转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。     

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.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

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.Abbreviations hCG human Chorionic Gonadotrophin - TfR Transferrin Receptor     

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.     

The effect of different iron compounds on transferrin receptor expression in term human cytotrophoblast cells

During pregnancy, the mother is faced with an increased food demand. A good example of this increased demand is iron (Fe). Fe is needed in all growing cells. During pregnancy, the Fe transport to the fetus increases enormously. This amount can easily induce an Fe deficiency in the mother. Fe suppletion is very important for her, but not for the Fe status of the fetus, which is protected against Fe toxicity as well as deficiency. The placenta seems to be autonomous in Fe uptake. Likely there is a regulation mechanism. The human placenta is hemomonochorial. The cell layer of the fetus in contact with the maternal blood is formed by syncytiotrophoblasts. Fe is transported to the placenta by transferrin. Transferrin binds to a transferrin receptor on the trophoblast membrane and is internalized via an endocytic pathway. During this cycle, Fe is released from transferrin and the transferrin-transferrin receptor complex is recycled to the membrane. Isolated trophoblast cells from term placentas form a syncytium in vitro, and transferrin receptors are expressed. Expression depends on the number of cells in culture, culture time, the amount of Fe available, and the Fe compound. By regulation of the number of transferrin receptors, trophoblasts are able to control their Fe uptake.     

Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices

Summary Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells. This work was supported by an operating grant from the Medical Research Council of Canada.     

Influence of transferrin glycans on receptor binding and iron-donation

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, β-galactosidase or N-Acetyl-β-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions. This revised version was published online in November 2006 with corrections to the Cover Date.     

Iron regulation of transferrin synthesis in the human hepatoma cell line HepG2

In human beings, serum transferrin levels increase during iron deficiency and decrease with iron overload. Yet, whether or not iron levels actually affect the synthesis of transferrin in human liver cells is not known. In previous studies, iron was shown to suppress the expression of chimeric human transferrin genes in livers of transgenic mice. The goal of this study was to determine if iron suppresses intact endogenous human transferrin synthesis by testing the effects of changes in iron levels on synthesis of transferrin in a human hepatoma cell line HepG2. In HepG2 cells, normalized(35)S-metabolically labeled transferrin synthesis was consistently less following iron treatment with hemin or ferric citrate, than following treatment with an iron-chelator deferroxamine. Thus, this study provides new evidence that iron can regulate synthesis of intact endogenous human transferrin.     

Abnormal iron delivery to the bone marrow in neonatal hypotransferrinemic mice

Hypotransferrinemic (HP) mice have a splicing defect inthe transferrin gene, resulting in <1% of the normal plasma levels of transferrin. They have severe anemia, suggesting that transferrin is essential for iron uptake by erythroid cells in the bone barrow. To clarify the significance of transferrin on iron delivery to the bone marrow, iron concentration and ⁵⁹Fe distribution were determined in 7-day-old HP mice. Iron concentration in the femur, bone containing the bone marrow, of HP mice was approximately twice higher than in wild type mice. Twenty-four h after injection of ⁵⁹FeCl₃, ⁵⁹Fe concentration in the bone and bone marrow of HP mice was also twice higher than in wild type mice. The present findings indicate that iron is abnormally delivered to the bone marrow of HP mice. However, the iron seems to be unavailable for the production of hemoglobin. These results suggest that transferrin-dependent iron uptake by erythroid cells in the bone marrow is essential for the development of erythrocytes.     

Tertiary structural changes associated with iron binding and release in hen serum transferrin: a crystallographic and spectroscopic study

The iron binding and release of serum transferrin are pH-dependent and accompanied by a conformational change between the iron-bound (holo-) and iron-free (apo-) forms. We have determined the crystal structure of apo-hen serum transferrin (hAST) at 3.5A resolution, which is the first reported structure to date of any full molecule of an apo-serum transferrin and studied its pH-dependent iron release by UV-vis absorption and near UV-CD spectroscopy. The crystal structure of hAST shows that both the lobes adopt an open conformation and the relative orientations of the domains are different from those of apo-human serum transferrin and human apolactoferrin but similar to that of hen apo-ovotransferrin. Spectroscopic analysis reveals that in hen serum transferrin, release of the first iron starts at a pH approximately 6.5 and continues over a broad pH range (6.5-5.2). The complete release of the iron, however, occurs at pH approximately 4.0. The near UV-CD spectra show alterations in the microenvironment of the aromatic residues surrounding the iron-binding sites.     

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 m Pb²⁺ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.     

Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes

Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor.     

Pumping iron: the strange partnership of the hemochromatosis protein, a class I MHC homolog, with the transferrin receptor

People suffering from hereditary hemochromatosis (HH) can not regulate the uptake of iron properly and gradually accumulate iron in their body over their lifetime. The protein involved in HH, HFE, has been recently identified as a class I major histocompatibility complex (MHC) homolog. The wild-type HFE associates and co-traffics with the transferrin receptor (TfR). The mutation responsible for 83% of HH (C260Y) results in the failure of HFE to form a critical disulfide bond, bind β₂ microglobulin, bind TfR, and traffic to the cell surface. In non-polarized cells, the partnership of HFE and TfR results in decreased iron uptake into cells. The mechanism whereby a class I MHC homolog modifies the function of a membrane receptor and how this dynamic complex of molecules regulates iron transport across intestinal epithelial cells is the subject of this review.     

The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain

Brain iron transport and distributional pattern of divalent metal transporter I (DMT1) were studied in homozygous Belgrade rats (b/b) which suffer from a mutation in the DMT1 gene. In adult rats, brain uptake of transferrin-bound iron injected intravenously (i.v.) was significantly lower compared with that in heterozygous Belgrade (+/b) and Wistar rats, whereas transferrin uptake was identical. The difference in iron uptake was not apparent until 30 min after injection. The brain iron concentration was lower, and neuronal transferrin receptor-immunoreactivity higher, in adult b/b rats, thus confirming their iron-deficient stage. Antibodies targeting different sites on the DMT1 molecule consistently detected DMT1 in neurones and choroid plexus at the same level irrespective of strain, but failed to detect DMT1 in brain capillary endothelial cells (BCECs), or macro- or microglial cells. The absence of DMT1 in BCECs was confirmed in immunoblots of purified BCECs. DMT1 was virtually undetectable in neurones of rats aged 18 post-natal days irrespective of strain. Neuronal expression of transferrin receptors and DMT1 in adult rats implies that neurones at this age acquire iron by receptor-mediated endocytosis of transferrin followed by iron transport out of endosomes mediated by DMT1. The existence of the mutated DMT1 molecule in neurones suggests that the low cerebral iron uptake in b/b rats derives from a reduced neuronal uptake rather than an impaired iron transport through the blood-brain barrier.     

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.     

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.