A variant of human transferrin with abnormal properties.
Ontology highlight
ABSTRACT:
SUBMITTER: Evans RW
PROVIDER: S-EPMC1163605 | biostudies-other | 1982 Jan
REPOSITORIES: biostudies-other
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Project description:1. A new human erythrocyte variant (Ph) is described. The variant contains an unusual sialic acid-rich glycoprotein in addition to the blood-group-MN([unk])- and blood-group-Ss(delta)-active sialoglycoproteins found in normal erythrocytes. 2. The unusual component Ph has an apparent mol.wt. of 32000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Ph component is not degraded during trypsin treatment of intact erythrocytes. 3. The Ph component was labelled by lacto-peroxidase-mediated radioiodination of intact erythrocytes and was found to be present in amounts approximately equimolar to alpha-sialoglycoprotein in the variant erythrocytes. 4. The Ph component had receptors for the lectins from Maclura aurantiaca (osage orange) and Triticum vulgaris (wheat-germ), but lacked a receptor for the Phaseolus vulgaris (red kidney bean) lectin, suggesting that it carries only O-linked oligosaccharides. 5. The presence of the Ph component in these erythrocytes does not correspond to any of the known blood-group-MNSs-related antigens examined. 6. We suggest that this component may be a hybrid polypeptide containing the N-terminal portion derived from normal delta-sialoglycoprotein, and the C-terminal portion from normal alpha-sialoglycoprotein, in a manner similar to the anti-Lepore haemoglobin.
| S-EPMC1161815 | biostudies-other
Project description:Transferrins are secreted proteins that bind iron. The well-studied transferrins are mammalian serum transferrin, which is involved in iron transport, and mammalian lactoferrin, which functions as an immune protein. Lactoferrin and lactoferrin-derived peptides have bactericidal activity, and the iron-free form of lactoferrin has bacteriostatic activity due to its ability to sequester iron. Insect transferrin is similar in sequence to both serum transferrin and lactoferrin, and its functions are not well-characterized; however, many studies of insect transferrin indicate that it has some type of immune function. The goal of this study was to determine the specific immune functions of transferrin from Manduca sexta (tobacco hornworm). We verified that transferrin expression is upregulated in response to infection in M. sexta larvae and determined that the concentration of transferrin in hemolymph increases from 2 μM to 10 μM following an immune challenge. It is also present in molting fluid and prepupal midgut fluid, two extracellular fluids with immune capabilities. No immune-induced proteolytic cleavage of transferrin in hemolymph was observed; therefore, M. sexta transferrin does not appear to be a source of antimicrobial peptides. Unlike iron-saturated lactoferrin, iron-saturated transferrin had no detectable antibacterial activity. In contrast, 1 μM iron-free transferrin inhibited bacterial growth, and this inhibition was blocked by supplementing the culture medium with 1 μM iron. Our results suggest that M. sexta transferrin does not have bactericidal activity, but that it does have a bacteriostatic function that depends on its iron sequestering ability. This study supports the hypothesis that insect transferrin participates in an iron withholding strategy to protect insects from infectious bacteria.
| S-EPMC5292288 | biostudies-literature
Project description:Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.
| S-EPMC1153923 | biostudies-other
Project description:Transferrin-binding protein B (TbpB) is one component of a bipartite receptor in several gram-negative bacterial species that binds host transferrin and mediates the uptake of iron for growth. Transferrin and TbpB are both bilobed proteins, and the interaction between these proteins seems to involve similar lobe-lobe interactions. Synthetic overlapping peptide libraries representing the N lobe of TbpB from Moraxella catarrhalis were prepared and probed with labeled human transferrin. Transferrin-binding peptides were localized to six different regions of the TbpB N lobe, and reciprocal experiments identified six different regions of the C lobe of transferrin that bound TbpB. Truncations of the N lobe of TbpB that sequentially removed each transferrin-binding determinant were used to probe an overlapping peptide library of the C lobe of human transferrin. The removal of each TbpB N-lobe transferrin-binding determinant resulted in a loss of reactivity with peptides from the synthetic peptide library representing the C lobe of transferrin. Thus, individual peptide-peptide interactions between ligand and receptor were identified. A structural model of human transferrin was used to map surface regions capable of binding to TbpB.
| S-EPMC152632 | biostudies-literature
Project description:Block polypeptides are an emerging class of materials that have the potential to be used in many biomedical applications, including the field of drug delivery. We have previously developed a negatively charged block copolypeptide, poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20), which forms spherical vesicles in aqueous solution. Since these vesicles are negatively charged, they are minimally toxic toward cells. However, the negative charge also inhibits these vesicles from effectively being internalized by cells, which can be problematic as many therapeutics have intracellular targets. To overcome this limitation of the E60L20 vesicles, transferrin (Tf) was conjugated onto the vesicle surface, since the receptor for Tf is overexpressed on cancer cells. The enhanced uptake of the Tf-conjugated vesicle was verified through confocal microscopy. Furthermore, endocytosis and immunostaining experiments confirmed that the Tf conjugated on the vesicle surface plays a critical role in the internalization and subsequent intracellular trafficking behavior of the vesicles.
| S-EPMC4022909 | biostudies-literature
Project description:Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.
| S-EPMC3267578 | biostudies-literature
Project description:The salt dependence of the iron-binding properties of transferrin was studied by urea/polyacrylamide-gel electrophoresis. The distribution of iron between the N-terminal and C-terminal binding sites under equilibrium conditions and the rates of release of iron from the two sites were studied. It was found that salt increases the thermodynamic stability of iron binding in the N-terminal site relative to the C-terminal site. Similar behaviour is observed for the kinetics of iron release, where salt retards the rate of removal of iron from the N-terminal site but facilitates removal from the C-terminal site.
| S-EPMC1163679 | biostudies-other
Project description:The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.
| S-EPMC2547852 | biostudies-literature
Project description:Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe.
| S-EPMC2860049 | biostudies-literature
Project description:Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.
| S-EPMC3156180 | biostudies-literature