Molecular and pathological basis of aceruloplasminemia

Aceruloplasminemia is an autosomal recessive neurodegenerative disease characterized by iron accumulation in the brain as well as visceral organs. It is a loss-of-function disorder caused by mutations in the ceruloplasmin gene. Clinically, this disease consists of the triad of adult-onset neurological disease, retinal degeneration and diabetes mellitus. Massive iron accumulation and extensive loss of neurons are observed in the basal ganglia. The elevated iron concentration is associated with increased lipid peroxidation in the brains of aceruloplasminemia patients. Enlarged or deformed astrocytes and spheroid-like globular structures are characteristic neuropathological findings in aceruloplasminemia. Moreover, deformed astrocytes and globular structures react positively to anti-4-hydroxynonenal antibody, suggesting that increased oxidative stress is involved in neuronal cell death in aceruloplasminemia brain. More than 30 aceruloplasminemia-causing mutations in the ceruloplasmin gene have been identified. We examined the biosynthesis of two missense ceruloplasmin proteins that result from a Japanese P177R mutation and a Dutch G631R mutation, using Chinese hamster ovary cell expression system. The P177R mutant protein is retained in the endoplasmic reticulum. The G631R mutant protein, predicted to alter the interactions at a single type I copper-binding site, prevented incorporation of copper into apoceruloplasmin and resulted in the synthesis and secretion only of apoceruloplasmin. Molecular analysis of missense mutations showed different structure-function relationships in ceruloplasmin protein. The investigation of mutant ceruloplasmin reveals new insights into molecular pathogenesis of aceruloplasminemia as well as biosynthesis, trafficking, and function of ceruloplasmin.     

Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin

To examine the mechanisms of copper incorporation during ceruloplasmin biosynthesis, we developed methods to resolve and identify apo and holoceruloplasmin. The identity of holoceruloplasmin was confirmed by oxidase activity staining, immunoblotting, 67Cu-ligand exchange, and 67Cu-ligand blotting. Following metabolic labeling of human liver and lung cell lines with 67Cu, newly synthesized holoceruloplasmin was detected in the culture media as two species with apparent molecular masses of 84 and 79 kDa. Pulse-chase studies demonstrate that exogenous copper is readily available for incorporation into newly synthesized ceruloplasmin and that the kinetics of apo and holoceruloplasmin synthesis and secretion are identical. Inhibition of N-linked glycosylation did not affect the rate or amount of copper incorporated into newly synthesized ceruloplasmin but did result in the secretion of a single 68-kDa holoceruloplasmin moiety. Despite differences in the kinetics of copper uptake between cell lines a linear rate of copper incorporation into newly synthesized ceruloplasmin was observed with no evidence of copper exchange following biosynthesis. Under the conditions studied, holoceruloplasmin accounted for less than 5% of the total ceruloplasmin synthesized and secreted by each cell line. The data indicate that copper is incorporated into newly synthesized ceruloplasmin early in the course of biosynthesis by a process independent of N-linked carbohydrate addition. This process of copper incorporation results in an apparent conformational change in the ceruloplasmin molecule which does not affect the secretory rate of the protein.     

Biochemical analysis of a missense mutation in aceruloplasminemia.

Aceruloplasminemia is an inherited neurodegenerative disease characterized by parenchymal iron accumulation secondary to loss-of-function mutations in the ceruloplasmin gene. To elucidate the molecular pathogenesis of aceruloplasminemia, the biosynthesis of a missense mutant ceruloplasmin (P177R) occurring in an affected patient was examined. Chinese hamster ovary cells transfected with cDNAs encoding secreted and glycosylphosphatidylinositol (GPI)-linked wild-type or P177R human ceruloplasmin were examined by pulse-chase metabolic labeling. These experiments, as well as immunofluorescent analysis and N-linked glycosylation studies, indicate that both the secreted and GPI-linked forms of the P177R mutant are retained in the endoplasmic reticulum (ER). The P177R mutation resides within a novel motif, which is repeated six times in human ceruloplasmin and is conserved in the homologous proteins hephaestin and factor VIII. Analysis of additional mutations in these motifs suggests a critical role for this region in ceruloplasmin trafficking and indicates that substitution of the arginine residue is critical to the ER retention of the P177R mutant. Metabolic labeling of transfected Chinese hamster ovary cells with (64)Cu indicates that the P177R mutant is retained in the ER as an apoprotein and that copper is incorporated into both secreted and GPI-linked ceruloplasmin as a late event in the secretory pathway. Taken together, these studies reveal new insights into the determinants of holoceruloplasmin biosynthesis and indicate that aceruloplasminemia can result from retention of mutant ceruloplasmin within the early secretory pathway.     

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.     

Rat albumin inhibits the formation of apoceruloplasmin during storage

Purified rat ceruloplasmin is extraordinarily unstable in storage at –70 °C. In a 20 mM phosphate buffer, pH 7.0, the ferroxidase and amine oxidase of ceruloplasmin are over 90% inactivated within two weeks. Holoceruloplasmin stored for three months in a 20 mM barbital buffer (or acetate buffer), pH 7.0 (or pH 5.5) was transformed into an apo-protein and amine (o-dianisidine) oxidase of ceruloplasmin was inactivated by 50–55%. The patterns of ferroxidase activity loss were similar to those of amine oxidase activity loss. On the contrary, when holoceruloplasmin was mixed with rat serum albumin, transformation into apoceruloplasmin was significantly prevented in a 20 mM barbital buffer, pH 7.0 (or 20 mM acetate buffer, pH 5.5). Consequently, ferroxidase and amine oxidase activities of ceruloplasmin were not inactivated and the immunochemical reactivity was not changed. These results can be applied for laboratorial and clinical purposes.     

The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin

M98Q amicyanin is isolated with zinc bound to its type 1 copper-binding site. The influence of the axial ligand of the type 1 copper site on metal specificity is strongest prior to the completion of protein folding and adoption of the final type 1 site geometry. The preference for zinc over copper correlated with the selectivity of apoamicyanin in vitro in the partially folded, rather than the completely folded state. These results suggest that metal incorporation in vivo occurs during protein folding in the periplasm and not to a preformed type 1 site.     

Relationships between the copper and iron systems in hemodialysis patients and variables affecting these systems

The copper-binding protein ceruloplasmin oxidizes ferrous iron to ferric iron, an action that is critical for the binding of iron to transferrin in plasma. Ceruloplasmin, in common with ferritin and transferrin, is an acute-phase protein that is altered by inflammation. We sought to identify interrelationships between the copper and iron systems by measuring copper, ceruloplasmin, ferroxidase, ferritin, transferrin, iron, and iron-binding capacity in a group of hemodialysis patients. We looked for evidence of inflammation and free-radical injury by assaying for protein carbonyl groups, protein pyrrolation, di-tyrosine, and advanced oxidation protein products. Our findings were compatible with an active inflammatory state that affected both iron and copper metabolism. Transferrin levels were low, whereas ceruloplasmin levels were elevated compared to normal. Copper concentration was increased proportional to ceruloplasmin. Several variables including ceruloplasmin and transferrin were observed to correlate significantly with the level of pyrrolated protein. The data suggest that posttranslational modification of circulating proteins may affect their structural, enzymatic, and ligand-binding properties. Abnormalities in copper metabolism and their influence on iron handling in renal failure are complex and will require additional study before their importance can be defined.     

Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein

Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an approximately 72 amino acid consensus motif and the first four of these domains, denoted WLN1-4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5-6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5-6, and 6, denoted by WLN1, WLN3, WLN4, WLN5-6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.     

Copper transport and metabolism are normal in aceruloplasminemic mice.

Ceruloplasmin is an abundant serum glycoprotein containing greater than 95% of the copper found in the plasma of vertebrate species. Although this protein is known to function as an essential ferroxidase, the role of ceruloplasmin in copper transport and metabolism remains unclear. To elucidate the role of ceruloplasmin in copper metabolism, the kinetics of copper absorption, transport, distribution, and excretion were examined utilizing (64)Cu in wild-type and aceruloplasminemic mice. No differences in gastrointestinal absorption, hepatic uptake, or biliary excretion were observed in these animals. Furthermore, steady state measurements of tissue copper content utilizing (64)Cu and atomic absorption spectroscopy revealed no differences in the copper content of the brain, heart, spleen, and kidney. Consistent with these findings, the activity of copper-zinc superoxide dismutase in these tissues was equivalent in wild-type and ceruloplasmin-deficient mice. Hepatic iron was elevated 3.5-fold in aceruloplasminemic mice because of the loss of ferroxidase function. Hepatic copper content was markedly increased in aceruloplasminemic mice. As no differences were observed in copper absorption or biliary copper excretion, these data suggest that in these animals, hepatocyte copper intended for ceruloplasmin incorporation is trafficked into a compartment that is less available for biliary copper excretion. Taken together, these data reveal no essential role for ceruloplasmin in copper metabolism and suggest a previously unappreciated complexity to the subcellular distribution of this metal within the hepatocyte secretory pathway.     

Immunochemical and enzymatic study of ceruloplasmin in rheumatoid arthritis

Forty adult patients (30 women and 10 men) with rheumatoid arthritis (RA), treated with nonsteroidal anti-inflammatory drugs, were studied. Serum levels of immunoreactive ceruloplasmin, oxidase activity of the ceruloplasmin and total copper, as well as the specific oxidase activity (enzyme activity per unit of mass) and the copper/immunoreactive ceruloplasmin relationship were significantly higher in the group of patients than in the healthy control group (p < 0.001). However, no significant difference was found for the concentration of non-ceruloplasmin copper between both groups. A statistically significant negative correlation was obtained for the concentration of serum thiobarbituric acid-reacting substances with the immunoreactive ceruloplasmin and its oxidase activity in the group of patients (p < 0.005). These results suggest that in RA increases of serum copper are produced at the expense of the fraction linked to the ceruloplasmin, diminishing the proportion of apoceruloplasmin and other forms poor in copper. Although the increase in the serum concentration of ceruloplasmin might offer an additional safeguard against oxidative stress. it does not appear to have a beneficial effect upon the activity of the illness as evaluated by means the biological inflammation markers C-reactive protein, erythrocyte sedimentation rate and sialic acid.     

Stoichiometry of Fe(II) oxidation during ceruloplasmin-catalyzed loading of ferritin.

Ceruloplasmin catalyzed the incorporation of iron into apoferritin with a stoichiometry of 3.8 Fe(II)/O2. This value remained the same when ferritin containing varying amounts of iron was used. Contrary to the "crystal growth" model for ferritin formation, no iron incorporation into holoferritin was observed in the absence of ceruloplasmin. Fe(II)/O2 ratios close to 2 were obtained for iron incorporation into apo- and holoferritin in Hepes buffer, in the absence of ceruloplasmin, indicating the formation of reduced oxygen species. Sequential loading of ferritin in this buffer resulted in increasing oxidation of the protein as measured by carbonyl formation. Sequential loading of ferritin using ceruloplasmin did not result in protein oxidation and a maximum of about 2300 atoms of iron were incorporated into rat liver ferritin. This corresponded to the maximum amount of iron found in rat liver ferritin in vivo after injection with iron. These results provide evidence for ceruloplasmin as an effective catalyst for the incorporation of iron into both apo- and holoferritin. The possibility that these findings may have physiological significance is discussed.     

Biological effects of mutant ceruloplasmin on hepcidin-mediated internalization of ferroportin

Ceruloplasmin plays an essential role in cellular iron efflux by oxidizing ferrous iron exported from ferroportin. Ferroportin is posttranslationally regulated through internalization triggered by hepcidin binding. Aceruloplasminemia is an autosomal recessive disorder of iron homeostasis resulting from mutations in the ceruloplasmin gene. The present study investigated the biological effects of glycosylphosphatidylinositol (GPI)-linked ceruloplasmin on the hepcidin-mediated internalization of ferroportin. The prevention of hepcidin-mediated ferroportin internalization was observed in the glioma cells lines expressing endogenous ceruloplasmin as well as in the cells transfected with GPI-linked ceruloplasmin under low levels of hepcidin. A decrease in the extracellular ferrous iron by an iron chelator and incubation with purified ceruloplasmin in the culture medium prevented hepcidin-mediated ferroportin internalization, while the reconstitution of apo-ceruloplasmin was not able to prevent ferroportin internalization. The effect of ceruloplasmin on the ferroportin stability was impaired due to three distinct properties of the mutant ceruloplasmin: namely, a decreased ferroxidase activity, the mislocalization in the endoplasmic reticulum, and the failure of copper incorporation into apo-ceruloplasmin. Patients with aceruloplasminemia exhibited low serum hepcidin levels and a decreased ferroportin protein expression in the liver. The in vivo findings supported the notion that under low levels of hepcidin, mutant ceruloplasmin cannot stabilize ferroportin because of a loss-of-function in the ferroxidase activity, which has been reported to play an important role in the stability of ferroportin. The properties of mutant ceruloplasmin regarding the regulation of ferroportin may therefore provide a therapeutic strategy for aceruloplasminemia patients.     

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.     

Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)

The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hepatocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular trafficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mutation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the constitutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.     

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.     

Cloning and expression analysis of the sheep ceruloplasmin cDNA.

The cDNA encoding sheep ceruloplasmin (sCP) was isolated from a sheep liver cDNA library. The cDNA contig was 3530 nucleotides in length and encoded a protein of 1048 amino acids. The deduced amino acid sequence showed a high degree of conservation (87%) when compared to the human ceruloplasmin (hCP) sequence. Northern blot analysis of sheep tissue revealed that the sheep ceruloplasmin gene (sCP) was expressed primarily in the liver, but low levels of mRNA were detected in the hypothalamus, spleen and uterus. No sCP mRNA was detected in the cortex, heart, intestine or kidney. Expression was not significantly affected by hepatic copper content. Northern blot analysis of sheep liver during development demonstrated little sCP expression during fetal life, but significant levels of mRNA were observed after birth. Significantly, the developmental expression pattern of sCP was closely correlated with that of the sheep Wilson disease gene (sATP7B), suggesting that the expression of the two genes may be coordinated to ensure that copper is supplied to apoceruloplasmin. Overall, the structure and expression of sCP appeared similar to other mammals, suggesting that abnormalities in CP were not responsible for the unusual sheep copper phenotype.     

Effects of the Pathological Q212P Mutation on Human Prion Protein Non-Octarepeat Copper-Binding Site

Prion diseases are a class of fatal neurodegenerative disorders characterized by brain spongiosis, synaptic degeneration, microglia and astrocytes activation, neuronal loss and altered redox control. These maladies can be sporadic, iatrogenic and genetic. The etiological agent is the prion, a misfolded form of the cellular prion protein, PrP(C). PrP(C) interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. The physiological implication of this interaction is still unclear, as is the role of metals in the conversion. Since prion diseases present metal dyshomeostasis and increased oxidative stress, we described the copper-binding site located in the human C-terminal domain of PrP-HuPrP(90-231), both in the wild-type protein and in the protein carrying the pathological mutation Q212P. We used the synchrotron-based X-ray absorption fine structure technique to study the Cu(II) and Cu(I) coordination geometries in the mutant, and we compared them with those obtained using the wild-type protein. By analyzing the extended X-ray absorption fine structure and the X-ray absorption near-edge structure, we highlighted changes in copper coordination induced by the point mutation Q212P in both oxidation states. While in the wild-type protein the copper-binding site has the same structure for both Cu(II) and Cu(I), in the mutant the coordination site changes drastically from the oxidized to the reduced form of the copper ion. Copper-binding sites in the mutant resemble those obtained using peptides, confirming the loss of short- and long-range interactions. These changes probably cause alterations in copper homeostasis and, consequently, in redox control.     

Cox17 is functional when tethered to the mitochondrial inner membrane

Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.