Iron release from transferrin induced by mixed ligand complexes of copper(II)

Summary Copper(II) complexes CuL₁L₂ with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25°C. Half-times of the reaction with Cu(PG)(en)^– were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS) ₂ ^4– and virtually none with Cu(serine)(en) nor Cu(PS)(2,2-bipyridyl)^–, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.Abbreviations PG phosphoglycerate - PS phosphoserine - en ethylenediamine - Fe2Tf diferric transferrin - Fe_cTf and Fe_NTf transferrin with iron bound to the lobe containing the C- or N-terminus, respectively - apoTf apotransferrin - K-3 all-cis-1,3,5-tris(trimethylammonio)-2,4,6-cyclo-hexanetriol - NTA nitrilotriacetic acid; bipy, 2,2-bipyridine; mal, malonate     

Role of transferrin in iron uptake by the brain: a comparative study

Summary The role of specific transferrin (Tf) and Tf receptor interaction on brain capillary endothelial cells in iron transport from the plasma to the brain was investigated by using Tf from several species of animals labeled with ⁵⁹Fe and ¹²⁵I, and 15-day and adult rats. The rate of iron transfer was much greater in the 15-day rats. It was greatest with Tf from the mammals, rat, rabbit and human, but much lower with chicken ovotransferrin and quokka (a marsupial), toad, lizard, crocodile, and fish Tf. The uptake of Tf by the brain showed a similar pattern, except for a very high uptake of ovotransferrin (ovo Tf). Iron uptake by the femurs (a source of bone marrow) was also high with Tf from the mammalian species and low with the other types of Tf, but showed little change with aging of the animals. It is concluded that iron transport into the brain is dependent on the function of Tf receptors, probably on capillary endothelial cells, and that these receptors show the same type of species specificity as the receptors on immature erythroid cells. Also, the decrease in iron uptake by the brain as rats age from 15 days to adulthood is specific for the brain and is not a general effect of the aging process.Abbreviations Tf transferrin - ovo Tf ovotransferrin     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Interaction of rat asialotransferrin with adult rat hepatocytes: its relevance for iron uptake and protein degradation

Comparative studies with rat transferrin (rTf) and asialotransferrin (asialo-rTf) were performed on suspended adult rat hepatocytes with the aim of elucidating the mechanism of enhanced hepatic Fe acquisition from asialo-rTf observed previously in vivo. At low ligand concentrations (0.05-20 micrograms/ml), the cells bound more asialo-rTf than rTf. However, the excess binding was abolished by incubation either in the presence of 1.55 mg/ml of diferric rTf or of 1 mg/ml of asialomucin. Following either treatment, asialo-rTf and rTf were bound to comparable extents. These findings indicate that both transferrin receptors and the hepatic galactose recognition system (lectin) are essential for preferential binding of asialo-rTf by hepatocytes. The possibility is considered that the lectin facilitates capture of asialo-rTf by the same binding sites that are normally available for rTf rather than that it functions as an alternative pathway. In agreement with this view, asialo-rTf could not be channeled into the lectin-mediated degradative pathway by blocking Tf receptors with human Tf. Enhanced Fe uptake from asialo-rTf was fully prevented by asialomucin and partially prevented by human Tf. The incomplete efficacy of human Tf in this regard supports reports in the literature about Fe uptake by the liver in a manner that is independent of Tf receptors. Rhesus asialo-Tf was deployed to show that no recognition mechanism exists for heterologous asialo-Tf in rat hepatocytes. The importance of using undenatured labeled proteins for studies with cells is demonstrated.     

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN₃, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl₂, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the sameThus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.     

Species specificity of transferein binding,endocytosis and iron internalization by cultured chick myogenic cells

Summary The ability of unlabelled heterologous transferrin to interact with transferrin receptors on developing chick myogenic cells was investigated by measuring their capacity to inhibit the surfacebinding and internalization of¹²⁵I-and⁵⁹Fe-labelled ovotransferrin. Transferrins from rat, rabbit, human, and a species of kangaroo (Macropus fuliginosus) were unable to inhibit either surfacebinding or internalization of labelled ovotransferrin even at concentrations ten times the molar concentration of the ovotransferrin. Transferrins isolated from the serum of a toad (Bufo marinus) and a lizard (Teliqua rugosa), when added at high concentrations, were found to reduce surface-binding of¹²⁵I-Tf by 20–25% but did not inhibit internalization of either¹²⁵I-Tf or⁵⁹Fe. This suggests that the effects of toad and lizard transferrins are due to non-specific binding to the myogenic cells. In contrast, inhibition of both surface-binding and internalization of labelled ovotransferrin was found when myogenic cells were incubated in the presence of the homologous transferrin (ovotransferrin). The species-specificity of transferrin binding, endocytosis and iron internalization did not vary with the state of proliferation or differentiation of the myogenic cells. However, the intracellular iron utilization was found to differ between differentiating presumptive and terminally differentiated myotubes. Internalized⁵⁹Fe was fractioned by gel filtration. In dividing and non-dividing presumptive myoblasts⁵⁹Fe was found to elute in three peaks, two with elution volumes corresponding to ferritin and transferrin and one at greater elution volume than that of myoglobin. In myotubes the same fractions occurred, and in addition some⁵⁹Fe was eluted at the same volume as myoglobin.Abbreviations Tf-Fe ₂ differic transferrin - BSA bovine serum albumin - BSS balanced salt solution - MEM Eagle's modified minimum essential medium - MW molecular weight - BUdR bromodeoxyuridine - Ara-C cytosine arabinoside     

Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells.

The effects of brefeldin A (BFA) on transferrin (Tf) transcellular transport, Tf receptor (TfR) distribution, and TfR-mediated endocytosis in filter-grown Madin-Darby canine kidney (MDCK) cells were studied. BFA (1.6 micrograms/ml) markedly enhanced the transcytosis of 125I-labeled Tf (125I-Tf) in both apical-to-basal and basal-to-apical directions; yet, BFA did not enhance the transcytosis of either native horseradish peroxidase (HRP) or membrane-bound HRP-poly(L-lysine) conjugates. Furthermore, this enhanced transcytosis of 125I-Tf was abolished either by competition with excess unlabeled Tf or by incubation at temperatures less than or equal to 25 degrees C. In addition, BFA treatment to MDCK cells: (a) increased 125I-Tf specific binding to the apical membrane and decreased 125I-Tf specific binding to the basal membrane; (b) decreased TfR recycling at the basolateral membrane; (c) altered the apical/basolateral distribution of TfRs in favor of the apical side; and (d) markedly increased 59Fe extraction, but not transcytosis, from apically endocytosed 59Fe-loaded Tf. These effects are consistent with a model in which BFA alters the traffic pattern of internalized Tf by decreasing basolateral TfR recycling, while diverting the nonrecycled fraction to the apical side of the cell. Our results indicate that, unlike the reported inhibition of polymeric IgA transcytosis (Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627), BFA can enhance the transcytosis of Tf in MDCK cells. Thus, by altering the intracellular traffic of ligand-receptor complexes, BFA can elicit either a decrease or an increase in transcytosis depending on the nature of the intracellular receptor processing.     

Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate.

The effects of ferric ammonium citrate (FAC) and desferrioxamine (DFO) on iron (Fe), and transferrin (Tf) uptake have been investigated using SK-MEL-28 human melanoma cells, which express the Tf homologue, melanotransferrin, in high concentrations. Previously we demonstrated two separate Fe uptake mechanisms from Tf, viz. a specific process mediated by the transferrin receptor (TfR) and a nonspecific process (Richardson, D. R., and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12). Cells exposed to DFO demonstrated up-regulation of the TfR with a concurrent increase in the rate of Fe uptake. Desferrioxamine also stimulated the nonspecific process of Fe uptake, resulting in a further increase in accumulation of Fe over Tf after saturation of the specific TfR. Ferric ammonium citrate had two effects. First, it resulted in down-regulation of the TfR. Second, and paradoxically, it markedly stimulated the rate of Fe uptake from Tf by the nonspecific process without increasing the rate of nonspecific Tf uptake. These data conclusively demonstrate that two entirely different mechanisms of iron uptake from Tf exist in melanoma cells and that ferric ammonium citrate may be a useful experimental tool to further characterize the specific and nonspecific mechanisms of Fe uptake from Tf.     

Novel diaroylhydrazine ligands as iron chelators: coordination chemistry and biological activity

The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health of patients suffering diseases such as β-thalassemia major. Herein, we report the syntheses and characterization of some new members of a series of N-aroyl-N′-picolinoyl hydrazine chelators (the H₂IPH analogs). Both 1:1 and 1:2 Fe^III:L complexes were isolated and the crystal structures of Fe(HPPH)Cl₂, Fe(4BBPH)Cl₂, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H₂PPH=N,N′-bis-picolinoyl hydrazine; H₂APH=N-4-aminobenzoyl-N′-picolinoyl hydrazine, H₂3BBPH=N-3-bromobenzoyl-N′-picolinoylhydrazine and H₂4BBPH=N-(4-bromobenzoyl)-N′-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The Fe^III complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization are reported. Five of these new chelators, namely H₂BPH (N-(benzoyl)-N′-(picolinoyl)hydrazine), H₂TPH (N-(2-thienyl)-N′-(picolinoyl)-hydrazine), H₂PPH, H₂3BBPH and H₂4BBPH, showed high efficacy at mobilizing ⁵⁹Fe from cells and inhibiting ⁵⁹Fe uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator, pyridoxal isonicotinoyl hydrazone (H₂PIH). The ability of the chelators to inhibit ⁵⁹Fe uptake could not be accounted for by direct chelation of ⁵⁹Fe from ⁵⁹Fe–Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease.     

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding

Summary A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4°C, after initial incubation with⁵⁹Fe-¹²⁵I-Tf at 37°C (Richardson and Baker, 1992a). The release of this internalized⁵⁹Fe could be markedly reduced if the cells were treated with proteases or incubated at 37°C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular⁵⁹Fe release increased linearly as a function of preincubation time with⁵⁹Fe-Tf subsequent to mechanical detachment at 4°C using a spatula. These data suggest that the⁵⁹Fe released was largely composed of end product(s) and was not an “intermediate Fe pool.” When the Fe(II) chelator, dipyridyl (DP), was incubated with⁵⁹Fe-Tf and the cells, it prevented the accumulation of⁵⁹Fe that was released following mechanical detachment at 4°C. Other chelators had much less effect on the proportion of⁵⁹Fe released. Examination of the⁵⁹Fe released showed that after a 4-h preincubation with⁵⁹Fe-Tf, approximately 50% of the⁵⁹Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4°C resulted in membrane disruptions that allow the release of high M, molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4°C resulted in pronounced membrane damage. In contrast, when cells were detached at 37°C, or at 4°C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37°C. The evidence also indicates that caution is required when interpreting data from expriments where cells have been mechanically detached at 4°C.     

Pinocytic uptake of divinyl ether-maleic anhydride (pyran copolymer) and its failure to stimulate pinocytosis

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of ¹²⁵I-labelled PVP and colloidal ¹⁹⁸Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [¹⁴C]DIVEMA and ¹²⁵I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.     

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.     

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC ₅₀ of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.     

Cu loading alters expression of non-IRE regulated, but not IRE regulated, Fe dependent proteins in HepG2 cells

This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using ¹²⁵I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein - iron responsive element regulation mechanism.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

Pinocytic capture and exocytosis of rat immunoglobulin IgG-N-(2-hydroxy-propyl)methacrylamide copolymer conjugates by rat visceral yolk sacs culturedin vitro

Rat immunoglobulin (IgG) was covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via glycylglycyl spacer. The resultant conjugate, free IgG and HPMA copolymer (containing a low percentage of tyrosinamide to facilitate radiolabelling) were radioiodinated, and their rates of pinocytic uptake, intracellular degradation and exocytic release by rat visceral yolk sacs culturedin vitro were determined. Free IgG was pinocytosed rapidly by the yolk sac and some IgG was subject to intracellular proteolysis. In comparison the IgG-HPMA copolymer conjugate was captured more slowly, but faster than unmodified HPMA. IgG was also exocytosed rapidly by the yolk sac following pinocytic capture and similarly IgG-HPMA copolymer had a much higher rate of release than unmodified H PMA. Measurement of tissue accumulation of¹²⁵I-labelled IgG-H PMA copolymer in the presence of increasing concentrations of non-radiolabelled IgG showed competition for membrane binding sites between the free, and polymer-bound immunoglobulin. These experiments indicate that immunoglobulins can be covalently bound to a soluble polymer developed as a drug-carrier in such a way that they can still interact with specific membrane receptors and they are subsequently subjected to specific cellular transport mechanisms.     

The interaction of transferin with isolated hepatocytes

Freshly isolated rat heptocytes display about 36 700 high-affinity sites to which deferric transferrin may bind with an apparent association constant of 1.62·10⁷ 1·mol^?1.Uptake of iron from diferric transferrin by hepatocytes is linear with time and is accelerated at increased differric transferrin concentrations.Apotransferrin is able to decrease net iron uptake by hepatocytes from diferric transferrin by a process not dependent on the apotransferrin concentrations used or on the rate at which the cells take up iron. Immunoprecipitation of the apotransferrin during these incubations indicates that iron is being released from the cells to apotransferrin at the same time as iron is being taken up from diferric transferrin. The simultaneous uptake and release of iron, and the insensitivity to apotransferrin concentration, suggest that the processes of iron uptake and release occur via separate mechanisms. The effect of apotransferrin on net retention of iron may be one way in which the in vivo distribution of iron between sites of storage and utilization is controlled.     

Influence of transferrin glycans on receptor binding and iron-donation

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, β-galactosidase or N-Acetyl-β-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions. This revised version was published online in November 2006 with corrections to the Cover Date.