Acquisition of the functional properties of the transferrin receptor during its biosynthesis

The properties of the newly synthesized and partially glycosylated forms of the transferrin receptor were examined to determine which co- and post-translational modifications are necessary for the acquisition of transferrin binding activity and transport of the receptor to the cell surface. The nascent transferrin receptor containing core-glycosylated asparagine-linked oligosaccharides does not possess complete intersubunit disulfide bonds, sediments predominantly as a monomer in sucrose density gradients, and shows reduced binding to transferrin-agarose. Within 20-30 min after synthesis, the transferrin receptor acquires the ability to bind to a transferrin-linked affinity column. Intersubunit disulfide bond formation occurs slowly throughout the transit of the receptor to the cell surface. These results indicate that core glycosylation of the receptor may be necessary but is not sufficient for the acquisition of the ability of the receptor to bind transferrin and that intersubunit disulfide bond formation is a post-translational event. Inhibition of complex carbohydrate synthesis by either swainsonine (1 micrograms/ml) or deoxynojirimycin (4 mM) does not inhibit the ability of this receptor to form intersubunit disulfide bonds or to be transported to the cell surface. The partially glycosylated receptor, however, does show an approximately 3-fold reduced affinity for transferrin.     

Inhibition of 2,4-dichlorophenoxyacetic Acid conjugation to amino acids by treatment of cultured soybean cells with cytokinins

Kinetin, and all other cytokinins tested, inhibited the conjugation of [¹⁴C]2,4-dichlorophenoxyacetic acid (2, 4-D) to amino acids when supplied simultaneously with the 2,4-D to cultured soybean cells. Upon transfer to hormone-free medium, the cytokinin-treated cells released more of their [¹⁴C]2,4-D than did the control cells. Initial exposure to low 2,4-D and high kinetin levels resulted in the greatest release of 2,4-D upon subsequent transfer. The observed alteration in 2,4-D metabolism did not seem to be correlated with growth rate. Appropriate treatment of soybean cells with kinetin resulted in 2,4-D metabolism that resembled the 2,4-D metabolism of embryogenic carrot cells. However, no new morphological structures were observed in these soybean cultures, indicating that other factors are related to the failure of soybean cells to regenerate in culture.     

Human transferrin receptor contains O-linked oligosaccharides

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.     

Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis

The plant signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses that are activated upon pathogen or insect attack. Cross-talk between SA- and JA-dependent signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but suppression of the JA response by SA should be lethal in combination with the herbicide. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. SA effectively suppressed the expression of the PDF1.2::BAR transgene. However, suppression of the BAR gene did not result in suppression of herbicide resistance. Hence, a screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants. Here, we demonstrate that the PDF1.2::GUS reporter will be excellently suited in this respect.Key words: plant defense, salicylic acid, jasmonic acid, cross-talk, mutant screen, Arabidopsis     

Neogenin-mediated hemojuvelin shedding occurs after hemojuvelin traffics to the plasma membrane

HFE2 (hemochromatosis type 2 gene) is highly expressed in skeletal muscle and liver hepatocytes. Its encoded protein, hemojuvelin (HJV), is a co-receptor for the bone morphogenetic proteins 2 and 4 (BMP2 and BMP4) and enhances the BMP-induced hepcidin expression. Hepcidin is a central iron regulatory hormone predominantly secreted from hepatocytes. HJV also binds neogenin, a membrane protein widely expressed in many tissues. Neogenin is required for the processing and release of HJV from cells. The role that neogenin plays in HJV trafficking was investigated, using HepG2 cells, a human hepatoma cell line. Knockdown of endogenous neogenin markedly suppresses HJV release but has no evident effect on HJV trafficking to the plasma membrane. The addition of a soluble neogenin ectodomain to cells markedly inhibits HJV release, indicating that the HJV shedding is not processed before trafficking to the cell surface. At the plasma membrane it undergoes endocytosis in a dynamin-independent but cholesterol-dependent manner. The additional findings that HJV release is coupled to lysosomal degradation of neogenin and that cholesterol depletion by filipin blocks both HJV endocytosis and HJV release suggest that neogenin-mediated HJV release occurs after the HJV-neogenin complex is internalized from the cell surface.     

The molecular mechanism for receptor-stimulated iron release from the plasma iron transport protein transferrin

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.     

Integrity of actin fibers and microtubules influences metastatic tumor cell adhesion

Tumor cell adhesion within host organ microvasculature, its stabilization and invasion into host organ parenchyma appear to be important steps during formation of distant metastasis. These interactions of circulating tumor cells with the host organs occur in the presence of fluid shear forces and soluble and cellular environmental conditions of the blood that can modulate their cellular responses and possibly their metastatic efficiency. Cytoskeletal components, such as actin filaments and microtubules, can regulate biophysical characteristics and cellular signaling of the circulating cells. Therefore, we investigated the role of these cytoskeletal structures for early steps during metastasis formation in vivo and in vitro. Using an intravital observation technique, tumor cell adhesion of colon carcinoma cells within the hepatic microcirculation of rats and their invasion into liver parenchyma was observed. Disruption of actin filaments increased cell adhesion, whereas tubulin disruption inhibited adhesive interactions in vivo. The impairment of the cytoskeleton modulated adhesion-mediated cell signaling via focal adhesion kinase (FAK) and paxillin under flow conditions in vitro. In the presence of fluid flow, focal adhesions were enlarged and hyperphosphorylated, whereas stress fibers were reduced compared to static cell adhesion. Disruption of microtubules, however, partially inhibited these effects. Combining the in vivo and in vitro results, our study suggested that changes in cell rigidity and avidity of cell adhesion molecules after disruption of cytoskeletal components appear to be more important for initial adhesive interactions in vivo than their interference with adhesion-mediated cellular signal transduction.     

Thermoregulatory responses of Holstein and Brown Swiss Heat-Stressed dairy cows to two different cooling systems

Thirty-seven Holstein and 26 Brown Swiss dairy cows were used to evaluate the effect of two different cooling systems on physiological and hormonal responses during the summer. A control group of cows had access only to shade (C). A second group was cooled with spray and fans (S/F) and the third group was under an evaporative cooling system called Korral Kool (KK). The maximum temperature humidity index during the trial was from 73 to 85. Rectal temperatures and respiration rates of the C group were higher (P < 0.05) than those of the S/F and KK groups in both Holstein and Brown Swiss cows. Triiodothyronine levels in milk were higher (P < 0.05) in the KK group than in the S/F and C groups, while cortisol levels were lower (P < 0.05) in the C group than in S/F and KK. There was no significant difference in the hormonal response of the two breeds. These results demonstrate that both cooling systems may be used increase the comfort of Holstein and Brown Swiss cows during summer in hot, dry climates.     

Expression of the hereditary hemochromatosis protein HFE increases ferritin levels by inhibiting iron export in HT29 cells

Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport.     

Expression of epithelial cell iron-related genes upon infection by Neisseria meningitidis

Infection by the obligate human pathogens Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) reduces the expression of host epithelial cell transferrin receptor 1 (TfR-1) (Bonnah et al., 2000, Cellular Microbiology 2: 207-218). In addition, the rate and pattern of TfR-1 cycling is altered, leading to diminished uptake of Tf-iron by infected host cells. As Tf-iron is important for maintaining iron homeostasis in the eukaryotic cell, these findings raised the possibility that Neisseria infection might affect further pathways of epithelial cell iron metabolism. We used a specialized cDNA microarray platform, the 'IronChip', to investigate the expression of genes involved in iron transport, storage and regulation. We show that mRNA expression of several host genes involved in iron homeostasis is altered. Surprisingly, the general mRNA expression profile of infected cells closely resembled that of uninfected cells grown in an iron-limited environment. An important exception to this profile is TfR-1, the mRNA level of which is strongly reduced. Low TfR-1 expression may be explained in part by decreased activity of the iron-regulatory proteins (IRPs) in MC-infected cells, which may result in the destabilization of TfR-1 mRNA. Intriguingly, low IRP activity contrasts with the decrease in H-ferritin protein levels in infected cells. This finding suggests that low IRP activity may be responsible in part for the decrease in TfR-1 mRNA levels. A discussion of these novel findings in relation to MC infection and virulence is provided.