Red cell ferritin and iron storage during chick embryonic development

Ferritin, the iron storage protein, is at least 10 times as abundant in the circulating primitive red cells of the chick embryo as in the circulating definitive red cells of adult roosters. The decline in the ferritin content of the circulating red cells in the embryo corresponded to the replacement of primitive red cells by definitive red cells, monitored by the disappearance of primitive and embryonic hemoglobins. Iron concentrations in the yolk, the major nutrient storage site, changed little during the period when ferritin was lost from the circulating red cells. The storage of iron in the ferritin of the primitive red cells and the preferential loss of the stored red cell iron that was observed in chickens also occur in mice and bullfrogs, which suggests a special role for red cell ferritin in developing animals.     

The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis

The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.     

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.     

Receptor-mediated endocytosis and exocytosis of transferrin in Concanavalin A-stimulated rat lymphoblasts

Iron-loaded transferrin has been shown to be necessary for the support of cell proliferation in culture. This function depends upon interaction of transferrin with a specific high-affinity cell surface receptor. The present report is directed toward determining the consequences of the interaction of transferrin with this receptor on Concanavalin A-stimulated rat lymphocytes. Three specific questions have been posed: (a) Is transferrin endocytosed following binding to its specific receptor in a temperature-dependent fashion? (b) Following endocytosis, is the carrier protein released from the cell in a structurally and functionally intact form? and (c) Is the cell surface transferrin receptor also endocytosed following ligand binding? The results provide affirmative answers to all questions. Using two independent probes of the cell surface versus intracellular location of transferrin we observed that cell-bound transferrin moved from the cell surface to the inside of the cell and subsequently back to the medium. This process occurred in a temperature-dependent fashion. When cells containing only intracellular transferrin were further incubated at 37°C approximately 80% of cell-bound transferrin was released to the medium. Nearly all of this material retained reactivity with antibody to transferrin. In addition, exocytosed transferrin exhibited qualitatively and quantitatively equivalent binding reactivity with the transferrin receptor and showed identical electophoretic mobility on SDS gel electrophoresis. Finally, using similar methodology to that employed with transferrin itself, we provide evidence that the specific receptor is also endocytosed.     

Plasma cell antigen PC-1 and the transferrin receptor in mouse, rat, and hamster: serologic and biochemical analysis

The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin are high m.w., developmentally regulated proteins consisting of two similar or identical disulfide-bonded subunits. In this paper, we report the results of a serologic and biochemical analysis of these proteins in various strains of inbred mice, and in rats and hamsters. A monoclonal antibody against the PC-1a allelic product is shown to detect an antigenic determinant on the PC-1 molecule that has the same strain distribution as the antigen previously detected with polyclonal alloantisera. The mouse PC-1 protein was purified from plasma cells of the PC-1a genotype and was used to generate polyclonal rabbit anti-PC-1 antibodies. These antibodies precipitated a homologous protein from plasmacytoma cells derived from PC-1- congenic mice, demonstrating that PC-1b is not a "null" allele. The PC-1b allelic product had a slightly lower apparent m.w. than the PC-1a product, and had a slightly more basic isoelectric point. Rabbit anti-mouse PC-1 antibodies also precipitated a homologous protein from immunoglobulin-secreting cells of rat and hamster origin, but did not show detectable cross-reaction with the transferrin receptor. Disulfide bonding between chains was conserved in both PC-1 and the transferrin receptor in all species examined, but transferrin receptors from mouse cells had a significantly higher apparent m.w. than those of rat, hamster, or human cells.     

Ontogenetic changes of circulating erythroid cells and haemoglobin components in Esox lucius

Using light microscopy the morphology, the mitotic index and levels of erythroid cell types were detected from 48 h pike Esox lucius embryos before hatching to adult specimens. At the same developmental stages, the haemoglobins and globin chains expressed were electrophoretically characterized. The erythroid cells of the primitive generation were the most abundant from 48 h before hatching until 15–20 days after hatching, then their number decreased and only rare cells remained in the 3 month‐old juvenile specimens. These cells divided and differentiated in the blood and were substituted by the definitive erythrocyte series. As in other vertebrates, the immature cells of the two generations differed in morphological properties and in the synthetized haemoglobin. The circulating erythroid cells of the definitive population cell lineage were, at all differentiation stages, smaller than those of the primitive generation. The definitive erythrocytes appeared in blood smears of 7 days post‐hatching larvae, they increased rapidly and at 20 days they represented the predominant red blood cell population in the circulation of young pike. Electrophoretic analysis of haemolysates obtained from different developmental stages indicated the presence of distinct embryonic, larval and adult haemoglobins. The embryonic haemoglobins differed from those of the older larva and juvenile specimens and were detectable within the first week of post‐hatching development when only primitive erythrocytes were present in the blood.     

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

Co-migration and internalization of transferrin and its receptor on K562 cells

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC- transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.     

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

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

Turnover of the transferrin receptor is not influenced by removing most of the extracellular domain

We treated intact cells with trypsin to remove most of the external domain of the transferrin receptor and investigated what effect the absence of the external domain had on the turnover of the fragment that remained associated with the cells. To detect the cell-associated tryptic fragment, which contains a small amount of the external domain, the transmembrane domain, and the cytoplasmic domain, we prepared an anti-peptide antibody against a segment of the cytoplasmic domain. This antibody specifically immunoprecipitated the intact transferrin receptor as well as a 21-kDa peptide from trypsin-treated HeLa cells. Several lines of evidence indicated that the 21-kDa peptide was the cell-associated tryptic fragment of the transferrin receptor. The fragment was only present in trypsin-treated cells; the fragment migrated as a dimer in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as it should if it were derived from the transferrin receptor; a goat antibody prepared to the purified human transferrin receptor also precipitated the 21-kDa peptide from trypsinized cells. In addition, treating the tryptic fragment with neuraminidase increased the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting the fragment contained O-linked carbohydrate. When cells were trypsinized and then incubated at 37 degrees C, the half-life of the tryptic fragment (15 +/- 4 h) was not significantly different than the half-life of the intact receptor (19 +/- 6 h). This indicates that removing 95% of the external domain of the transferrin receptor has little effect on processes operating in the turnover of the receptor.     

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.     

A monoclonal antibody D51 recognizes the transferrin-receptor structure

A monoclonal antibody D51 has been obtained during immunization against human fetal thymus. The antibody binds to a variety of leukemic cells. This is similar to the staining pattern of the mAb OKT9 and L 5.1, which recognize the transferrin receptor. However, there are some differences. The antibody immunoprecipitates a protein present on Molt-4 cells. Under reducing conditions this protein has a molecular weight of 90 K dalton. Under non-reducing conditions it has a molecular weight of 180 K dalton. This suggests a dimeric structure. The consecutive immunoprecipitation with D51 and OKT9, respectively, demonstrates that both antibodies recognize the structure of the transferrin receptor.     

Detection of a molecular complex between ras proteins and transferrin receptor

Immunoprecipitation of extracts of human carcinoma cell lines with three different monoclonal antibodies generated against ras proteins revealed the coprecipitation of a 90,000 dalton protein. The coprecipitated protein was identified as the transferrin receptor by comigration in both reducing and nonreducing SDS-polyacrylamide gels, by absorption with a monoclonal antibody directed against transferrin receptor, and by analysis of partial proteolysis products. Coprecipitation of the transferrin receptor with three monoclonal antibodies with differing specificities to ras proteins, as well as the inability to coprecipitate the transferrin receptor from cell extracts from which ras proteins were depleted by preabsorption, indicates that ras proteins and the transferrin receptor form a molecular complex. This complex is disrupted by addition of transferrin to cell extracts. These findings suggest that ras proteins function in regulation of cell growth via interaction with the cell surface receptor for transferrin.     

Characterization of the human transferrin receptor produced in a baculovirus expression system

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.     

The p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor.

Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22-qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.     

Alterations in the rate of hemoglobin synthesis during chick embryogenesis

The rate of synthesis of different hemoglobin (Hb) species — embryonic, primitive, definitive, and adult — in the circulating red cells of the chick embryo have been measured. On the fifth day of chick embryogenesis, there is a marked decrease in the capability of these red cells to synthesize Hb. The rates of synthesis of embryonic and primitive Hb decrease markedly compared to that of definitive and adult Hb. On the fourth day of embryogenesis nucleic acid synthesis drops sharply in these cells. These decreases in metabolic activity preceed the disappearance of the primitive erythrocyte from the chick embryo circulation. It appears that the primitive cell line is specifically shut off with respect to its metabolic activities and possibly destroyed at this time.     

Characterization of Transferrin Receptor in an Immortalized Cell Line of Rat Brain Endothelial Cells,RBE4

Abstract

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell mono-layers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (K_D) of 17.1 ± 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 ± 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Developmental changes in the expression of high mobility group chromosomal proteins

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-14a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.