that was provided iron-containing chow until the moment of sacrifice. Light and electron microscopic immunolocalization of transferrin and transferrin receptor in proximal small intestinal absorptive cells was limited to basolateral membranes and coated pits of cells predominantly in the crypts and basal regions of the villi. Transferrin and transferrin receptor were not detected in apical microvillous brush border membranes of these enterocytes. In parallel immunolocalization protocols designed to show the ability to immunodetect other antigens at these locations, maltase and proteoglycan were demonstrated in apical microvillous brush border membranes and in basolateral membranes, respectively, in absorptive cells of small intestinal villous tip, base, and crypt regions. Furthermore, transferrin and transferrin receptor were immunolocalized in hepatocyte sinusoidal microvillus membranes. We conclude that food does not induce the appearance of immunodetectable transferrin and transferrin receptor in the apical microvilli of small intestinal absorptive cells and, therefore, that these iron transport proteins are not involved in the apical microvillous membrane transport of luminal dietary iron.     

Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.     

Iodination significantly influences the binding of human transferrin to the transferrin receptor

The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor's ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor-ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of Kd=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of Kd=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3-5-fold to Kd=0.66 nM and Kd=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.     

Rapid endocytosis of the transferrin receptor in the absence of bound transferrin

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.     

The evolution of transferrin

A theory of the evolution of the iron-binding protein transferrin is proposed. Particular importance is attached to the pattern of disulphide bridges as a phylogenetic marker.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

Iodogen-catalyzed iodination of transferrin

Transferrin (human, rabbit) labels at low efficiency (1%-10%) with 125I when reaction of 0.5-0.7 ng of I- (8-10 microCi) with 20 micrograms of the protein is catalyzed by iodogen in a constant volume of 0.1 ml. Microiodination by this technique was therefore analyzed with regard to the relative proportions of the reactants, oxidant requirement, and timing. In vials giving a reaction volume-to-active surface ratio of 0.88, efficiency was independent of the amount of iodogen in the range from 1 microgram to 15 micrograms, and prolongation of the reaction beyond 1 min failed to improve yields. In contrast, the amount of I- present was decisive. Butanol/NH4OH chromatograms of iodination reactions carried out with 0.6 ng or 20 ng of I- showed 3-4 radioactivity peaks, the relative proportions of which markedly depended on the amount of I- present originally. A link was established between labeling efficiency and chromatographic profile of the I- derivatives formed during oxidation. Dual-label experiments in rats showed that transferrin (20 micrograms) can be labeled using iodogen (1-5 micrograms, 1 min) to behave indistinguishably from its IC1-labeled counterpart. However, prolonged exposure to more oxidant progressively damaged the protein. The damage was independent of substituting I and it manifested itself in increased protein binding to the anion exchange resin, Dowex 1-X8. Over 99.5% of the labeled residues in iodotransferrin were mono- and diiodotyrosines (MIT, DIT). DIT content of the protein increased linearly with the number of I atoms substituted. At comparable levels of substitution, more label was present as MIT after using iodogen than after using IC1. Electrophoretic data are presented regarding homogeneity of the label as obtained after iodinating transferrin by different methods and to varying extents.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.