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.     

Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta

The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin.     

Brefeldin a down-regulates the transferrin receptor in K562 cells

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance k_e, from 0.28 min^–1 to 0.43 min^–1, while BFA had little effect (K_e=0.20 min^–1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a slow-release, monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.Abbreviations BFA brefeldin A - ARF ADP-ribosylation factor - HRP horseradish peroxidase - Tf transferrin - PMA phorbol 12-myristate 13-acetate - DMSO dimethyl sulfoxide - PBS phosphate-buffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin - FITC-Tf fluorescein isothiocyanate-labelled transferrin     

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.     

Purification and molecular identification of the human hyaluronan receptor for endocytosis

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.     

Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.     

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.     

Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology

The plant CDK inhibitor ICK1 was identified previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauliflower mosaic virus 35S promoter. In comparison to control plants, growth was significantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modifications in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.     

Expression of the chicken hepatic glycoprotein receptor in Xenopus oocytes: conservation of ligand and receptor targeting signals.

We have obtained expression of the beta-N-acetylglucosamine-binding receptor from chicken hepatocytes in Xenopus oocytes by injecting mRNA synthesized in vitro from a full length cDNA cloned into an expression vector (Mellow et al: J. Biol Chem 263: 5468-5473, 1988). Immunoprecipitation of the receptor after labeling of oocytes with [35S]-methionine for times ranging from 6 to 72 h revealed 4-5 closely spaced bands of 25-30 kDa after SDS-PAGE. Although these bands were largely resistant to endoglycosidase H cleavage, endoglycosidase F reduced the size of all bands to a single species at 23-24 kDa, indicating that they resulted from heterogeneity in glycosylation of a single polypeptide. Incubation of oocytes expressing this receptor with [125I]-GlcNAc-BSA resulted in 1.8 to 10 x higher levels of cell-associated ligand in mRNA-injected vs. water-injected control oocytes, 2-35% of cell-associated counts was removed by EGTA rinse at 20 degrees C, suggesting that most ligand was inaccessible (presumably intracellular). Immunoprecipitation of sucrose gradient fractions detected receptor molecules predominantly in a light organelle at 1.09-1.12 g/cc (the density of early endosomes and plasma membrane vesicles), with no evidence of the receptor in much heavier yolk platelet fractions even in the presence of ligand. In contrast, internalized [125I]-GlcNAc-BSA was found either at the top of the gradients or in organelles at 1.09-1.17 g/cc and in yolk platelets. TCA precipitation indicated that much intracellular ligand was degraded to acid-soluble fragments. Addition of vitellogenin (the yolk protein precursor) to the medium together with the [125I]-GlcNAc-BSA shifted much of the ligand into yolk platelets. These data indicate that the chicken glycoprotein receptor expressed in oocytes mediates binding and internalization of this ligand into an organelle in which ligand-receptor dissociation occurs, allowing for separation of these two molecules into different compartments. The behavior of ligand in Xenopus oocytes expressing the chicken receptor closely resembles its behavior in hepatocytes.     

Distribution of transferrin and transferrin receptor of the eype 1 in the process of formation of the rat eye retina in early postnatal ontogenesis

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type 1 (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layer (in the neuroblast layer-NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retina layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retina is discussed.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.     

A human transferrin-binding protein of Staphylococcus aureus is immunogenic in vivo and has an epitope in common with human transferrin receptor

To understand human immune responses against the human transferrin-binding protein of Staphylococcus aureus (SA-tbp), we examined cell wall proteins from S. aureus ATCC 6538 using human convalescent sera, and a monoclonal antibody specific for human transferrin receptor (McAb-HTR). The SA-tbp, detected by immunoblot assay, was iron-repressible, reacted with the convalescent sera, and cross-reacted with McAb-HTR. Immunoelectron microscopy probed with McAb-HTR showed a reaction zone around the test strain from the deferrated BHI. After being preincubated with an S. aureus -bacteremic serum, the electroblot of the SA-tbp still reacted with McAb-HTR, but not with human transferrin-horseradish peroxidase conjugate. We conclude, there are at least two kinds of epitopes in the SA-tbp; one able to bind to human transferrin is immunogenic in humans, but the other sharing epitopes common with human transferrin receptor is not immunogenic in humans.     

Characterization of lactoferrin receptor in brain endothelial capillary cells and mouse brain

We present, herein, the evidence for lactoferrin (Lf) binding sites in brain endothelial capillary cells (BCECs) and mouse brain. The results from confocal microscopy showed the presence of Lf receptors on the surface of BCECs and the receptor-mediated endocytosis for Lf to enter the cells. Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs (high affinity: dissociation constant (K (d)) = 6.77 nM, binding site density (B (max)) = 10.3 fmol bound/mug protein; low affinity: K (d) = 4815 nM, B (max) = 1190 fmol bound/mug protein) and membrane preparations of mouse brain (high affinity: K (d) = 10.61 nM, B (max) = 410 fmol bound/mug protein; low affinity: K (d) = 2228 nM, B (max) = 51641 fmol bound/mug protein). The distribution study indicated the effective uptake of (125)I-Lf in brain after intravenous administration. The present study provides experimental evidence for the application of Lf as a novel ligand for brain targeting.     

Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responses

The trafficking of receptor tyrosine kinases (RTKs) to distinct subcellular locations is essential for the specificity and fidelity of signal transduction and biological responses. This is particularly important in the PNS and CNS in which RTKs mediate key events in the development and maintenance of neurons and glia through a wide range of neural processes, including survival, proliferation, differentiation, neurite outgrowth, and synaptogenesis. The mechanisms that regulate the targeting of RTKs to their subcellular destinations for appropriate signal transduction, however, are still elusive. In this review, we discuss evidence for the spatial organization of signaling machinery into distinct subcellular compartments, as well as the role for ligand specificity, receptor sorting signals, and lipid raft microdomains in RTK targeting and the resultant cellular responses in neural cells.     

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.     

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.     

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.