Ceruloplasmin carries the anionic glycan oligo/poly alpha2,8 deaminoneuraminic acid

Oligo/poly alpha2,8 deaminoneuraminic acid (KDN), a unique posttranslational protein modification, was found on megalin and a not yet characterized 150 kDa glycoprotein. We purified this glycoprotein from rat testis and identified it as ceruloplasmin. Furthermore, immunoprecipitated ceruloplasmin from rat thymus, ovary, blood serum, and postnatal day 2 but not adult lung and brain was immunoreactive for oligo/poly alpha2,8 KDN. The immunoreactivity for oligo/poly alpha2,8 KDN on purified serum ceruloplasmin was abolished by N-glycosidase F treatment but not by beta-elimination, indicating that it is present on N-glycosidically linked oligosaccharides. However, the copper binding activity of ceruloplasmin was independent of the presence of the anionic glycan. By immunohistochemistry, ceruloplasmin was detectable in histologically defined regions in rat ovary, thymus, and spleen. Likewise, by RT-PCR, ceruloplasmin expression was found in various non-hepatic rat tissues and showed a developmentally regulated pattern. Thus, ceruloplasmin, in addition to megalin, represents a glycoprotein carrying oligo/poly alpha2,8 KDN.     

Ceruloplasmin gene expression in human cancer cells

The copper transport protein, ceruloplasmin, is suggested to have a role in cancer since it is involved in angiogenesis and neovascularization. In order to understand the role of ceruloplasmin in malignant cells, we have recently isolated and sequenced a human ceruloplasmin cDNA clone. In the present study, we have investigated the ceruloplasmin gene expression in human colon and breast cancer cell lines. The poly (A) RNA from human colon (WiDr) and human breast (MCF-7) cancer cell lines was analyzed for the presence of ceruloplasmin mRNA. The Northern blot analysis revealed the presence of a 3.7 kb band of ceruloplasmin mRNA in these cell lines. Dot blot analysis revealed that ceruloplasmin mRNA is at least three fold more abundant in tumor cells as compared to normal rat liver.     

Joint Chromatographic Purification of Bovine Serum Ceruloplasmin and Amineoxidase

Abstract

A purification procedure leading to a joint separation of two serum copperenzymes: ceruloplasmin (EC 1.16.3.1) and amineoxidase (EC 1.4.3.6), is described. Both enzymes are obtained in electrophoretically homogeneous form and their specific activities are higher than those obtained by previously described purification techniques. Two common steps: precipitation of bovine plasma proteins with ammonium sulphate (at 35 % and 55 % saturation) followed by column chromatography on AE-Agarose (obtained by treatment of agarose beads with 1-chloro-2-ethylamine), lead to an electrophoretically homogeneous ceruloplasmin. At the same time, the ceruloplasmin-free protein preparation eluted in a first peak, following further Q-Sepharose and Con A-Sepharose chromatography, leads to purified bovine serum amine oxidase (BSAO) with an improved yield. The emphasis was given to a mutual improving effect as a consequence of the integration of the two enzymes purification procedures.     

Horse Plasma Ceruloplasmin Molecular Weight and Subunit Analysis

Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin (1). There are several reports demonstrating that ceruloplasmin is made up of multiple chains (2-3-4-5-6-7). Ryden (8) has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial preparations of the protein. By introducing aminocaproic acid, a general protease inhibitor, at the beginning of the enzyme preparation, Ryden proposed a single-chain structure for ceruloplasmin. On the contrary the results presented by Freeman and Daniel (9) showed that human ceruloplasmin is a multichain protein.

In this paper we report a new purification method for horse ceruloplasmin which furnishes a homogeneous protein preparation in high yield and with good reproducibility.

This procedure allowed to determine with greater accuracy the molecular mass of the protein, of 120000 dal-tons by gel cromatography and 115000 daltons by SDS gel electrophoresis. The protein is composed of one unit only and contains 6 copper atoms. Horse cerulopla-smin is a glycoprotein containing about 20% carbohydrate by weight.     

Ceruloplasmin, an Indicator of Copper Status

For clinical purposes, the non-ceruloplasmin copper fraction is routinely derived on the basis that ceruloplasmin binds six Cu atoms. However, this approach is limited because the actual ceruloplasmin copper binding is unclear. We performed direct measurement of the total serum copper and ceruloplasmin in 790 healthy individuals. We used an immunoprecipitation technique to separate ceruloplasmin and determined Cu content. With these values, we calculated the Cu/ceruloplasmin (Cp) ratio and thus generated data to support or discard the theoretical calculation of the non-ceruloplasmin fraction. Average of serum Cu and Cp levels were 18.4 +/- 4.4 micromol/l and 390 +/- 100 mg/l, respectively. The immunoprecipitation procedure allowed us to calculate a Cu/Cp ratio of 5.8, respectively, which supports the methodology of calculation that assigns a mean of six copper atoms to each ceruloplasmin molecule. With these values, we calculated that, in apparently normal adults, the non-ceruloplasmin copper (NCPC) fraction is lower than 1.3 micromol/l of Cu. In this report, we examine the Cp/Cu ratio by using Cp immunoprecipitation procedure. Our in vitro and in vivo studies indicate that, as a mean, there are 5.8 atoms of Cu per Cp molecule and that <1.3 micromol/l of Cu would correspond to the NCPC.     

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: Deceptive implications for free radical detection

Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification.     

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe^V redox intermediate of myeloperoxidase, to Compound II, which has Fe^IV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.     

Effect of colchicine and demecolcine on the serum ceruloplasmin level in rats.

Six hours after the administration of demecolcine (0.3 mg/100 g b.w.), a decrease in the ceruloplasmin level was recorded in rat serum. The time course of the decrease in the rat serum ceruloplasmin concentration after the same dose of colchicine was then studied. After two to three hours, the serum ceruloplasmin concentration fell significantly in both male and female rats. Eight hours after the administration of colchicine, the serum ceruloplasmin level began to rise again in female rats, but not in male rats. Mortality among females was lower than among males.     

Ceruloplasmin in human plasma and its relationships with the copper-albumin complex.

The electron paramagnetic spectrum of human plasma is dominated, in the g = 2 region, by resonances from copper atoms bound to ceruloplasmin, and does not reveal the fraction of copper normally associated with albumin, except in a few cases, where a copper-albumin signal increases with time after blood withdrawal. This copper-albumin complex is responsible for a resonance at a g value below g = 2 in the spectrum of human serum, which has been recently attributed to a modified form of type 2 copper bound to ceruloplasmin [Rylkov, V.V., Tarasiev, M.Y. & Moshkov, K.A. (1991) Eur. J. Biochem. 197, 185-189]. In the plasma, copper associated to albumin comes from ceruloplasmin. Purified ceruloplasmin is unable to exchange copper with albumin, either purified or in plasma. It can not be ruled out that some serum components trigger the metal exchange, in a defence mechanism operating when ceruloplasmin leaks, by unknown processes, its copper content before discharging the metal into the various organs.     

Ceruloplasmin decreases respiratory burst reaction during pregnancy

Testing of pregnant women reveals weakening of neutrophil-mediated effector functions, such as reactive oxygen species generation. This study provides data confirming the phenomenon, gained through application of the flow cytometry technique. Key factors influencing neutrophil functional activity in blood plasma of pregnant women have not been detected so far. At the same time, concentration of ceruloplasmin – a copper-containing glycoprotein – is known to increase in blood significantly during pregnancy. We observed the negative correlation between ceruloplasmin concentration in blood plasma of pregnant women and the intensity of respiratory burst of neutrophils. Fractionation of plasma using gel-filtration revealed that ceruloplasmin-containing fraction demonstrated suppression of the respiratory burst reaction. Partial elimination of ceruloplasmin from the blood of pregnant women, performed with the help of specific antibodies and followed by immunoprecipitation, leads to an increased respiratory burst reaction. On the contrary, addition of ceruloplasmin to blood samples of healthy donors noticeably decreases the respiratory burst reaction. The results presented prove that change in ceruloplasmin level in plasma is necessary and sufficient for modulating the ability of neutrophils to produce reactive oxygen species during pregnancy.     

Ceruloplasmin and metallothionein induction by zinc and 13-cis-retinoic acid in rats with adjuvant inflammation

The induction of ceruloplasmin and metallothionein was investigated in rats with the early inflammatory phase of adjuvant arthritis. When examined at the peak of the acute inflammatory response, 5 days after adjuvant treatment, zinc given daily (2 mg/kg, intraperitoneally) increased serum ceruloplasmin levels by 2.0 times that found in nonarthritic rats and 1.2 times that found in non-zinc-treated arthritic rats. 13-cis-Retinoic acid (160 mg/kg, orally) given daily increased serum ceruloplasmin 2.2 and 2.7 times that found in nontreated arthritic rats when given alone and with zinc (2 mg/kg, intraperitoneally), respectively. Reduction in the inflammatory response was measured by weight of the adjuvant-injected paw, 5 days after adjuvant was administered. The reduction in inflammation was 13 and 19-20% for 13-cis-retinoic acid and zinc, respectively, when given alone, and between 26 and 31% when the treatments were combined. Zinc markedly increased liver metallothionein levels whereas 13-cis-retinoic acid was a much less potent inducer of the protein in liver. The results are discussed in light of the probable physiological roles of both ceruloplasmin and metallothionein.     

The Ceruloplasmin Transferrin Ratio in the Blood of Patients at Different Stages of Parkinson’s Disease

The ratio of the concentrations of Cu²⁺-ceruloplasmin/Fe³⁺-transferrin in the blood plasma of 54 patients at different stages of Parkinson’s disease treated and not treated with L-DOPA was estimated by EPR-spectroscopy. It was established that in patients who suffer from Parkinson’s disease, the value of ceruloplasmin/ transferrin increased by 157% in comparison with the control group of clinically healthy people of the same age group. In patients with Parkinson’s disease, the ratio of ceruloplasmin/transferrin increased at stage 1 of the disease by 119%, at stage 2 by 117%, and at stage 3 by 135% in comparison with the control group. There was no statistically significant difference between the ratio of ceruloplasmin/transferrin in patients who received and did not recive L-DOPA replacement therapy. These data reveal changes in the functioning of the ceruloplasmin: transferrin system, which decreases the content of toxic ions of Fe²⁺ in the plasma of patients with Parkinson’s disease. These changes are a pathogenetically significant factor of Parkinson’s disease at all stages of the disease.     

Ceruloplasmin Is a Potential Biomarker for aGvHD following Allogeneic Hematopoietic Stem Cell Transplantation

Acute graft-versus-host-disease (aGvHD) is the major cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recently, diagnostic biomarkers for aGvHD have been shown to play important roles in evaluating disease status and mortality risk after allo-HSCT. To identify plasma biomarkers for aGvHD with high sensitivity and specificity, a quantitative proteomic approach using 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) was employed to screen differentially expressed proteins in peripheral blood before and after the onset of aGvHD. Four target proteins, ceruloplasmin (CP), myeloperoxidase (MPO), complement factor H (CFH), and alpha-1-acid glycoprotein (AGP), were chosen for preliminary validation with enzyme linked immunosorbent assay (ELISA) in 20 paired samples at both the time of diagnosis of aGvHD and the time of complete response. The most promising candidate, ceruloplasmin, was further validated at fixed time points after allo-HSCT and during aGvHD. The plasma ceruloplasmin levels were significantly increased during the period of aGvHD onset and were markedly decreased as aGvHD resolved. The plasma ceruloplasmin levels at different time points post-transplant in the aGvHD (+) group were significantly higher than those in the aGvHD (−) group (p<0.001). The elevation of ceruloplasmin level in patients with active aGvHD was independent of infection status. Patients whose ceruloplasmin levels were elevated above 670 μg/ml at 7, 14 and 21 days after allo-HSCT had a remarkably increased probability of subsequently developing aGvHD. In conclusion, our results suggest that plasma ceruloplasmin is a potential plasma biomarker of aGvHD, and it also has prognostic value for risk-adapted prophylaxis during the consecutive time points monitored in the first month after allo-HSCT.     

Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human ⁶⁴Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the ⁶⁴Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The ⁶⁴Cu in ceruloplasmin purified from plasma of ⁶⁴Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived ⁶⁴Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent ⁶⁴Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of ⁶⁴Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.     

Ceruloplasmin elevation and synthesis in rats with transplantable tumors.

In rats with transplantable mammary or hepatic tumors, plasma ceruloplasmin oxidase activity was increased 50--200%. This occurred progressively with tumors weighing 0.3% of body weight of more, and did not occur upon sham operation or implantation of normal tissue. Incorporation of [3H]-leucine indicated a specific enhancement of ceruloplasmin synthesis in the tumor-bearing rats, and a greater state of activation of the enzyme was also observed. The mechanism of the increase in ceruloplasmin levels in rats and humans with cancer thus appears to involve increased synthesis and activation of the enzyme.     

Ceruloplasmin fragmentation is implicated in 'free' copper deregulation of Alzheimer disease

A dysfunction in copper homeostasis seems to occur in Alzheimer’s disease (AD). We recently demonstrated that an excess of non-ceruloplasmin-copper (i.e. ‘free’ copper) correlates with the main functional and anatomical deficits as well as the cerebrospinal markers of the disease, thus suggesting that copper contributes to AD neurodegeneration. Aim of this study was to investigate the profile of serum ceruloplasmin isoforms immunoreactive protein in relation to copper dysfunction in AD. Twenty-five AD patients and 25 controls were included in the study. All subjects underwent individual measurements of serum ceruloplasmin and copper concentrations, and the amount of ‘free’ copper was computed for each copper and ceruloplasmin pair. Serum samples were also pooled and analyzed by two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blot analysis. The mean concentration of ’free’ copper resulted higher in AD patients than in controls. Ceruloplasmin 2-D PAGE western blot analysis of pooled sera showed in the AD samples low-molecular-weight spots in the &lt;50 kDa range that were not detected in controls’ pooled sera (p &lt; 0.029). Our data indicate a ceruloplasmin fragmentation in the serum of AD patients, possibly related to ‘free’ copper deregulation in this disease.     

Production of Melanins by Ceruloplasmin

It was shown that ceruloplasmin, apart from the known oxidative conversion of dopamine into melanin, can also produce (DHI)-melanin from 5,6-dihydroxyindole and THP-melanin from tetrahydropapaveroline. Ceruloplasmin acts as an oxidase and the kinetic parameters for these oxidative reactions are reported. Since these ceruloplasmin-catalyzed reactions occur also at pH 7.4, they could have a significant physiological impact. This ceruloplasmin-oxidasic activity is enhanced by copper ions and inhibited by chelators, such as ethylenediaminetetraacetic acid (EDTA) and desferoxamine (DEF). Some possible implication of melanin production in blood are discussed.     

Ceruloplasmin stimulates NADH oxidation of pig liver plasma membrane.

NADH oxidation by pig liver plasma membranes is stimulated by ceruloplasmin (CUP) reaching a maximal value at 50 U/ml of CUP. NADH oxidation activated by CUP is proportional to the amount of protein. Concanavalin A (Con A) which recognizes the glucidic residues of the CUP required for binding to the receptor inhibits the NADH oxidation in a dose-responsive manner. Both adriamycin and bathophenantroline disulfonate (BPS), previously reported as transplasma membrane electron transport inhibitors, also inhibit the CUP-stimulated NADH oxidation of pig liver plasma membranes. Our results show a clear interaction between CUP and the NADH oxidase of plasma membrane, which supports an oxidative role for CUP in its growth effect.     

A comparison of two procedures used for complexing Fe(III) with human apotransferrin: I. Physicochemical properties of the Fe(III) . transferrin products

Samples of human Fe.transferrin (Fe.HTr) were prepared from a single batch of apotransferrin (apo.HTr) by either the Fe(III)-citrate or the Fe(II)-ceruloplasmin (ferroxidase) method. By using 55Fe, 55Fe.HTr prepared by the citrate method and 55Fe.HTr prepared by the ceruloplasmin method contained 2.2-2.3 and 2.0 Fe/mol, respectively. For both 55Fe.HTr preparations, the isotope was shown to be associated with the protein from the measurement of absorbance at 465 nm and dialysis studies. However, passage of the 55Fe.HTr (ceruloplasmin) reaction mixture through DEAE-cellulose caused 55-60% of 55Fe to be lost from the protein, although no decrease in absorbance at 465 nm was observed. Ion-exchange chromatography of 55Fe.HTr (citrate) did not induce loss of 55Fe. Absorbance measurements showed significant differences between the two Fe.HTr preparations with respect to the ratios A212/A278 and A463/A278. Using an excitation wavelength of 275 nm, the fluorescence intensity ratios relative to apo.HTr were 0.275 and 0.309 for Fe.HTr (citrate) and Fe.HTr (ceruloplasmin), respectively. Electron spin resonance (ESR) measurements confirmed that Fe.HTr (citrate) and Fe.HTr (ceruloplasmin) were saturated with Fe. Hyperfine coupling constants and other features of the resonance profile revealed distinct differences between the two Fe.HTr preparations. Dialysis against H2O caused Fe.HTr (citrate), but not Fe.HTr (ceruloplasmin), to lose absorbance at 465 nm. The ESR profile of Fe.HTr (citrate), after dialysis against H2O, was reduced to multiple splittings and a lack of resolution of the central hyperfine structure. Addition of Na2CO3 restored the absorbance (465 nm) and the ESR pattern of Fe.HTr (citrate). In contrast, these properties of Fe.HTr (ceruloplasmin) were little affected by dialysis against H2O. However, the addition of trisodium citrate to Fe.HTr (ceruloplasmin) caused a reduction in absorbance at 465 nm and a change in ESR profile to resemble that of Fe.HTr (citrate) after dialysis in H2O; these changes, caused by citrate binding to Fe.HTr (ceruloplasmin), were restored to normal by the addition of Na2CO3. The data indicate that different protein conformations result from complexing Fe(III) with apo.HTr by these two different procedures. The two Fe.HTr products may differ, conceivably, in their abilities to transfer Fe to cells.     

Ceruloplasmin serum level in post-menopausal women treated with oral estrogens administered at different times.

The liver is an estrogen-responsive organ and the administration of estrogens in humans increases the hepatic synthesis of many proteins. The existence of a circadian rhythm of estrogen receptors in the liver has been proved by different authors. We studied the presence of a different responsiveness of the human liver to the estrogens in two groups of post-menopausal women by evaluating the changes in ceruloplasmin serum level. Conjugated equine estrogens were administered at different times (A: 8 a.m. and B: 8 p.m.). The replacement therapy increased ceruloplasmin serum levels both in group A and B, but the increase was higher in group B than in group A. These data reflect indirectly the presence of a circadian rhythm of hepatic responsiveness to the estrogens.