Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid

The human transferrin receptor is post-translationally modified by the covalent attachment of palmitic acid to Cys62 and Cys67 via a thio-ester bond. To investigate the role of the acylation of the transferrin receptor, Cys62 and Cys67 were substituted with serine and alanine residues. The properties of the mutant receptors were compared with wild-type receptors after expression in Chinese hamster ovary cells that lack endogenous transferrin receptors. Rapid incorporation of [3H]palmitate into the wild-type transferrin receptor was observed, but the mutant receptors were found to be palmitoylation-defective. The kinetics of endocytosis and recycling of the wild-type and mutant receptors were compared. It was observed that the rate of endocytosis of the palmitoylation-defective transferrin receptors was significantly greater than the rate measured for the wild-type transferrin receptor. In contrast, the mutation of Cys62 and Cys67 was found to have no significant effect on the rate of transferrin receptor recycling. Consistent with these observations, it was found that cells expressing palmitoylation-defective transferrin receptors exhibited an increased rate of accumulation of [59Fe]diferric transferrin. Together, these data indicate that the palmitoylation of the transferrin receptor is associated with an inhibition of the rate of transferrin receptor endocytosis. Addition of insulin to cultured cells causes an increase in the palmitoylation of cell surface transferrin receptors and a decrease in the rate of transferrin receptor internalization. It was observed that the effect of insulin to inhibit the endocytosis of the acylation-defective [Ala62 Ala67]transferrin receptor was attenuated in comparison with the wild-type receptor. The decreased effectiveness of insulin to inhibit the internalization of the acylation-defective transferrin receptor is consistent with the hypothesis that palmitoylation represents a potential mechanism for the regulation of transferrin receptor endocytosis.     

Localization of transferrin and transferrin receptors in rat testes

One of the major proteins secreted by rat Sertoli cells in culture is a transferrin-like protein (Skinner and Griswold, 1980). The purpose of this study was to quantitate the amount of testicular transferrin in fluids isolated from the testis by the use of a radioimmunoassay and to determine the location of transferrin and transferrin receptors in the testis by indirect immunofluorescence. Seminiferous tubule fluid, rete testis fluid, and testicular lymph were collected from rat testes and were found to contain 141 micrograms, 47 micrograms and 3.7 mg transferrin per ml of fluid, respectively. Serum was found to contain 3.7 mg/ml transferrin. Paraffin sections of rat testis were incubated with rabbit anti-rat transferrin, biotinylated goat anti-rabbit and fluorescein-conjugated avidin. Immunoreactive transferrin was thus localized on the proacrosome and nuclear cap of developing spermatids. Late spermatids showed transferrin over the entire region of the head but mature testicular spermatozoa exhibited little fluorescence. The interstitial tissue between seminiferous tubules fluoresced brightly, indicating a large amount of transferrin in this area. By pretreating sections with rat transferrin, the receptor for the protein was localized on and in spermatocytes and early round spermatids. Dividing germ cells were brightly fluorescent.     

In vivo behaviour of rat transferrin bearing a hybrid glycan and its interaction with macrophages.

Production of rat transferrin containing a single hybrid glycan was induced by treating rats with swainsonine, an inhibitor of alpha-mannosidase II. The principal component of this variant transferrin containing one sialic acid residue per mole of protein was separated from other forms of transferrin by anion-exchange chromatography, followed by lectin affinity chromatography. Transferrin bearing the hybrid glycan was degraded in vivo with a half-life of 14 h as compared with 40 h for transferrin containing a standard diantennary glycan. By using 125I-labelled tyramine-cellobiose, a label whose discharge from lysosomes is strongly retarded, organs rich in reticuloendothelial elements (liver, bone marrow, lungs, and spleen) were identified as the major sites of catabolism of the transferrin variant. The liver took up more 59Fe from the variant (26% of the dose in 90 min) than from control rat transferrin (12%). The excess iron uptake was reduced by the intravenous injection of either human transferrin or ovalbumin, and it was abolished by administering both. Macrophages from bone marrow and lungs degraded the transferrin variant in vitro. The degradation was significantly enhanced when transferrin receptors were blocked by human transferrin, and it was significantly reduced by ovalbumin and methyl glucopyranoside.     

Purification and characterization of testicular transferrin secreted by rat Sertoli cells.

Sertoli cells synthesize and secrete a transferrin-like protein (testicular transferrin) [Skinner & Griswold (1980) J. Biol. Chem. 255, 1923-1925]. The purpose of the present study was to purify and characterize testicular transferrin and to compare it with serum transferrin. Testicular transferrin was obtained from the medium of cultured rat Sertoli cells, whereas serum transferrin was obtained from rat serum. Both proteins were purified with the use of phenyl-Sepharose hydrophobic chromatography and transferrin immunoaffinity chromatography. The purified proteins were shown to have similar molecular masses (75 000 Da) and amino acid compositions. The pattern of tryptic peptides from testicular and serum transferrin were found to be essentially the same when analysed by reverse-phase high-pressure liquid chromatography. The carbohydrate composition of both transferrins was determined by several colorimetric assays and g.l.c. Testicular transferrin, isolated from cell culture medium, had increased amounts of glucose, galactose and glucosamine. Serum transferrin that was incubated with cell culture medium also had a large amount of associated glucose. The results show that testicular transferrin and serum transferrin are structurally very similar and are possibly products of the same gene expressed in two different tissues, the testis and liver. However, the amount of carbohydrate associated with these two proteins is different.     

Antifungal activity of transferrin.

Inhibitory effects of transferrin on fungal growth were successfully estimated by measuring fungal ATP content. By this method, it was demonstrated that both human and rabbit transferrin possessed the inhibitory effect in the absence of any other factor on yeast-like and filamentous fungi. However, rabbit stimulation factor enhanced the inhibitory effect. The inhibitory effect of transferrin was nonspecific and correlated with unsaturated iron binding capacity (UIBC) of transferrin. Human transferrin was more inhibitory than rabbit transferrin.     

The biosynthesis of rat transferrin. Evidence for rapid glycosylation, disulfide bond formation, and tertiary folding

The transit time of newly synthesized transferrin in the liver is markedly longer than that of albumin. We sought to learn the basis of this difference by the use of labeled leucine and mannose in vivo and by isolation of newly formed transferrin from rough microsomes of rat liver. Albumin and alpha 1-antitrypsin, a second glycoprotein, were also studied for comparison. Minimal hepatic transit times were 17, 23, and 31 min for albumin, alpha 1-antitrypsin, and transferrin, respectively. The delay in the case of transferrin was found to occur chiefly in the rough endoplasmic reticulum and to be paralleled by an increase in the amount of transferrin relative to albumin in that organelle. Initial glycosylation of transferrin was as rapid as that of alpha 1-antitrypsin, and essentially all of the transferrin in the rough endoplasmic reticulum contained glycans which bound to concanavalin A and were removed by endoglycosidase H. Only 3% of the transferrin isolated from the rough microsomes came from the plasma by endocytosis or adsorption. Rapidity of disulfide bond formation in rough microsomes was evident from the presence of only 1.3 cysteine thiols/molecule of rough microsomal transferrin (total of 19 cystines) and the absence of mixed disulfides. Peptide patterns upon mild proteolysis were consistent with a native configuration of disulfide bond pairing. The ability of rough microsomal transferrin to bind and deliver iron through interaction with transferrin receptors on reticulocytes suggests that considerable tertiary structure is present. Thus, initial glycosylation, disulfide bridging, and tertiary folding are all rapid processes. The cause for the slow release of transferrin from the rough endoplasmic reticulum may lie with a rate-limiting transfer mechanism.     

Levels and patterns of 125I-labeled transferrin binding in mouse embryonic teeth and kidneys at various developmental stages

The iron-transporting serum glycoprotein, transferrin, is necessary for the cell proliferation, morphogenesis, and differentiation of mouse embryonic teeth and kidneys in organ culture. The stimulatory effect of transferrin is mediated by the binding of transferrin to its specific cell-surface receptor and by receptor-mediated endocytosis. Since, in both teeth and kidneys, the requirement for and responsiveness to transferrin depend on the developmental stage of the organ, we studied the binding of transferrin at various stages of tooth and kidney development by incubating tissues with 125I-labeled transferrin. The amount of bound transferrin was determined by measuring the tissue-incorporated radioactivity, and the binding sites were localized by autoradiography. During tooth development in vitro, the requirement for exogenous transferrin is lost as the teeth proceed from the early cap stage to the bell stage. The level of transferrin binding was found to decrease simultaneously, and in bell-stage teeth, the transferrin receptors were concentrated in the areas of most active cell proliferation. In kidneys, the number of transferrin receptors was highest at the stage during which the undifferentiated kidney mesenchyme becomes responsive to transferrin. These receptors were located in both the ureter epithelium and the metanephric mesenchyme, and they dramatically decreased in number with advancing kidney differentiation. The results of the present study indicate that, during the embryonic development of teeth and kidneys, the amount and localization of transferrin binding are correlated with cell proliferation. The number of transferrin receptors is highest during the developmental stages when cell proliferation is most active, and decreases with advancing differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the forms of iron-transferrin released from rabbit reticulocytes

Measurement of the distribution of the four species of transferrin, viz, apotransferrin, diferric transferrin and the two monoferric transferrin, before and after incubation of iron-rich rabbit transferrin with rabbit reticulocytes showed that not all transferrin released from the cells were in the form of apotransferrin. Instead, a mixture of all four species of the protein was released with apotransferrin and C-terminal monoferric transferrin being the major fractions. The buffer solution containing 125I-labelled transferrin showed a continuous gain in percentages in apotransferrin and C-terminal monoferric transferrin after each incubation with reticulocytes. The N-terminal monoferric transferrin, however, remained unchanged suggesting that in the process of transferrin uptake by cells, the diferric transferrin releases its iron from the acid-labile site at N-domain first before the other iron from the acid-stable site.     

Transferrin receptors during rabbit reticulocyte maturation

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

The distribution of cerebral expression of the transferrin gene is species specific.

Various plasma proteins, for example, transferrin, are synthesized not only in the liver, but also in the brain. The proportion of transferrin mRNA in total RNA from different regions of brains from various mammalian species was studied by Northern blot analysis. Absolute amounts of transferrin mRNA were determined in brain, choroid plexus, and liver from rats, sheep, and pigs by hybridization in solution followed by ribonuclease protection assay. Corrections for differences in yields of RNA were made using internal RNA standards. Large proportions of transferrin mRNA in total RNA and high absolute levels of transferrin mRNA in choroid plexus were found only in rats. Small proportions of transferrin mRNA were observed in RNA from choroid plexus from mice, dogs, and rabbits, while no transferrin mRNA at all was detected in choroid plexus from humans, sheep, pigs, cows, and guinea pigs. In further analysis of sheep and pigs, various amounts of transferrin mRNA were found in many parts of the brain, in contrast to the absence of transferrin mRNA from choroid plexus. In conclusion, a striking species specificity was observed for the pattern of cerebral expression of the transferrin gene.     

Transferrin receptors during rabbit reticulocyte maturation.

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

A high level of transferrin mRNA in the liver of analbuminemic rats

By means of immunological screening, a cDNA clone bearing the mRNA sequence for rat transferrin was isolated from a cDNA library of rat liver mRNA. The amounts of transferrin mRNA in livers of analbuminemic rats (NAR, Nagase analbuminemia rats) and normal rats were determined by RNA blot hybridization using a cloned transferrin cDNA probe. The level of transferrin mRNA in the NAR liver was about 1.7 times that in the normal rat liver. These findings suggest that the enhanced synthesis of transferrin in the NAR liver resulted from an increase in the transferrin mRNA level.     

The bacterial receptor protein, transferrin-binding protein B, does not independently facilitate the release of metal ion from human transferrin.

Pathogenic Gram-negative bacteria of the Pasteurellaceae and Neisseriaceae acquire iron for growth from host transferrin through the action of specific surface receptors. Iron is removed from transferrin by the receptor at the cell surface and is transported across the outer membrane to the periplasm. A periplasmic binding protein-dependent pathway subsequently transports iron into the cell. The transferrin receptor is composed of a largely surface-exposed lipoprotein, transferrin binding protein B, and a TonB-dependent integral outer membrane protein, transferrin binding protein A. To examine the role of transferrin binding protein B in the iron removal process, complexes of recombinant transferrin binding protein B and transferrin were prepared and compared with transferrin in metal-binding and -removal experiments. A polyhistidine-tagged form of recombinant transferrin binding protein B was able to purify a complex with transferrin that was largely monodisperse by dynamic light scattering analysis. Gallium was used instead of iron in the metal-binding studies, since it resulted in increased stability of recombinant transferrin binding protein B in the complex. Difference absorption spectra were used to monitor removal of gallium by nitrilotriacetic acid. Kinetic and equilibrium binding studies indicated that transferrin binds gallium more tightly in the presence of transferrin binding protein B. Thus, transferrin binding protein B does not facilitate metal ion removal and additional components are required for this process.     

Pulmonary transvascular flux of transferrin

We compared the pulmonary transvascular fluxes of transferrin and albumin in the intact sheep lung. Anesthetized sheep were prepared with lung lymph fistulas. The vascular blood pool was marked with 99mTc-erythrocytes, autologous transferrin was labeled with 113mIn, and albumin was labeled with 125I. Samples of blood, plasma, lymph, and lung were obtained up to 180 min after tracer infusion. Lymph tissue radioactivities were corrected for the intravascular component and expressed as extravascular-to-plasma concentration ratios. Clearance of transferrin and albumin from the plasma space followed a two-compartment model. The clearance rate constant was 2.1 +/- 0.1 x 10(-3) min for albumin and 2.4 +/- 0.1 x 10(-3) min for transferrin (P less than 0.05). Lymph-to-plasma ratios for albumin and transferrin were not different. However, the extravascular-to-plasma ratio for albumin was greater than transferrin (P less than 0.05). The lymph and lung data were deconvoluted for the plasma input function and fit to a two-compartment model. The results indicate that albumin and transferrin have similar permeabilities across the vascular barrier but have different pulmonary circulation to lymph kinetics because the extravascular volume of distribution of albumin is greater than transferrin.     

Serum transferrins of Japanese macaques: Comparison with other species of monkeys

Sera from seven species of monkeys and apes were electrophoresed in starch gels for examining their serum transferrin variations. A single transferrin component was observed in 130 individuals ofMacaca fuscata, while other three macaque species showed multiple components of transferrin. The transferrin ofMacaca fuscata was electrophoretically identical to one of the transferrin components of other macaque species.     

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

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

Seminal transferrin and spermatogenic capability in the bull

The objective of this study was to determine the relationship between seminal transferrin and sperm output in ejaculates from mature dairy bulls. Caudal sperm reserves in mature Holstein bulls (n = 15) were depleted by 8 successive ejaculations during a 50-70-min period (depletion phase). Bulls were then ejaculated 6 times per week for a period of 4 weeks (6X phase). Weekly sperm output (WSO) and weekly transferrin output (WTfO) were the sums of sperm and transferrin levels in 6 ejaculates taken in 1 week of the study. Mean WSO ranged from 20.7 billion to 39.6 billion and mean WTfO ranged from 334 micrograms to 1872 micrograms among the bulls. Regression analysis of sperm and transferrin levels in ejaculates collected during the depletion phase indicated that approximately 40% of seminal transferrin was not related to sperm output and probably was from accessory fluids. A relationship between total seminal transferrin and total sperm in ejaculate was observed (p less than 0.01, r = 0.54). This relationship was stronger when the transferrin was corrected for accessory fluid contribution (p less than 0.01, r = 0.65). The relationship between WSO and WTfO corrected for accessory fluid transferrin contribution (cWTfO) was significant (p less than 0.01, r = 0.64). The relationship between WSO and cWTfO can be interpreted to reflect the relationship between actual testicular sperm production and transferrin from testicular or epididymal origin.     

Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells

The mechanisms by which the duodenal mucosa absorbs iron are unknown. Insorption into absorptive cells of luminal iron bound to transferrin via receptor-mediated endocytosis has been hypothesized, but transferrin and transferrin receptor are absent in apical microvillous brush borders of small bowel biopsies taken from fasted patients and normal volunteers. We hypothesized that a normal iron-containing diet might induce the transient appearance of transferrin and transferrin receptor in apical brush borders of small intestinal absorptive cells in a normal mouse that was provided iron-containing chow until the moment of sacrifice. Light and electron microscopic immunolocalization of transferrin and transferrin receptor in proximal small intestinal absorptive cells was limited to basolateral membranes and coated pits of cells predominantly in the crypts and basal regions of the villi. Transferrin and transferrin receptor were not detected in apical microvillous brush border membranes of these enterocytes. In parallel immunolocalization protocols designed to show the ability to immunodetect other antigens at these locations, maltase and proteoglycan were demonstrated in apical microvillous brush border membranes and in basolateral membranes, respectively, in absorptive cells of small intestinal villous tip, base, and crypt regions. Furthermore, transferrin and transferrin receptor were immunolocalized in hepatocyte sinusoidal microvillus membranes. We conclude that food does not induce the appearance of immunodetectable transferrin and transferrin receptor in the apical microvilli of small intestinal absorptive cells and, therefore, that these iron transport proteins are not involved in the apical microvillous membrane transport of luminal dietary iron.     

Amonabactin-mediated iron acquisition from transferrin and lactoferrin by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> : direct measurement of individual microscopic rate constants

 The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe. The ability of A. hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments. The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis. The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored. Addition of transferrin or lactoferrin promoted bacterial growth. Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing. Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube. Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin. The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access. Taken together, these results support an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin. The implications of these findings for an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed. Received: 8 August 1999 / Accepted: 22 October 1999     

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.