A systems biology approach to the blood-aluminium problem: the application and testing of a computational model

Transport and distribution of systemic aluminium are influenced by its interaction with blood. Current understanding is centred upon the role played by the iron transport protein transferrin which has been shown to bind up to 90% of serum total aluminium. We have coined what we have called the blood-aluminium problem which states that the proportion of serum aluminium which, at any one moment in time, is bound by transferrin is more heavily influenced by kinetic constraints than thermodynamic equilibria with the result that the role played by transferrin in the transport and distribution of aluminium is likely to have been over estimated. To begin to solve the blood-aluminium problem and therewith provide a numerical solution to the aforementioned kinetic constraints we have applied and tested a simple computational model of the time-dependency of a putative transferrin ligand (L) binding aluminium to form an Al-L complex with a probability of existence, K(E), between 0% (no complex) and 100% (complex will not dissociate). The model is based upon the principles of a lattice-gas automaton which when ran for K(E) in the range 0.1-98.0% demonstrated the emergence of complex behaviour which could be defined in the terms of a set of parameters (equilibrium value, E(V), equilibrium time, E(T), peak value, P(V), peak time, P(T), area under curve, AUC) the values of which varied in a predictable way with K(E). When K(E) was set to 98% the model predicted that ca. 90% of the total aluminium would be bound by transferrin within ca. 350 simulation timesteps. We have used a systems biology approach to develop a simple model of the time-dependency of the binding of aluminium by transferrin. To use this approach to begin to solve the blood-aluminium problem we shall need to increase the complexity of the model to better reflect the heterogeneity of a biological system such as the blood.     

Isolation of transferrin from porcine gastric mucosa: comparison with porcine serum transferrin

1. An iron-binding glycoprotein has been purified to homogeneity from porcine gastric mucosa. 2. The molecular weight (80,000), amino acid composition, carbohydrate content, N-terminal amino acid sequence, tryptic map, stoichiometry of iron binding (2 mol/mol), visible absorption spectrum of the ferric complex and chromatographic behaviour of the gastric protein are all strikingly similar to the corresponding properties of porcine serum transferrin. 3. The quantity of the gastric protein (1.3 mg/g wet weight) present in the gastric mucosa suggests that it is not serum transferrin (plasma concentration 1.8 mg/ml) contaminating the tissue. 4. A role for transferrin in the uptake of dietary iron by the gastrointestinal tract is proposed.     

Aluminum site structure in serum transferrin and lactoferrin revealed by synchrotron radiation X-ray spectroscopy

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site.     

Chromosome 3q (22-ter) encodes the human transferrin receptor

The human transferrin receptor is an integral membrane glycoprotein of 180,000 molecular weight (mol. wt.) formed from two subunits of 90,000 mol. wt. A clone panel of Chinese hamster-human somatic cell hybrids was screened using a single cell plating cytotoxicity assay and rabbit antiserum raised to purified human transferrin receptor. Chromosome 3 displayed the highest rate of concordance with the presence of human transferrin receptor, as assayed by cytotoxicity. Antitransferrin receptor serum-resistant segregants of chromosome 3 positive, receptor-positive hybrids were selected, using antiserum and complement. The segregants consistently lost chromosome 3. 125I human transferrin binding studies confirmed synteny between the functional human transferrin receptor and chromosome 3. Examination of hybrids with either translocated or deleted chromosome 3's allows regional mapping to 3q(22-ter).     

Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.     

Characterization and localization of the transferrin receptor on rat reticulocytes

1. A further characterization and localization of the membrane receptor for transferrin on rat reticulocytes is described. PAGE studies with a purified membrane complex B2, from which the functional role in transferrin binding and iron uptake has been shown previously, showed that the transferrin receptor is localized on a membrane protein with a mol. wt of approximately 70-80.10(3). 2. Selective solubilization of the rat reticulocyte membrane has shown that this receptor protein belongs to one of the minor integral membrane polypeptides, embedded in the lipid bilayer of the membrane. 3. Proteolipid complexes, glycolipids and sialoglycoproteins of the rat reticulocyte membrane play no direct role in the binding capacity of the receptor.     

转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。     

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.     

Aluminium toxicity and binding to Escherichia coli

The toxicity and binding of aluminium to Escherichia coli has been studied. Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6–6.8. In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures. Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a K _m of 0.4 mM and a capacity of 0.13 mol (g dry wt)^-1. In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity. Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations. It is concluded that aluminium toxicity is related to pH-dependent speciation [with Al(H₂O) ₆ ³⁺ probably being the active species] and chelation of aluminium in the medium. Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.     

Gallium-67 as a Potential Marker for Aluminium Transport in Rat Brain: Implications for Alzheimer''s Disease

Evidence of a link between aluminium and Alzheimer's disease, parkinsonism-dementia of Guam, and dialysis encephalopathy raises questions regarding the role of this element in the pathogenesis of these conditions. Therefore, we have investigated the use of gallium-67 (67Ga) as a marker for brain uptake of aluminium. The binding of 67Ga to plasma proteins has been studied, and the blood-brain barrier permeability and autoradiographic distribution of this isotope in rat brain determined in vivo. The autoradiographic distribution of 125I-Fe-transferrin receptors in rat brain has also been determined in vitro. Results show that 67Ga was bound to plasma transferrin, entered the brain with a blood-brain barrier permeability of 2.48 x 10(-6) ml/min/g, and showed a marked regional distribution that was very similar to that of 125I-Fe-transferrin receptors. Our data suggest that the vulnerability of the hippocampus, amygdala, and cerebral cortex in conditions such as those mentioned above may be partly due to an increased uptake and deposition of aluminium in these regions by the iron transport system.     

铽(Ⅲ)与人血清脱铁转铁蛋白结合的荧光光谱研究

在ｐＨ７．４０．１ｍｏｌ／ＬＨｅｐｅｓ及室温条件下,使用荧光光谱进行了Ｔｂ３＋对人血清脱铁转铁蛋白的滴定．结果表明Ｔｂ３＋与人血清脱铁转铁蛋白结合后,其５４９ｎｍ处的荧光强度增强约１０５倍．在５４９ｎｍ处Ｔｂ３＋－脱铁转铁蛋白络合物的摩尔荧光强度是（９．６５±０．０５）×１０４ｍｏｌ－１Ｌ,Ｔｂ３＋可占据脱铁转铁蛋白的两个金属离子结合部位,优先占据脱铁转铁蛋白的Ｃ端结合部位,条件平衡常数是ｌｇＫＣ＝９．９６±０．２０,ｌｇＫＮ＝６．３７±０．１６．Ｔｂ３＋与Ｒ３＋Ｅ（ＲＥ＝Ｎｄ、Ｓｍ、Ｅｕ和Ｇｄ）间的线性自由能关系表明稀土离子占据脱铁转铁蛋白的Ｃ端结合部位时受离子大小的影响     

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.     

A variant of human transferrin with abnormal properties.

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.     

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)     

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.     

Transferrin receptors of isolated rat seminiferous tubules bind both rat and human transferrin

The binding and uptake of rat and human transferrin by isolated rat seminiferous tubules was studied. During the isolation and incubation of the tubules, the blood-testis barrier remained intact. Iron-saturated and iron-free (apo-) transferrin use the same binding sites on the surface of the tubules, but the dissociation constant is about two times higher for apotransferrin than for iron-saturated transferrin. The affinity of the receptors is equal for rat and human transferrin, but human transferrin binds to more surface binding sites (2.6 X 10(10) per 10 cm tubule length) than rat transferrin (1.1 X 10(10) per 10 cm tubule length) at 0 degrees C. At 33 degrees C equal numbers of human and rat transferrin molecules are taken up (about 8 X 10(10)) per 10 cm tubule length. The quantitative difference between 0 degrees C and 33 degrees C is caused by the fact that at 33 degrees C receptor-mediated endocytosis and recycling occur. As a consequence, both surface and intracellular transferrin receptors are detected at 33 degrees C. The dissociation constants are not temperature-dependent.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

TDPAC studies of the metal-binding sites in serum transferrin: comparison between 181Hf-labeled human- and rat-serum transferrin

The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding 181Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species.     

A comparison of the interaction of Chinese hamster fibroblasts with human and bovine transferrins, and conalbumin (chick-egg transferrin)

The iron requirement of a cell line of Chinese hamster fibroblasts is met more efficiently by human transferrin than by bovine transferrin or conalbumin. One possible explanation is that the binding of these transferrins to the Chinese hamster V79 cells may differ. Binding studies now show that the affinity of V79 cells for human transferrin is about 40 times greater than for bovine transferrin. Conalbumin has no detectable affinity for the human transferrin binding sites. Human apotransferrin has approximately one-sixth the affinity for the transferrin binding sites. The binding constant for the relation of human transferrin with the V79 cell is about 2.3·10⁶1· mole⁻¹, and the approximate number of binding sites per cell is 9 · 10⁵.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.