Aluminum exchange between citrate and human serum transferrin and interaction with transferrin receptor 1

The kinetics and thermodynamics of Al(III) exchange between aluminum citrate (AlL) and human serum transferrin were investigated in the 7.2-8.9 pH range. The C-site of human serum apotransferrin in interaction with bicarbonate removes Al(III) from Al citrate with an exchange equilibrium constant K1 = (2.0 +/- 0.6) x 10(-2); a direct second-order rate constant k1 = 45 +/- 3 M(-1) x s(-1); and a reverse second-order rate constant k(-1) = (2.3 +/- 0.5) x 10(3) M(-1) x s(-1). The newly formed aluminum-protein complex loses a single proton with proton dissociation constant K1a = (15 +/- 3) nM to yield a first kinetic intermediate. This intermediate then undergoes a modification in its conformation followed by two proton losses; first-order rate constant k2 = (4.20 +/- 0.02) x 10(-2) s(-1) to produce a second kinetic intermediate, which in turn undergoes a last slow modification in the conformation to yield the aluminum-loaded transferrin in its final state. This last process rate-controls Al(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau3(-1) = (6 +/- 1) x 10(-5) x s(-1). The affinities involved in aluminum uptake by serum transferrins are about 10 orders of magnitude lower than those involved in the uptake of iron. The interactions of iron-loaded transferrins with transferrin receptor 1 occur with average dissociation constants of 3 +/- 1 and 5 +/- 1 nM for the only C-site iron-loaded and of 6.0 +/- 0.6 and 7 +/- 0.5 nM for the iron-saturated ST in the absence or presence of CHAPS, respectively. No interaction is detected between receptor 1 and aluminum-saturated or mixed C-site iron-loaded/N-site aluminum-loaded transferrin under the same conditions. The fact that aluminum can be solubilized by serum transferrin in biological fluids does not necessarily imply that its transfer from the blood stream to cytoplasm follows the receptor-mediated pathway of iron transport by transferrins.     

Comparison of interaction between ceruloplasmin and lactoferrin/transferrin: to bind or not to bind

The year 2016 marked the 50th anniversary of the discovery by S. Osaki who first showed that ceruloplasmin (CP, ferro:O₂-oxidoreductase or ferroxidase) is capable of oxidizing Fe(II) to Fe(III) and favors the incorporation of the latter into transferrin (TF). However, much debate remains in the literature concerning the existence of a complex between the enzyme oxidizing iron and the protein facilitating its transport in plasma. We studied CP in exocrine fluids and demonstrated its high-affinity interaction with transferrin found in breast milk and in lacrimal fluid, i.e. with lactoferrin (LF). Here we present data obtained by comparing the interaction of CP with LF and TF using surface plasmon resonance and Hummel–Dreyer chromatography. Binding of apo-LF within the range of concentrations 1.6-51.3 μM with CP immobilized on a CM5-chip is characterized by K _D = 1.07 μM. Under similar conditions, the K _D for apo-TF was measured and appeared to be higher than 51.3 μM. Hummel–Dreyer chromatography of CP with 51 μM apo-LF/apo-TF in the effluent demonstrated the absence of interaction between apo-TF and CP in solution, contrary to efficient interaction between apoLF and CP. In contrast to LF, the interaction of apo-TF with CP is probably not stable within the physiological range of concentrations of TF.     

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.     

Linkage between the loci for transferrin and ceruloplasmin in pigs

Summary. Evidence for genetic linkage between the loci for transferrin ( Tf ) and ceruloplasmin ( Cp ) in pigs was presented. The results were based on a study of a single sire family comprising 35 informative offspring. No recombinants were observed. The recombination frequency was estimated to be in the range of 0 to 8%. This indicated that the recombination frequency between Tf and Cp loci in pigs may be much lower than that reported previously between these two loci in cattle and in human.     

Linkage between the loci for transferrin and ceruloplasmin in pigs

Evidence for genetic linkage between the loci for transferrin (Tf) and ceruloplasmin (Cp) in pigs was presented. The results were based on a study of a single sire family comprising 35 informative offspring. No recombinants were observed. The recombination frequency was estimated to be in the range of 0 to 8%. This indicated that the recombination frequency between Tf and Cp loci in pigs may be much lower than that reported previously between these two loci in cattle and in human.     

In vivo and in vitro studies of hafnium-binding to rat serum transferrin

The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy.     

EPR study of serum ceruloplasmin and iron transferrin in myocardial infarction.

Electron paramagnetic resonance (EPR) analysis of frozen serum from myocardial infarction patients has been conducted. Signal at g=4.3 was found definitively attributable to iron(III)-transferrin complex. Imcrease of serum ceruloplasmin as compared to normal was confirmed, with a concomitant decrease of iron-transferrin content. A mechanism for such correlated variation is hypothesized.     

In vitro biosynthesis of the human cell surface receptor for transferrin

The human cell surface receptor for transferrin is a transmembrane phosphoglycoprotein composed of two disulphide linked and apparently identical subunits of Mr 90 000. Using an affinity purified, polyclonal rabbit antibody, we have studied the in vitro biosynthesis of this receptor. The primary translation product, synthesised in a rabbit reticulocyte lysate programmed with human placental RNA, appears to have the same Mr (78 000) as the unglycosylated molecule immunoprecipitated from tunicamycin-treated cells. In the presence of a dog pancreatic microsomal system the cell free system accurately reproduces the glycosylation and the asymmetric transmembrane integration.     

The interaction between cholesterol and human serum albumin

The interaction between cholesterol and Human Serum Albumin (HSA) was studied by fluorescence technique. Addition of cholesterol causes decreasing of the fluorescence intensity of HSA and the mechanism can be attributed to static quenching. Both negative enthalpy and entropy change indicate this binding was an "enthalpy-driven" reaction. The number of binding site and distance between residues and ligands were also calculated: n = 0.98, r = 3.84 nm. UV-vis spectra showed HSA molecules unfolded to some extent and the hydrophobicity was decreased in the presence of cholesterol.     

Characterization of an interaction between protein C and ceruloplasmin

Coagulation factors V and VIII are substrates for activated protein C. Binding sites for the protease have been localized to homologous sequences within the terminal A domains of these proteins. Since ceruloplasmin contains significant sequence homology to these domains, a study was undertaken to determine whether ceruloplasmin was an activated protein C-binding protein. Ceruloplasmin was observed to inhibit the activated protein C-catalyzed inactivation of both factor Va and factor VIII. Searches of the ceruloplasmin sequence revealed a decapeptide sequence, HAGMETTYTV (residues 1028-1037) that shares 60 and 40% sequence identity with the activated protein C binding sequence in factors VIII and V, respectively. This peptide also inhibited factor Va inactivation and in addition was observed to enhance the amidolytic activity of activated protein C. The ferrous oxidase activity of ceruloplasmin was stimulated 5-fold by activated protein C, and this effect was negated by the peptide HAGMETTYTV. These results indicate that these conserved sequences of ceruloplasmin and factors V and VIII interact with activated protein C and suggest that this region may be important in the regulation of this anticoagulant protein.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

Periodate inactivation of ovotransferrin and human serum transferrin

Azari and Phillips (Azari, P., and Phillips, J. L. 1970 Arch. Biochem. Biophys. 138, 32-38) reported that periodate treatment of iron-free ovotransferrin causes a rapid loss of iron-binding activity and an oxidation of 3 to 5 tyrosines and 1 tryptophan. Rapid inactivation and loss of tyrosine in ovotransferrin has been confirmed, and the work extended to human serum transferrin and effects of denaturing concentrations of urea. Extensive (> 80%) inactivations of both ovotransferrin and human serum transferrin were observed when approximately 4 tyrosines were destroyed. Amino acid analysis and 360-MHz 1H NMR spectra confirmed that tyrosines are the only residues rapidly oxidized; the correlation of tyrosine loss with the loss of iron-binding activity suggests strongly that the tyrosines involved are those that function as ligands to metal ions bound to the protein. NMR spectra also showed that periodate oxidation causes local changes of structure in ovotransferrin (presumably at the metal-binding sites) but does not grossly alter the conformation. The addition of 5 to 8 M urea greatly retarded the inactivation and losses of tyrosine.