Identification and characterization of the transferrin receptor from Neisseria meningitidis

Expression of the meningococcal transferrin receptor, detected by assay with human transferrin conjugated to peroxidase, was regulated by the level of iron in the medium. The transferrin receptor was identified by SDS-PAGE and Western blot analysis, as a 71,000 molecular weight iron-regulated outer membrane protein in Neisseria meningitidis B16B6. Growth studies with iron-deficient cells and competition binding experiments demonstrated that the meningococcal receptor was species-specific for human transferrin. Reciprocal competitive binding experiments and limited proteolysis of intact cells indicated that the transferrin and lactoferrin receptors are distinct.     

Isolation and characterization of transferrin receptor from embryonic chicken red cell

Transferrin receptor is isolated from the plasma membrane of chicken embryo red cell by affinity chromatography on transferrin-Sepharose 4B matrix. The molecular weight of the protein is approximately 58,000. The purified antibody to this protein is capable of agglutinating chicken embryo red cells, and the purified Fab fragments derived from this antibody are capable of inhibiting the antibody-induced agglutination, as well as the complement-induced hemolysis of chicken embryo red cells. The Fab fragments also inhibit the transferrin-mediated uptake of iron by chicken embryo red cells.     

Identification and isolation of the Leishmania transferrin receptor.

In a previous report, we have presented several lines of evidence, derived from widely different methodologies, suggesting that Leishmania has specific receptors for transferrin with a Kd similar to the mammalian transferrin receptor. This paper describes the identification, purification, and biochemical characterization of Leishmania transferrin receptor. The Leishmania transferrin receptor, detected on intact parasites by immunoperoxidase staining, was first identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using 125I-transferrin, as a 70-kDa protein. It has been isolated initially from Leishmania infantum promastigotes using affinity chromatography on a transferrin-Sepharose column and, subsequently, from Leishmania major promastigotes. The use of polyclonal antisera to the purified 70-kDa Leishmania transferrin receptor and to the purified rat transferrin receptor showed that the two receptors are antigenically distinct. The 70-kDa Leishmania transferrin receptor was subsequently characterized as an integral membrane glycoprotein. The monomeric state of the Leishmania transferrin receptor was demonstrated by gel filtration of purified receptor complexed with 125I-transferrin. Thus, the Leishmania transferrin receptor, unlike the mammalian receptor, is not a disulfide-linked dimer but a single 70-kDa polypeptide.     

Identification and characterization of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae

All six strains of Actinobacillus pleuropneumoniae screened for the ability to use different transferrins as a source of iron for growth were capable of using porcine but not human, bovine, or avian transferrins. A specific binding activity for porcine transferrin (pTf) was expressed in cells grown in the presence of specific iron-chelators and was repressed by addition of excess iron. Two iron-repressible outer-membrane proteins of 105 and 56 kD were specifically isolated from serotype 1, 2 and 7 strains of A. pleuropneumoniae by an affinity-isolation method using biotinylated porcine transferrin and streptavidin-agarose.     

Isolation and characterization of the transferrin receptor from human placenta.

The transferrin receptor has been isolated from human placenta using immunochromatography and affinity chromatography. The receptor is a glycoprotein and has a Mr = 90,000 on sodium dodecyl sulfate-gel electrophoresis in the presence of 2-mercaptoethanol. The isolated receptor is immunologically related to the transferrin receptor on the reticulocyte cell surface.     

Solubilization and characterization of the chicken oocyte vitellogenin receptor.

This paper describes the biochemical characterization of the chicken oocyte plasma-membrane receptor for one of the major lipid-carrying yolk proteins, vitellogenin (VTG). The receptor was extracted from oocyte membranes with the non-ionic detergent octyl-beta-D-glucoside and visualized by ligand blotting, with 125I-VTG as a protein with an apparent Mr of 96000, under non-reducing conditions. It exhibited high affinity for native chicken VTG (Kd 2 X 10(-7) M) but was unable to bind VTG with reductively methylated lysine residues or phosvitin (the phosphoserine-rich intracellular cleavage product of VTG). Polyclonal antibodies to the 96 kDa protein inhibited VTG binding to the receptor and were able to precipitate functional VTG-receptor activity from oocyte-membrane detergent extracts with a concomitant removal of the 96 kDa protein. Antibodies directed against the mammalian receptor for low-density lipoprotein showed cross-reactivity with the chicken oocyte VTG receptor, raising the possibility that lipoprotein receptors in birds are structurally related to those in mammalian species.     

Identification of the O-linked glycosylation site of the human transferrin receptor.

The human transferrin receptor is a glycoprotein containing three N-linked and one O-linked glycosylation sites. Tryptic digestion of the receptor, followed by chromatography on BioGel P-2 and reverse-phase HPLC, yields a glycopeptide (amino acids 101-120) containing the O-linked site. Amino acid sequence analysis reveals that the site of O-glycosylation is Thr-104. Mass spectral analysis is consistent with the presence of a Gal-GalNAc core with predominantly two sialic acid residues.     

Identification and characterization of a chicken alpha-globin enhancer.

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.     

Molecular and cellular characterization of transferrin receptor 2

Iron is an essential component of many biological processes. However, an excess of iron in the body is also toxic; thus, the levels of this element are tightly regulated. Our knowledge of the mechanism by which iron levels are maintained has been bolstered by the dramatic increase in the discovery of novel molecules implicated in iron homeostasis. The transferrin receptor-transferrin pathway is the main mechanism by which cells take up iron. The recently identified homolog of transferrin receptor, its characterization and its role in iron metabolism is the subject of this review.     

Physical characterization of the transferrin receptor in human placentae

The physical properties and binding characteristics of the solubilized transferrin receptor isolated from the placental brush-border membrane of a human trophoblast cell were investigated. The receptor protein was isolated from solubilized 125I-labeled membranes by immunoprecipitation with anti-human transferrin in the presence of saturating amounts of human transferrin. Gel filtration on acrylamide agarose (AcA-22) at 23 degrees C in the absence of transferrin indicates the transferrin receptor has a Stokes radius of 4.6 nm. In the presence of transferrin, the Stokes radius of the receptor shifts to 6.3 nm. Sucrose density centrifugation studies indicate that it has a sedimentation coefficient of 9.8 S in the absence of transferrin and 11.2 S in the presence of transferrin. The molecular weight for the transferrin free receptor is calculated to be 213,000. Upon incubation with transferrin, it increases to 364,000. This is consistent with the idea that the active form of the solubilized receptor is a dimer and the dimer is in turn capable of binding two transferrin molecules.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

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.     

Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site.

Structural studies of the human transferrin receptor have shown that the molecule is a disulfide-bonded dimer consisting of two identical subunits (Mr = 95,000) which are post-translationally modified by the addition of a fatty acyl moiety. Oligonucleotide site-directed mutagenesis has been used to obtain mutant molecules in which each of the four cysteines, residues 62, 67, 89 and 98, clustered within or adjacent to the membrane-spanning region were modified to serine. By first preparing mutants with only one of these cysteine residues modified to serine and then obtaining additional mutants in which different combinations of two cysteine residues were modified, we have shown that both cysteine 89 and cysteine 98, which are located in the extracellular domain of the receptor, are involved in intermolecular disulfide bonds. Further, we have identified cysteine 62 as the major site of acylation. Each of the mutant molecules is synthesized and transported to the cell surface when the modified human transferrin receptor cDNAs are transiently expressed in simian Cos cells. It should therefore now be possible to design experiments to determine whether these modified receptors bind transferrin normally and mediate iron uptake.     

Selection and characterization of transferrin receptor mutants using receptor-specific antibodies

Lymphoma cell lines were selected by growth in transferrin receptor-specific antibodies and in transferrin receptor-specific antibody coupled to ricin toxin. Sequential selections were used to isolate lines with multiple mutations affecting the transferrin receptor molecule. Mutant cell lines were characterized by their growth in antibody and their antibody-binding properties. Two basic types of mutations were found. One type resulted in the loss of a binding determinant for the antibody used for selection on one of the two transferrin receptor allelic products. The other type of mutation resulted in the loss of cell-surface expression of the entire gene product of one of the transferrin receptor alleles.     

Internalization efficiency of the transferrin receptor.

Quantitative ultrastructural and biochemical methods have allowed us to obtain a coherent set of data on the internalization efficiency of the transferrin receptor (TfR). In confluent cell cultures we find that (1) the initial internalization rate of transferrin is approximately 10% per minute, and (2) around 10% of cell-surface TfRs are present in coated pits. From these data a lifetime of coated pits of ca. 1 min is derived. Furthermore, we show that coated pits constitute 1.1-1.4% of the plasma membrane area in confluent cell cultures. Thus, the TfR is concentrated six- to ninefold in coated pits compared to resident plasma membrane proteins. Moreover, we show that the concentration of TfRs in coated pits is cell density dependent, since only around 5% of the receptors are present in coated pits in low-density cultures. Correspondingly, the internalization of TfRs in high-density cell cultures is roughly twice as efficient as that in low-density cell cultures. The reduced TfR internalization efficiency at low cell density is accounted for by a concomitant decrease to 0.55% in the relative surface area occupied by coated pits.