Horse Spleen Ferritin Research Articles

Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta.

Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two-hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co-immunoprecipitation. The pET-28a-FER147 prokaryotic expression vector was constructed. The expressed recombinant Fer147 was then isolated, purified, and refolded. When ferritins were treated by different heavy metals, several detection methods, including scanning electron microscopy (SEM), circular dichroism (CD), and inductively coupled plasma-mass spectrometry (ICP-MS) were applied to examine the structures and functions of the new protein Fer147, recombinant P. esculenta ferritin (Rferritin), and natural horse-spleen ferritin (Hferritin). SEM revealed that the three ferritin aggregates changed obviously after different heavy metals treatment, meanwhile, a little different in aggregates were detected when the ferritins were trapped by the same heavy metal. Hence, changes in aggregation structure of the three proteins are related to the nature of the different heavy metals and the interaction between the heavy metals and the three ferritins. CD data suggested that the secondary structure of the three ferritins hardly changed after different heavy metals were trapped. ICP-MS revealed that the ferritins exhibit different enrichment capacities for various heavy metals. In particular, the enrichment capacity of the recombinant Fer147 and Rferritin is much higher than that of hferritin.

Synthesis and characterization of a novel electron conducting biocomposite as biofuel cell anode

This study is based on the construction of an enzymatic bioanode adopting the exclusively reported layer-by-layer (LBL) assembly of Ppy-Ag-GO/ferritin (Frt)/glucose oxidase (GOx). The glassy carbon (GC) electrode was immobilised with the conducting polypyrrole (Ppy)-silver nanoparticles (Ag)-graphene oxide (GO) based biocomposite as electron transfer elevator, horse spleen ferritin (Frt) protein as electron transfer mediator and glucose oxidase (GOx) enzyme in layer by layer configuration. The fabricated bioanode exhibited good electrochemical performance with a maximum current response of 5.7mAcm−2 accompanied with biocompatibility and environmental stability because of the synergistic effect between outstanding properties of PPy, silver and GO, thereby, showing superior catalytic efficiency for the oxidation of glucose.

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.

X-ray Structure of the Carboplatin-Loaded Apo-Ferritin Nanocage.

The second-generation Pt anticancer agent carboplatin (CBDCA) was encapsulated within the apo horse spleen ferritin (AFt) nanocage, and the X-ray structure of the drug-loaded protein was refined at 1.49 Å resolution. Two Pt binding sites, different from the one observed in the cisplatin-encapsulated AFt, were identified in Ft subunits by inspection of anomalous electron density maps at two wavelengths and difference Fourier electron density maps, which provide the necessary sensitivity to discriminate between Pt from CBDCA and Cd ions that are present in the crystallization conditions. Pt centers coordinate to the NE2 atom of His49 and to the NE2 atom of His132, both on the inner surface of the Ft nanocage.

Protein Metalation by Anticancer Metallodrugs: A Joint ESI MS and XRD Investigative Strategy.

Interactions of metal-based drugs with proteins and consequent adduct formation (the so-called "protein metalation" process) play a key role in the mode of action of several anticancer agents and in determining their toxicological profile. Through a novel investigative strategy grounded on the combined use of electrospray ionization mass spectrometry (ESI MS) and biological macromolecule X-ray crystallography we show that it is possible to clarify in depth the metalation process of small model proteins; a number of instructive examples are provided. Recently, this kind of investigative approach has been extended to bigger proteins such as human serum albumin and horse spleen ferritin, with rather encouraging results. Overall, by application of this strategy, metalation of proteins caused by anticancer metallodrugs can be disclosed in the molecular detail.

Heavy metal detoxification by recombinant ferritin from Apostichopus japonicus

Ferritin fromApostichopus japonicasshowed better ability in heavy metal detoxification than horse spleen ferritin.

Biocompatibility of ferritin-based nanoparticles as targeted MRI contrast agents

Ferritin is a naturally occurring iron storage protein, proposed as a clinically relevant nanoparticle with applications as a diagnostic and therapeutic agent. Cationic ferritin is a targeted, injectable contrast agent to measure kidney microstructure with MRI. Here, the toxicity of horse spleen ferritin is assessed as a step to clinical translation. Adult male mice received cationic, native and high dose cationic ferritin (CF, NF, or HDCF) or saline and were monitored for 3weeks. Transient weight loss occurred in the ferritin groups with no difference in renal function parameters. Ferritin-injected mice demonstrated a lower serum iron 3weeks after administration. In ferritin-injected animals pre-treated with hydrocortisone, there were no structural or weight differences in the kidneys, liver, lung, heart, or spleen. This study demonstrates a lack of significant detrimental effects of horse-derived ferritin-based nanoparticles at MRI-detectable doses, allowing further exploration of these agents in basic research and clinical diagnostics.

On the mineral core of ferritin-like proteins: structural and magnetic characterization.

It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magnetic study of the mineral core biomineralized by horse spleen ferritin (HoSF) and three prokaryotic ferritin-like proteins: bacterial ferritin (FtnA) and bacterioferritin (Bfr) from Escherichia coli and archaeal ferritin (PfFtn) from Pyrococcus furiosus. The prokaryotic ferritin-like proteins have been studied under native conditions and inside the cells for the sake of preserving their natural attributes. They share with HoSF a nanocrystalline structure rather than an amorphous one as has been frequently reported. However, the presence of phosphorus changes drastically the short-range order and magnetic response of the prokaryotic cores with respect to HoSF. The superparamagnetism observed in HoSF is absent in the prokaryotic proteins, which show a pure atomic-like paramagnetic behaviour attributed to phosphorus breaking the Fe-Fe exchange interaction.

Enrichment and characterization of ferritin for nanomaterial applications

Ferritin is a ubiquitous iron storage protein utilized as a nanomaterial for labeling biomolecules and nanoparticle construction. Commercially available preparations of horse spleen ferritin, widely used as a starting material, contain a distribution of ferritins with different iron loads. We describe a detailed approach to the enrichment of differentially loaded ferritin molecules by common biophysical techniques such as size exclusion chromatography and preparative ultracentrifugation, and characterize these preparations by dynamic light scattering, and analytical ultracentrifugation. We demonstrate a combination of methods to standardize an approach for determining the chemical load of nearly any particle, including nanoparticles and metal colloids. Purification and characterization of iron content in monodisperse ferritin species is particularly critical for several applications in nanomaterial science.

Preparation and representation of recombinant Mn-ferritin flower-like spherical aggregates from marine invertebrates.

Ferritin has important functions in the transition and storage of toxic metal ions, but its regulation and function in many invertebrate species are still largely unknown. In our previous work, the cDNA sequence of Sinonovacula constricta, Apostichopus japonicas and Acaudina leucoprocta were constructed and efficiently expressed in E. Coli BL21 under IPTG induction. In this follow-up study, the recombinant ferritins were exposed to heavy metal manganese. The manganese concentration levels in three recombinant ferritins were greater than horse spleen ferritin (HSF). Compared with HSF, the amount of manganese enrichment in the three recombinant ferritins was 1.75-fold, 3.25-fold and 2.42-fold increases in ScFER, AjFER, and AlFER, respectively. After phosphate stimulation, the concentration of manganese increased and was higher than the ordinary dialysis control groups. The ScFER was four times its baseline value. The AjFER and AlFER were 1.4- and 8-fold higher, respectively. The AlFER sample stimulated by phosphate was 22-fold that of HSF. The morphologies of the resulting Mn-Ferritin from different marine invertebrates were characterized with scanning electron microscopy. Surface morphologies were lamella flower-like and are consistent with changes in surface morphologies of the standard Mn-HSF. Invertebrate recombinant ferritin and HSF both can uptake manganese. We found that the structure of A. leucoproctarecombinant Mn-Ferritin aggregate changed over time. The surface formed lamella flower-like aggregate, but gradually merged to create a relatively uniform plate-like phase of aggregate spherically and fused without clear boundaries.

Acanthamoeba castellanii Proteases are Capable of Degrading Iron-Binding Proteins as a Possible Mechanism of Pathogenicity.

Acanthamoeba castellanii, a free-living amoeba, is an amphizoic organism that can behave as an opportunistic pathogen, causing granulomatous amoebic encephalitis in immunocompromised patients or infecting immunocompetent individuals via cutaneous lesions, sinusoidal infections, or amoebic keratitis. Therefore, this amoeba could be in contact with different iron-binding proteins, such as lactoferrin in tears and mucosa and transferrin and hemoglobin in blood. Iron is a vital and necessary element for host metabolism but also for parasite survival. Accordingly, parasites have developed iron uptake mechanisms, one of which is the utilization of proteases to degrade host iron-binding proteins. In this work, we performed a partial biochemical characterization of A. castellanii proteases at different pHs and utilizing protease inhibitors with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copolymerized with different iron-binding proteins. We describe for the first time the presence of several cysteine proteases in a total A. castellanii crude extract and in conditioned culture medium precipitated with ethanol. These amoebic peptidases degraded human holo-lactoferrin, holo-transferrin, hemoglobin, and horse spleen ferritin; some of these proteases were substrate specific, and others degraded multiple substrates. These proteases could be considered virulence factors that promote iron acquisition from the host.

The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma

BackgroundHorse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.FindingsAlthough fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.ConclusionsHeat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

Magnetic properties of ferritin and akaganeite nanoparticles in aqueous suspension

We have studied the magnetically induced optical birefringence Δn of horse spleen ferritin (HSF) and aqueous suspensions of several different-sized iron oxyhydroxide nanoparticles coated with different polysaccharides mimicking ferritin. The structure and dimensions of the akaganeite mineral core were characterized by XRD and TEM, respectively. The stability of the suspensions in the measurement temperature range from 278 to 358 K was confirmed by UV–Vis absorption spectroscopy. The values of optical polarizability anisotropy Δα, magnetic susceptibility anisotropy Δχ, and permanent magnetic dipole moment μ m of the akaganeite nanoparticles have been estimated on the basis of the temperature dependence of the Cotton–Mouton (C–M) constant. The magnetic birefringence of Fe-sucrose has been described tentatively by different types of Langevin function allowing another estimation of Δχ and μ m. The obtained permanent magnetic dipole moment μ m of the studied akaganeite nanoparticles proves small and comparable to that of HSF. The value of μ m is found to increase with decreasing nanoparticle diameter. Observed in a range spanning more than five orders of magnitude, the linear relation between the C–M constant and the iron concentration provides a basis for possible analytical application of the C–M effect in biomedicine. The established relation between the C–M constant and the nanoparticle diameter confirms that the dominant contribution to the measured magnetic birefringence comes from the magnetic susceptibility anisotropy Δχ. A comparison of the C–M constants of the studied akaganeite nanoparticles with the data obtained for HSF provides evidence that the ferritin core behaves as a non-Euclidian solid.

Scara5 involvement in retinal iron metabolism

AbstractPurpose Although transferrin is the principal source of iron in mammalian cells, recently, ferritin was proposed as a new iron transporter protein. Serum ferritin is composed mostly, but not exclusively, of L‐ferritin, and in the presence of even a single H‐ferritin chain, up to 4500 atoms of iron can be incorporated into one ferritin molecule. As each transferrin molecule only binds to two iron atoms, serum ferritin can be regarded as a potential deliverer of a considerable large amount of iron to tissues. Iron implication in oxidative damage has become clear, and it is well known that iron accumulation is associated in the retina with several retinopathies.Methods Scara5 and L‐ferritin expression in the retina were studied by means of laser confocal microscopy, rt‐PCR and Western Blotting in ICR adult mice and in a murine model of retinopathy. Horse spleen ferritin was intravenously injected in healthy ICR adult mice and alteration in iron handling proteins expression was analyzed.Results Scara5 receptors, that specifically bind to L‐ferritin, were found in retinal endothelial and perivascular cells. Intravenous injected ferritin crossed the blood‐retinal barrier (BRB), through Scara5 receptor binding, and accumulated in these cells, suggesting that serum ferritin, mostly composed by L‐ferritin, can be transported across the BRB into the retinal parenchyma. During retinopathy, alterations in L‐ferritin levels and Scara5 expression were evaluated.Conclusion Serum ferritin uptake could represent a new pathway of iron delivery to the retina and points out perivascular cells as a key element in retinal iron traffic and during retinal iron accumulation associated with some retinopathies.

Iron release from ferritin by flavin nucleotides

BackgroundExtensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo. MethodsIron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode. ResultsThe reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed. ConclusionsUnder normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin. General significanceThere is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi-quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co-immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co-immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin-binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.

Binding Analysis of Ferritin with Heme Using α-Casein and Biotinylated-Hemin: Detection of Heme-Binding Capacity of Dpr Derived from Heme Synthesis-Deficient <i>Streptococcus mutans</i>

Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to an α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Escherichia coli, did not bind to an α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After preincubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin and that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.

Potential dependence of ferritin monomer, dimer and oligomer adsorption on a polycrystalline Au surface

An electrochemical quartz crystal microbalance (EQCM) was used to measure the effects of in situ potential changes on the adsorption and desorption of ferritin on polycrystalline Au. As-received horse-spleen ferritin, and purified ferritin monomer, dimer and higher oligomer fractions separated by gel chromatography were compared. Experiments were carried out at pH 5 where the ferritin is expected to have a net negative charge. Despite electrostatic repulsion, significant adsorption was observed on Au even at the most negative potentials studied, though it could be suppressed by using a low ionic strength electrolyte. This adsorption was attributed to hydrophobic interactions caused by partial denaturation of the protein, and it was greater for as-received ferritin containing higher oligomers than for monomeric ferritin. The reversibility of the adsorption was also lower when higher oligomers were present. Potential dependent adsorption/desorption could be useful as a diagnostic method for protein solutions.

The Faraday effect of natural and artificial ferritins

Measurements of the Faraday rotation at room temperature over the light wavelength range of 300–680 nm for horse spleen ferritin (HSF), magnetoferritin with different loading factors (LFs) and nanoscale magnetite and Fe2O3 suspensions are reported. The Faraday rotation and the magnetization of the materials studied present similar magnetic field dependences and are characteristic of a superparamagnetic system. The dependence of the Faraday rotation on the magnetic field is described, excluding HSF and Fe2O3, by a Langevin function with a log-normal distribution of the particle size allowing the core diameters of the substances studied to be calculated. It was found that the specific Verdet constant depends linearly on the LF. Differences in the Faraday rotation spectra and their magnetic field dependences allow discrimination between magnetoferritin with maghemite and magnetite cores which can be very useful in biomedicine.

Blood‐retinal barrier serum ferritin transport in mouse retina

AbstractPurpose The general requirement for iron is due to its involvement in various heme and non‐heme‐containing enzymes, which are ubiquitously involved in metabolic pathways. Iron accumulation is associated with some degenerative diseases, and in the retina with several retinopathies. Iron transport and cell type involved in iron storage mechanisms in the retina are not completely understood.Methods The iron content was analysed by EDX. Retina ferritin, TIM2 and Scara5 receptors were studied by means of confocal microscopy, rt‐PCR and WB. Horse spleen ferritin (HSF) was intravenously injected in healthy ICR mice and in a murine model of blood‐retinal barrier (BRB) breakdown.Results EDX detected iron in perivascular cell lysosomes that significatively accumulate iron in comparison with other cellular compartments. In the lower pH of lysosomes iron is released from transferrin and transported to the cytoplasm. However, this mechanism cannot explain the referred iron lysosomal accumulation. Recently, ferritin was proposed as a new iron delivery protein. TIM2 and Scara5 receptors, that bind to H‐ and L‐ferritin, were expressed in mouse retina, suggesting that serum ferritin can be transported across the BRB into the retinal parenchyma. HSF was intravenously injected and, as expected, ferritin crossed the BRB, probably through TIM2 and Scara5 receptor binding, and accumulated in perivascular cells. In the presence of BRB breakdown injected ferritin was also found accumulated in TIM2 and Scara5 positive cells.Conclusion Serum ferritin uptake could represent a new pathway of iron delivery in the retina and points out perivascular cells as a key element in retinal iron storage.