Relationship between transferrin C2 and birth weight

In previous investigations transferrin C2 has been found to be associated with spontaneous abortion and premature birth. In a study of newborn infants from northern Sweden, no significant correlation was found between birth weight and transferrin C2 of the infants. Thus transferrin C2 appears to be associated with reproductive disturbances and with premature birth, but not with birth weight in mature infants.     

Subtypes of transferrin C.

Subtypes of transferrin C were studied by means of isoelectric focusing after complete desialylation of transferrin. Family data were consistent with an autosomal co-dominant mode of inheritance. Studies of serum samples from 75 individuals heterozygous for C and another (B or D) variant showed that the genes (C1 and C2) controlling the C subtypes are allelic to the B and D genes. The C2 gene frequency in Swedes and Swedish Lapps was similar to that found previously in Danes and Germans.     

Transferrin C2 and radiation-induced chromosomal damage

Radiation-induced chromosomal damage (after exposure to 1 Gy) in lymphocytes was studied in relation to transferrin C subtype (C1 vs. C2). In 72-hour lymphocyte cultures a significantly increased frequency of cells with radiation induced aberrations was observed in individuals with the transferrin type C2. Thus the results lend some support to the hypothesis that transferrin C2 may act as an enhancer of chromosomal damage.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

Transferrin subtypes and variants in Germany; Further evidence for a Tf null allele

Summary Isoelectric focusing (IEF) with carrier ampholytes was used for the determination of transferrin C subtypes and transferrin B and D variants in a sample of 1125 unrelated individuals from Southern Germany. The observed TfC allele frequencies were Tf*C1=0.7872, Tf*C2=0.1365, and Tf*C3=0.0675. The rare C subtype C6 was observed twice. A new C subtype, called C10, was observed and identified by IEF with immobilized pH gradients. The rare C subtypes C4 and C8 were also studied by this method. TfB and TfD variants were found with a heterozygous frequency of 1.53%. One new TfD was found which is located between D1 and D2 and therefore named D1-2. Evidence for a Tf null allele was obtained in a child and the putative father; they were considered to be heterozygous for an allele Tf0. The theoretical exclusion rate for paternity examinations was calculated for the Tf system and found to be 17.95%.     

Distribution of transferrin C subtypes among the Bedouin and non-Bedouin populations of Jordan

The distribution of transferrin C subtypes was studied in the population of Jordan. The sample comprised 121 Bedouins and 382 other Jordanians from Amman. The frequency of TfC2 was found to be 0.26 among the Bedouins and 0.23 among the non-Bedouin population. TfD was present in low frequency (0.005) among both the Bedouin and non-Bedouin populations.     

Transferrin C subtypes and occupational photodermatosis of the face

In a factory in northern Sweden where 120 workers were uniformly exposed to photoactive substances 73 developed occupational facial eczema while 47 showed no reaction. The workers were examined with respect to 16 genetic marker systems: HLA, blood groups (ABO, Rh, MNSs, P, K, Le and Fy) and serum groups (Hp, Tf, Gc, Pi, Bf, C3, C4 and C6). Between reactors and nonreactors the following differences were found: (1) a significant decrease (p less than 0.05) of HLA A11 among the reactors; (2) a significant increase (p less than 0.05) of the C3 FS type among the reactors; (3) a highly significant increase (p less than 0.001) of the transferrin C2 gene and of the C2 variant among the reactors. The association with Tf C2 remained significant also after correction for number of significance tests. Since transferrin (iron) is known to catalyze the formation of hydroxyl radicals we hypothesize that the Tf C2 variant is more efficient in promoting radical formation and thereby cell damage. Other results supporting the notion that transferrin C2 may be associated with an increased susceptibility to toxic damage are discussed.     

Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.     

Serum protein polymorphisms in a Liberian population

Serum protein variations were studied in a Liberian population living in Buchanan town. Of the alpha 1-antitrypsin genes only M1 and M3 were polymorphic. The frequencies of the haptoglobin and Gc genes were in accordance with earlier known estimates in African populations. There was, however, a relatively low frequency of Hp 0 which may be related to the low malarial parasite prevalence in this group. The transferrin C2 gene was found in a significantly lower frequency among Liberians compared to European and Asiatic populations. A new transferrin variant was observed by isoelectric focusing. This variant could not be identified with conventional starch or polyacrylamide electrophoresis.     

Transferrin subtypes in Iran

The frequency of transferrin Tf C subtypes has been determined by double one-dimensional electrophoresis of plasma samples from Moslems (n = 91), Zoroastrians (n = 97), Jews (n = 88) and Armenians (n = 88) of Iran. The Zoroastrians show the lowest frequency of TfC1 (0.4999) and highest frequencies of TfC2 and TfC3 (.02215, and 0.2783, respectively). The Jews have the highest TfC1- and the lowest TfC2- and TfC3 frequencies (0.8011, 0.1478, and 0.0512, respectively). It could be shown that the differences between Zoroastrians and Jews are highly significant (p less than 0.001). Arbitrary subtyping of transferrin Tf B and TfD phenotypes could be done on samples from three regional groups of Iran: North: n = 282, Central: n = 548, and South: n = 587 into Tf B (Iran 1, 2, 3 and 4) and Tf D (Iran 1, 2 and 3) was performed according to mobilities relative to the transferrin C protein during polyacrylamide gel electrophoresis and by relative pI deviations from the Fe2-transferrin C1 protein after isoelectric focussing. The allele frequencies found in the total sample (n = 1417) are: TfB1 = 0.0003, TfB2 = 0.0010, TfB3 = 0.0042, TfB4 = 0.0007; TfD1 = 0.0017, TfD2 = 0.0014, and TfD3 = 0.0010.     

Tyrphostin A8 stimulates a novel trafficking pathway of apically endocytosed transferrin through Rab11-enriched compartments in Caco-2 cells

The potential application of transferrin receptors as delivery vehicles for transport of macromolecular drugs across intestinal epithelial cells is limited by several factors, including the low level of transferrin receptor-mediated transcytosis, particularly in the apical-to-basolateral direction. The GTPase inhibitor, AG10 (tyrphostin A8), has been shown previously to increase the apical-to-basolateral transcytosis of transferrin in Caco-2 cells. However, the mechanism of the increased transcytosis has not been established. In this report, the effect of AG10 on the trafficking of endocytosed transferrin among different endosomal compartments as well as the involvement of Rab11 in the intracellular trafficking of transferrin was investigated. Confocal microscopy studies showed a high level of colocalization of FITC-transferrin with Rab5 and Rab11 in Caco-2 cells pulsed at 16 degrees C and 37 degrees C, which indicated the presence of apically endocytosed FITC-transferrin in early endosomes and apical recycling endosomes at 16 degrees C and 37 degrees C, respectively. The effect of AG10 on the accumulation of transferrin within different endosomal compartment was studied, and an increase in the transcytosis and recycling of internalized (125)I-labeled transferrin, as well as a decrease in cell-associated (125)I-labeled transferrin, was observed in AG10-treated Caco-2 cells pulsed at 37 degrees C for 30 min and chased for 30 min. Moreover, confocal microscopy showed that FITC-transferrin exhibited an increased level of colocalization with Rab11, but not with Rab5, in the presence of AG10. These results suggest an effect of AG10 on the later steps of transferrin receptor trafficking, which are involved in subsequent recycling, and possibly transcytosis, of endocytosed transferrin in Caco-2 cells.     

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.     

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.     

人血清转铁蛋白遗传多态性与疾病相关的研究

本文应用等电聚焦电泳技术,调查了广州地区128例白血病、80例原发性肝癌、52例系统性红斑狼疮和1456例正常人的Tf遗传多态性分布。与正常人组相比,急性粒细胞白血病组的Tf~(C_1)基因频率显著增高(P<0.05);TfC_1C_1表型频率也显著高于正常人组(P<0.05),TfC_1C_1人群的患病相对危险率为1.9。未发现急性淋巴细胞白血病、慢性粒细胞白血病、原发性肝癌疾病组的Tf表型和基因频率与正常人组有显著性统计学差异。此外,还发现系统性红斑狼疮组的Tf~(C_2)基因频率显著高于正常人(P<0.025),Tf~(C_1)频率则相应下降(P<0.05);表型频率TfC_1C_2显著增高(P<0.005),相对危险率为2.3,TfC_1C_2相应下降(P<0.01)。     

Large cooperativity in the removal of iron from transferrin at physiological temperature and chloride ion concentration

Iron removal from serum transferrin by various chelators has been studied by gel electrophoresis, which allows direct quantitation of all four forms of transferrin (diferric, C-monoferric, N-monoferric, and apotransferrin). Large cooperativity between the two lobes of serum transferrin is found for iron removal by several different chelators near physiological conditions (pH 7.4, 37 °C, 150 mM NaCl, 20 mM NaHCO₃). This cooperativity is manifested in a dramatic decrease in the rate of iron removal from the N-monoferric transferrin as compared with iron removal from the other forms of ferric transferrin. Cooperativity is diminished as the pH is decreased; it is also very sensitive to changes in chloride ion concentration, with a maximum cooperativity at 150 mM NaCl. A mechanism is proposed that requires closure of the C-lobe before iron removal from the N-lobe can be effected; the open conformation of the C-lobe blocks a kinetically significant anion-binding site of the N-lobe, preventing its opening. Physiological implications of this cooperativity are discussed.     

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

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.     

Effect of reduced atmospheric pressure and fasting on transferrin synthesis in the rat

The concentrations of transferrin and albumin in the blood serum and microsomal fraction of the liver and the incorporation of [¹⁴C] leucine into the proteins were measured in rats which were fasted while exposed to ambient atmospheric pressure or to a pressure of one-half atmosphere. The rates of protein synthesis were estimated in a relative manner from the ratio of ¹⁴C incorporation into the two proteins and in an absolute manner using the liver free ¹⁴C and leucine concentrations to measure the specific activity of the precursor pool. Fasting at ambient pressure was accompanied by a decrease in the serum and microsomal concentrations of transferrin but not of albumin and by a marked decrease in the relative and absolute synthesis rates of transferrin. By contrast, fasting at reduced ambient pressure was associated with an increase in the serum transferrin concentration and in the relative and absolute rates of synthesis of the protein. It is concluded that fasting in the rat produces a much greater decrease in the rate of synthesis of transferrin than of albumin and that exposure to reduced ambient pressure stimulates transferrin synthesis but not albumin synthesis.