Placental Transferrin Receptor Research Articles

Assessing erythroferrone and iron homeostasis in preeclamptic and normotensive pregnancies: A retrospective study

IntroductionPreeclampsia (PE) is a pregnancy-related disorder associated with maternal hypertension and placental dysfunction. A significant micronutrient during pregnancy is iron, which is important in cellular functions. While iron absorption increases in pregnancy, little is known about the exact mechanisms regulating maternal iron levels and transfer through the placenta in normal and complicated pregnancies. MethodsIn this retrospective study, we investigated the regulation of maternal and placental iron availability and storage, in normotensive and pregnancies complicated by early- or late-onset PE. Methods used were analysis of clinical records, ELISA analysis on plasma samples, immunofluorescent and Prussian Blue analysis on placenta biopsies. ResultsFocusing on erythroferrone (ERFE) as a new marker and hormonal regulator of iron, our results demonstrated altered maternal ERFE levels in PE. We are the first to report the expression of ERFE in trophoblasts and indicate its lower levels in early-onset PE placentas. These changes were associated with lower placental transferrin receptor 1 (TfR1) in syncytiotrophoblasts in both early- and late-onset PE. In addition, maternal plasma ERFE levels were elevated in both early- and late-onset PE and hepcidin levels reduced in early-onset PE. Unaltered maternal plasma IL-6 levels suggest mechanism other than inflammation being involved in altered iron regulation in PE pregnancy. DiscussionOur data supports a deregulation in maternal iron bioavailability in early- and late-onset PE vs normotensive pregnancies. The exact role of placental ERFE in regulating maternal-placental-fetal iron transport axis requires further investigation.

Prepregnancy Obesity Does Not Impact Placental Iron Trafficking

BackgroundIron is critical for fetal development. Neonates of obese women may be at risk for poor iron status at birth as a result of maternal inflammation-driven overexpression of hepcidin. ObjectivesThe objective of this study was to determine differences in placental transfer of oral iron (57Fe) and expression of placental transferrin receptor 1 (TFR1) and ferroportin (FPN) mRNA and protein and their association with maternal and neonatal iron-related parameters, including maternal hepcidin, among women with and without prepregnancy (PP) obesity. Methods57Fe ingested during the third trimester of pregnancy was recovered in venous umbilical cord blood among 20 PP obese [BMI (in kg/m2): 30.5–43.9] and 22 nonobese (BMI: 18.5–29.0) women aged 17–39 y. Placental TFR1 and FPN mRNA and protein expression were quantified via qPCR and Western blot. Maternal and neonatal markers of iron status and regulation, as well as inflammation, were measured. Descriptive and inferential statistical tests (e.g., Student t test, Pearson correlation) were used for data analysis. ResultsThere was no difference in cord blood enrichment of 57Fe or placental mRNA or protein expression of TFR1 or FPN among the women with and without PP obesity. Maternal hepcidin was not correlated with cord blood enrichment of 57Fe or placental FPN mRNA or protein expression. Maternal log ferritin (corrected for inflammation) was inversely correlated with log percent enrichment of 57Fe in cord blood (partial r = –0.50; P < 0.01, controlled for marital status) and protein expression of TFR1 (r = –0.43; P = 0.01). ConclusionsPlacental iron trafficking did not differ among women with and without PP obesity. Findings reinforce the importance of maternal iron stores in regulating placental iron trafficking.

Iron transport across the human placenta is regulated by hepcidin.

Background:Transport of iron across the placenta is critical for appropriate development of the fetus. Iron deficiency during pregnancy remains a major public health concern, particularly in low and middle-income countries, often exacerbated by infectious diseases leading to altered iron trafficking via inflammatory responses. Herein, we investigate the role of hepcidin, a master regulator of iron homeostasis, on regulation of iron transport across trophoblast cells.Methods:We utilized the Jeg-3 choriocarcinoma cell line, for analysis of expression of transferrin receptor, ferritin and ferroportin as well as the export of 59Fe in the presence of hepcidin. Placental tissue from human term pregnancies was utilized for immunohistochemistry.Results:Hepcidin treatment of Jeg-3 cells decreased expression of ferroportin and transferrin receptor (TfR) and reduced the cellular export of iron. Lower expression of TfR on the syncytiotrophoblast was associated with the highest levels of hepcidin in maternal circulation, and ferroportin expression was positively associated with placental TfR. Placentas from small-for-gestational-age newborns had significantly lower levels of ferroportin and ferritin gene expression at delivery.Conclusion:Our data suggest hepcidin plays an important role in the regulation of iron transport across the placenta, making it a critical link in movement of iron into fetal circulation.

Neonatal Iron: Factors Influencing its Level and Associated Complications - a Review Article.

Iron is an essential micronutrient which plays a significant role, particularly vital for early brain growth and function. Maternal iron condition influences the iron status of neonates since iron transferred from the mother is the only source for fetal iron. A depletion in iron as a result of rapid growth leads to iron deficiency which is common in neonates. Although there are inconsistencies with regard to the normal reference ranges for neonatal iron level, the current review summarized literature to provide compressive information about neonatal iron status and factors that influence its level. This is a narrative review on the basis of relevant literatures mainly on neonatal iron from peer-reviewed journals. Electronic databases such as PubMed, PMC, Scopus, Science Direct, Google scholar, Google, and Yahoo were used to retrieve relevant literatures using key terms like "newborn iron, neonatal iron, iron overload, maternal factors, complication, iron level and neonates" separately and in combination. Several factors had been postulated as factors associated with neonatal iron status. The current review figured out that maternal obesity, gestational diabetes mellitus, preterm delivery, placental transferrin receptor, inappropriate iron supplementation, use of iron fortified formula, uses of recombinant erythropoietin therapy, smoking, maternal iron deficiency anemia, umbilical cord clamping, and transfusion are the major factors which can influence neonatal iron level. These factors may have either positive or negative effects on neonatal iron level. Both iron deficiency and iron overload at some stage in the fetal development or at early stage of neonatal development cause abnormal functions of multiple organ system of neonates and subsequently contributed to neonatal and childhood morbidity and mortality. By one and other means insufficient, late and extra maternal iron supplementation, early and delayed umbilical cord clamping have negative effects on the iron level of neonates. Therefore, careful prenatal and antenatal follow-up need to be strengthened with due emphasis for maternal iron assessment.

The impact of maternal obesity on iron status, placental transferrin receptor expression and hepcidin expression in human pregnancy.

Obesity is associated with decreased iron status, possibly due to a rise in hepcidin, an inflammatory protein known to reduce iron absorption. In animals, we have shown that maternal iron deficiency is minimised in the foetus by increased expression of placental transferrin receptor (pTFR1), resulting in increased iron transfer at the expense of maternal iron stores. This study examines the effect of obesity during pregnancy on maternal and neonatal iron status in human cohorts and whether the placenta can compensate for decreased maternal iron stores by increasing pTFR1 expression. A total of 240 women were included in this study. One hundred and fifty-eight placentas (Normal: 90; Overweight: 37; Obese: 31) were collected at delivery. Maternal iron status was measured by determining serum transferrin receptor (sTFR) and ferritin levels at 24 and 34 weeks and at delivery. Hepcidin in maternal and cord blood was measured by ELISA and pTFR1 in placentas by western blotting and real-time RT-PCR. Low iron stores were more common in obese women. Hepcidin levels (ng ml(-1)) at the end of the pregnancy were higher in obese than normal women (26.03±12.95 vs 18.00±10.77, P<0.05). Maternal hepcidin levels were correlated with maternal iron status (sTFR r=0.2 P=0.025), but not with neonatal values. mRNA and protein levels of pTFR1 were both inversely related to maternal iron status. For mRNA and all women, sTFR r=0.2 P=0.044. Ferritin mRNA levels correlated only in overweight women r=-0.5 P=0.039 with hepcidin (r=0.1 P=0.349), irrespective of maternal body mass index (BMI). The data support the hypothesis that obese pregnant women have a greater risk of iron deficiency and that hepcidin may be a regulatory factor. Further, we show that the placenta responds to decreased maternal iron status by increasing pTFR1 expression.

Ovine uterine space restriction alters placental transferrin receptor and fetal iron status during late pregnancy

BackgroundFetal growth restriction is reported to be associated with impaired placental iron transport. Transferrin receptor (TfR) is a major placental iron transporter in humans, but is unstudied in sheep. TfR is regulated by both iron and nitric oxide (NO), the molecule produced by endothelial NOS (eNOS). We hypothesized that limited placental development downregulates both placental TfR and eNOS expression, thereby lowering fetal tissue iron.MethodsAn ovine surgical uterine space restriction (USR) model, combined with multifetal gestation, tested the extremes of uterine and placental adaptation. Blood, tissues, and placentomes from non-space restricted (NSR) singletons were compared to USR fetuses at 120 or 130 days of gestation (GD).ResultsWhen expressed proportionate to fetal weight, liver iron content did not differ while renal iron was higher in USR vs. NSR fetuses. Renal TfR protein expression did not differ, but placental TfR expression was lower in USR fetuses at GD130. Placental levels of TfR correlated to eNOS. TfR was localized throughout the placentome, including the hemophagous zone, implicating a role for TfR in ovine placental iron transport.ConclusionIn conclusion, fetal iron was regulated in an organ-specific fashion. In USR fetuses, NO-mediated placental adaptations may prevent the normal upregulation of placental TfR at GD130.

Placental Iron Transport and Maternal Absorption

The iron need in pregnancy is significantly higher in comparison to that in the nonpregnant state. The iron absorbed during pregnancy is used for expansion of the maternal erythrocyte mass, to fulfill the fetus’s iron needs, to create placenta, and to cope with blood loss at delivery. Term neonates have a total body store of about 1 g of iron, all derived from the mother. Despite the overall increase in nutritional requirements, biochemical, metabolic, and physiological adjustments of the maternal organism happen in order to meet the extra demands and to support the homeostasis of iron. In all healthy pregnant women with sufficient iron stores, the increased iron absorption is coupled with the mobilization of iron stores. Unfortunately, iron deficiency during pregnancy is alarmingly common. The function of placental transport determines the composition of umbilical cord blood providing nutrients and oxygen to the fetus to ensure appropriate fetal growth. Iron in the developing fetus is accumulated against a concentration gradient and, in the case of maternal iron deficiency, the placenta can protect the fetus significantly through the increased expression of placental transferrin receptor together with a rise in divalent metal transporter 1 (DMT1). Despite the resistance of the fetus to maternal deficiency, any stress that alters placental development or function may have consequences for the developing fetus. Despite its central importance in fetal development, little is known about the mechanism of iron transfer across the placenta. Consequently, it is crucial to understand the molecular basis of placental iron transport in order to optimize the iron intake recommendation, reducing adverse pregnancy outcomes for both the mother and her child.

Placental Transferrin Receptor Expression with Uterine Space Restriction in Ovine Pregnancy.

Placental Transferrin Receptor Expression with Uterine Space Restriction in Ovine Pregnancy.

Impact of maternal and neonatal iron status on placental transferrin receptor expression in pregnant adolescents

Objective To elucidate the role of maternal and neonatal iron status on placental transferrin receptor (TfR) expression. Study design and outcomes Ninety-two healthy pregnant adolescents (ages 14–18 years) were followed across pregnancy. Maternal iron status (hemoglobin, hematocrit, serum ferritin, TfR, and total body iron) was assessed in mid-gestation (21–25 wks) and at delivery in the mother and neonate. Placental TfR protein expression was assessed by western blot in placental tissue collected at delivery. Results Placental TfR expression was inversely associated with maternal iron status at mid-gestation (hemoglobin p = 0.046, R 2 = 0.1 and hematocrit p = 0.005, R 2 = 0.24) and at delivery (serum ferritin p = 0.02, R 2 = 0.08 and total body iron p = 0.02, R 2 = 0.07). Mothers with depleted body iron stores had significantly greater placental expression of TfR than mothers with body iron stores greater than zero ( p = 0.003). Neonatal iron stores were also inversely associated with the expression of placental TfR ( p = 0.04, R 2 = 0.06). Neonates with serum ferritin values ≤34 μg/L had significantly greater protein expression of placental TfR compared to neonates with cord serum ferritin values >34 μg/L ( p = 0.01). Conclusions Expression of placental TfR is associated with both maternal and neonatal iron demands. Increased expression of placental TfR may be an important compensatory mechanism in response to iron deficiency in otherwise healthy pregnant women.

Role of Maternal/Fetal Iron Status on Placental Transferrin Receptor Expression

The placenta plays an important role in mediating iron transfer to the fetus. However, the responsiveness of the placenta to maternal and fetal demands remains unclear. Placental transferrin receptor (TfR) is a key placental iron transport protein on the apical membrane. Placentas were collected from twelve pregnant adolescents (38‐40 wks gestation) and protein expression of TfR was examined. Iron status indicators were assessed from maternal blood obtained during the second trimester and at delivery. Cord blood samples were also collected at birth. There was a significant inverse relationship between cord serum ferritin (SF) and placental TfR expression (P=0.042). A non‐significant inverse trend was detected between maternal SF in the second trimester and placental TfR expression (P=0.075). Relationships between maternal SF in the second trimester and cord SF approached significance (P=0.055). However, maternal SF at birth was not significantly related to placental TfR expression or cord SF. Hemoglobin and TfR in both the infant and mother were not significantly related to placental TfR expression. In conclusion both maternal and fetal iron status appear to impact placental expression of TfR. Studies are on‐going to assess the impact of maternal iron status on neonatal iron stores at birth in the planned cohort of 300 pregnant teens.Grant Funding SourceSupported by USDA Grant no. 2008‐0857.

Structural and Functional Stability of the Mature Transferrin Receptor from Human Placenta

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65°C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.

Increased Placental Iron Regulatory Protein-1 Expression in Diabetic Pregnancies Complicated by Fetal Iron Deficiency

Placental transferrin receptor (TfR) protein expression is increased in diabetic pregnancies that are complicated by low fetal iron stores, suggesting regulation of placental iron transport by fetoplacental iron status. In cell culture, iron homeostasis is regulated by coordinate stabilization of TfR mRNA and translation inactivation of ferritin mRNA by iron regulatory proteins (IRP-1 and -2) which bind to iron-responsive elements (IREs) on the respective mRNAs. Concentrations of IRP-1, IRP-2 and TfR mRNA were measured in 10 placentae obtained from diabetic and non-diabetic human pregnancies with a wide range of fetoplacental iron status. IRP-1 activity was present in human placenta and correlated closely with TfR mRNA concentration (r=0.82;P=0.007). IRP-2 activity and protein were not detected. In a second experiment, placentae were collected from 12 diabetic pregnancies, six with low fetal cord serum ferritin and placental non-heme iron concentrations, and six with normal iron status. IRP-1 activity and TfRBmaxfor diferric transferrin were greater in the iron-deficient group (P<0.05). IRP-1 activity correlated inversely with cord serum ferritin (r=0.75;P<0.01) and placental non-heme iron (r=0.61;P=0.05) concentration. Placental IRP-1 activity is directly related to TfR mRNA concentration and is more highly expressed in iron-deficient placentae. The study provides direct in vivo evidence for IRP regulation of TfR expression in the human placenta.

Functional reconstitution of the human placental transferrin receptor into phospholipid bilayers leads to long tubular structures proceeding from the vesicle surface.

We have reconstituted the human placental transferrin receptor (hTfR) into phospholipid vesicles using either dialysis, dilution, or gel filtration. Several different detergents and phospholipids and a variety of lipid-to-protein weight ratios were tested. Preformed vesicles as well as detergent-solubilized phospholipids were used. Reconstituted mixtures were fractionated by sucrose density gradient centrifugation and screened for ferritransferrin binding activity, and peak fractions were analyzed by electron microscopy. The efficiency of reconstitution was strongly influenced by the choice of the phospholipids used and the reconstitution method. Best results were obtained when hTfR was dissolved with octylpolyoxyethylene, mixed with detergent-solubilized soy bean lecithin, and reconstituted by slow dialysis. The data show that hTfR capable of binding ferritransferrin was reconstituted into vesicles with an irregular surface and many protrusions. In addition the reconstitution of hTfR resulted in the formation of tubular structures proceeding from the vesicle surface, which may serve as an in vitro model for future studies on the relevance of self-assembly processes for cellular endocytosis.

Placental transferrin receptor in diabetic pregnancies with increased fetal iron demand.

Augmented fetal hemoglobin synthesis during diabetic pregnancy increases fetal iron demand. To study the effect of increased fetal iron demand on placental transferrin receptor (TR), we utilized a monoclonal antibody to localize placental TR immunoreactivity and 125I-labeled transferrin to study TR binding characteristics in 10 placentas from poorly controlled diabetic mothers with increased fetal iron demand and 10 placentas from nondiabetic mothers. The infants born to the diabetics had higher cord serum C-peptide, erythropoietin, and hemoglobin concentrations, indicating fetal hyperinsulinemia and hypoxia, with augmented erythropoiesis and iron demand. TR immunoreactivity was localized to the syncytiotrophoblast in both groups, was greater in the diabetic group, and was inversely correlated with fetal storage iron (r = -0.75; P < 0.001). Scatchard analysis of 125I transferrin binding data confirmed greater receptor number (Bmax 17.9 +/- 2.2 vs. 12.6 +/- 1.3 pM/mg protein, P = 0.05), but reduced binding affinity [dissociation constant (Kd) 7.6 +/- 0.9 vs. 5.4 +/- 0.4 nM/l, P = 0.03] in the diabetic group. The TR staining intensity, Bmax, and Kd were each correlated with cord C-peptide, suggesting either a primary or secondary role for fetal hyperinsulinemia in TR expression. This study provides in vivo evidence that fetal factors, such as iron demand or hyperinsulinemia, influence regulation of placental TR in humans. The increase in placental syncytiotrophoblastic TR expression associated with reduced cord serum ferritin concentration suggests that the fetus utilizes both increased placental iron transport and mobilization of fetal iron stores to support augmented fetal erythropoiesis.

The binding of human serum transferrin to its specific receptor reconstituted into liposomes

Human placental transferrin receptor (HPTR), purified following a procedure based on affinity chromatography step, was reconstituted by the detergent dialysis method into various kinds of phosphatidylcholine vesicles and the receptor ability to bind 125I-labeled human serum transferrin (HST) was then evaluated. In our experimental conditions, the binding of the labelled protein to its specific receptor showed several features, in particular: (1) in cholesterol/1-α-dipalmitoyphosphatidyl choline (CHO/DPPC) liposomes, a positive cooperativity of the transferrin binding resulted at the lowest cholesterol/phospholipids (C/P) ratio; 1-α-dioleylphosphatidyl choline (DOPC) and phosphatidic acid (PA) containing liposomes showed an opposite binding curve trend; (2) the apparent dissociation constant ( K′ d) did not change significantly as a function of the lipid composition, being always around 1.00 × 10 −6 M; (3) the encapsulation capacity of liposomes decreased from 27% to about 13% with increasing amounts of cholesterol and was around 20% in the presence of DOPC or PA; about 8–13% of this receptor was found to be functional; (4) receptor-loaded liposomes treated with polyclonal anti-HPTR rabbit antibodies showed a remarkable binding decrease for transferrin. All these results seem to point out the crucial role played by the environment in the binding behaviour of the transferrin receptor.

Enzymatic Modeling of the Oligosaccharide Chains of Glycoproteins Immobilized onto Polystyrene Surfaces

A method for the modification of the oligosaccharide moiety of even small amounts of purified glycoproteins by enzymatic glycosylation and deglycosylation is described. The method includes noncovalent immobilization of the glycoproteins onto the polystyrene surface of the wells of microtiter plates used as reaction tubes, deglycosylation or glycosylation by incubation either with exoglycosidases or endoglycosidases or with glycosyltransferases, and the characterization of the modified glycan structures by probing them with lectins. Placental transferrin receptor employed as a model glycoprotein was modified in amounts of as little as 100 ng removing sialic acid residues, hybrid-type glycans or all types of N-glycans with neuraminidase, endo-β-N-acetylglucosaminidase H or peptide-N4-(acetyl-β-glucosaminyl) asparagine amidase. Asialotransferrin receptor was α-2,6-sialylated with α-2,6-sialyltransferase from rat liver, but could not be α-2,3-sialylated with α-2,3-sialyltransferase from porcine liver. Changes in the structure and in the relative amount of the oligosaccharides could be monitored semiquantitatively with high sensitivity by the binding of digoxigenin-labeled lectins and anti-digoxigenin Fab fragments. The method is easy to use, does not require immobilization of the enzymes employed, offers simple separation of the enzymes and the product, and leaves the protein intact for further studies.

The human placental transferrin receptor: reconstitution into liposomes and electron microscopy.

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.

Structure of the N-linked oligosaccharides of the human transferrin receptor.

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (alpha 2-3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species.

トランスフェリン・レセプターの新しい精製方法とその生化学的性質

An improved method for the purification of human placental transferrin receptor (Tf-R) was developed. Fresh human placenta was homogenized in cold acetone and the acetone powder was prepared. After the acetone powder had been washed with HEPES buffer, the insoluble proteins containing Tf-R were separated by centrifugation and dissolved in Emulgen 109P-containing buffer. Tf-R was collected by affinity binding to Tf-Sepharose and extracted by consecutive treatment with 4 different kinds of buffers. Tf-R was eluted by buffer C (2 M KCl) and buffer D (0.5 M NaSCN). Tf-R was characterized as a 90-kDa monomer on gradient SDS-PAGE (4-20%) in the presence of 2-mercaptoethanol. Though there were several minor bands of 180- and above 205-kDa, all these bands were confirmed as Tf-R by Western blotting using an anti-Tf-R monoclonal antibody (OKT 9). The apparent molecular weights, measured by HPLC using a TSK-G 3,000 SW column, demonstrated that Tf-Rs eluted with buffer C were approximately 370-, 500- and above 500-kDa, but only a peak of above 500-kDa was found in Tf-R eluted with buffer D. Although the polymers of Tf-R with molecular weight of above 500-kDa were resistant to trypsin digestion, the Tf-R of 370-kDa was resistant to the enzyme only when it conjugated to the diferric Tf. The stability of the polymers of above 500-kDa to trypsin digestion suggested an advantage for the repeated use of Tf-R in the endocytosis of diferric Tf, which was performed by the translocation of Tf-R between cell surface and intracellular vesicles.

Serum transferrin receptor is a truncated form of tissue receptor.

Recent studies have provided immunological evidence for the existence of transferrin receptor in human serum and have revealed that its concentration is a sensitive measure of erythropoiesis and iron deficiency. The present study was undertaken to establish the molecular identity of this immunoreactive component. Purification from human serum was accomplished by immunoaffinity chromatography using, as the ligand, monoclonal antitransferrin receptor antibody. The receptor preparation contained two major components with Mr of 75,000 and 85,000, which were identified as transferrin and transferrin receptor, respectively. The physicochemical and immunochemical properties of the 85,000 serum receptor were compared with those established for intact placental transferrin receptor. The serum receptor exhibited an apparent Mr = 85,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, as compared with 190,000 for placental transferrin receptor. Upon reduction, the Mr of serum receptor was unaltered, whereas, the 190,000 placental receptor dimer decreased to the expected monomer value of 95,000. Amino-terminal amino acid sequence analysis revealed that residues 1-19 of serum receptor were identical to residues 101-119 of intact receptor. These findings provide physicochemical evidence for the existence of transferrin receptor in human serum, establish its molecular identity as a truncated form lacking the cytoplasmic and transmembrane domains (residues 1-100) of intact receptor, and demonstrate that it exists as a transferrin-receptor complex in serum.