A key structural role for active site type 3 copper ions in human ceruloplasmin

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.     

Mechanisms of copper incorporation into human ceruloplasmin

Ceruloplasmin is a multicopper oxidase essential for normal iron homeostasis. To elucidate the mechanisms of copper incorporation into this protein, holoceruloplasmin biosynthesis was examined by immunoblot analysis and (64)Cu metabolic labeling of Chinese hamster ovary cells transfected with cDNAs encoding wild-type or mutant ceruloplasmin. This analysis reveals that the incorporation of copper into newly synthesized apoceruloplasmin in vivo results in a detectable conformational change in the protein. Strikingly, despite the unique functional role of each copper site within ceruloplasmin, metabolic studies indicate that achieving this final conformation-driven state requires the occupation of all six copper-binding sites with no apparent hierarchy for copper incorporation at any given site. Consistent with these findings a missense mutation (G631R), resulting in aceruloplasminemia and predicted to alter the interactions at a single type I copper-binding site, results in the synthesis and secretion only of apoceruloplasmin. Analysis of copper incorporation into apoceruloplasmin in vitro reveals that this process is cooperative and that the failure of copper incorporation into copper-binding site mutants observed in vivo is intrinsic to the mutant proteins. These findings reveal a precise and sensitive mechanism for the formation of holoceruloplasmin under the limiting conditions of copper availability within the cell that may be generally applicable to the biosynthesis of cuproproteins within the secretory pathway.     

Role of copper in thermal stability of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six domains with one type-1 copper in each of domains 2, 4, and 6; the remaining coppers form a catalytic trinuclear cluster at the interface between domains 1 and 6. To assess the role of the coppers in CP thermal stability, we have probed the thermal unfolding process as a function of scan rate of holo- and apo-forms using several detection methods (circular dichroism, aromatic and 8-anilino-naphthalene-1-sulfonic acid fluorescence, visible absorption, activity, and differential scanning calorimetry). Both species of CP undergo irreversible thermal reactions to denatured states with significant residual structure. For identical scan rates, the thermal midpoint appears at temperatures 15-20° higher for the holo- as compared with the apo- form. The thermal data for both forms were fit by a mechanistic model involving two consecutive, irreversible steps (N → I → D). The holo-intermediate, I, has lost one oxidized type-1 copper and secondary structure in at least one domain; however, the trinuclear copper cluster remains intact as it is functional in oxidase activity. The activation parameters obtained from the fits to the thermal transitions were used to assess the kinetic stability of apo- and holo-CP at physiological temperatures (i.e., at 37°C). It emerges that native CP (i.e., with six coppers) is rather unstable and converts to I in <1 day at 37°C. Nonetheless, this form remains intact for more than 2 weeks and may thus be a biologically relevant state of CP in vivo. In contrast, apo-CP unfolds rapidly: the denatured state is reached in <2 days at 37°C.     

Milk ceruloplasmin is a valuable source of nutrient copper ions for mammalian newborns

This research focuses on the role of milk ceruloplasmin (Cp), the main extracellular copper-containing protein of vertebrates, as a source of copper for newborns. In the first part of the study, Cp concentration and Cp-associated copper were measured in human skimmed milk at the 1st and the 5th days postpartum. It was shown that most of the copper was associated with Cp and that the decrease in copper concentration during lactation was related to the drop of Cp levels. The following in vivo experiments demonstrated that milk [(125)I]Cp per os administered to 6-day-old rats (embryonic-type copper metabolism) was transported into their bloodstream. The electrophoretic mobility and relative molecular weight of [(125)I]Cp transferred through the cellular barrier remained unaltered. However, 22-day-old rats (adult-type copper metabolism) digested the administered milk [(125)I]Cp completely. In the final part of the study, newborn rats were fed with baby formula for 8d. It was found that these rats switched their copper metabolism from embryonic type to adult type earlier than their littermates fed by dams. Activation of Cp gene expression in the liver, increased Cp and copper concentrations in the blood, and reduced copper content of the liver were observed in the rats fed with baby formula. In the brain, no copper concentration change was observed, but Cp and copper concentrations were dramatically increased in the cerebrospinal fluid. The role of milk Cp as a source of copper adapted to embryonic-type copper metabolism is discussed.     

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.     

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.     

The participation of human serum albumin domains in chemical and thermal unfolding

Fluorescence spectroscopy and differential scanning calorimetry were used to follow local and global changes in human serum albumin domains during chemical and thermal denaturation of this protein. Results suggests that thermal and chemical treatments involved an unfolding pathway of at least two steps and that domain IIA is not homogeneous. Unfolding at site I exposes a larger hydrophobic area to the solvent than at site II. The bilirubin-binding site showed atypical behavior: a significant increase in the hydrophobic area was exposed to the solvent when its binding site was denatured by guanidine hydrochloride. This result might be due to the high specificity of the bilirubin-binding site, whose binding makes an extensive conformational change in the environment of this site.     

Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.     

Binding and uptake of copper from ceruloplasmin

Specific binding of [67Cu]ceruloplasmin to plasma membrane containing preparations from rat tissues was shown in the presence of an excess of nonradioactive Cu(II) or ceruloplasmin. With Cu(II) there was positive cooperativity and an apparent KD of 10(-7) M. The effects of both "cold" ligands was partly additive. No "specific" binding was shown with Zn(II), unrelated proteins and after boiling the membranes. Total and specific binding of [67Cu]ceruloplasmin were 2-7 fold greater for heart and brain than for liver preparations, per g tissue or per mg protein, +/- correction for yield of 5'-nucleotidase. Cu(II) also inhibited uptake of [67Cu] from ceruloplasmin by CHO cells, but monensin did not, suggesting uptake of ceruloplasmin Cu occurs at the cell surface.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.     

Embryotoxicity of silver ions is diminished by ceruloplasmin—further evidence for its role in the transport of copper

The effect of alimentary administration of silver salts upon embryogenesis in rats was studied. Feeding of female rats throughout the term on a regular diet supplemented with AgCl did not cause alterations of their physiological functions, despite the fact that enzymatically active copper-containing ceruloplasmin (CP) was eliminated from the blood plasma. However, developmental abnormalities of embryos, their prenatal death or the 100% mortality of the newborns in the first 24 h of life was seen. Copper content in placenta and fetal tissues was strongly diminished. Cu, Zn-superoxide dismutase (SOD) activity decreased in cytoplasm of embryonic cells along with a drop, though less pronounced, in the tissues of the pregnant females. Embryotoxicity of AgCl was seriously diminished by repetitive injections of native CP to the pregnant rats. Such treatment resulted in an increase of SOD activity in placenta and embryonic tissues. The mortality of the newborns also became less. It is suggested that the embryotoxic effect of AgCl is caused by its ability to interfere with copper metabolism, in particular by altering the copper-transporting function of CP.     

Protective effect of ceruloplasmin during human erythrocyte lysis induced by Fe2+ ions

In order to elucidate the nature of linkage between the oxidase activity and protective effect of ceruloplasmin during the Fe2(+)-induced lysis of erythrocytes, the both factors were identified in ceruloplasmin samples prepared from blood sera of healthy donors and patients with hepatocerebral dystrophy (HCD). It was found that the oxidase activity of healthy donor ceruloplasmin markedly exceeds that of HCD patients, whereas the protective effect of the HCD protein, contrariwise, markedly exceeds that of normal ceruloplasmin. The data obtained suggest that the protective effect of ceruloplasmin during Fe2(+)-induced erythrocyte lysis is not correlated with its oxidase (ferroxidase, in particular) activity.     

Modulation of the redox state of the copper sites of human ceruloplasmin by chloride

Incubation of human ceruloplasmin with physiological concentrations of chloride at neutral pH invariably caused dramatic changes of both the spectroscopic and the functional properties of the protein. The optical intensity at 610 nm increased up to 60%, with a concomitant decrease at 330 nm and the appearance of new bands between 410 and 500 nm. Signals previously undetectable appeared in the EPR spectrum. On the basis of computer simulations, they were interpreted as stemming from an oxidized type 1 copper site and from a half-reduced type 3 copper pair. Removal of chloride completely restored the original optical and EPR lineshapes. Hydrogen peroxide, added to ceruloplasmin in the presence of chloride, was able to capture the electron of the half-reduced type 3 site and to yield a protein insensitive to subsequent removal and readdition of the anion. As a whole, the spectroscopic data indicate that a blue site is partially reduced in the resting protein and that, upon binding of chloride, human ceruloplasmin undergoes a structural change leading to displacement of an electron from the reduced type 1 site to the type 3 site pair. Chloride dramatically affected the catalytic efficiency of human ceruloplasmin. At neutral pH, the anion was an activator of the oxidase activity, being able to enhance up to tenfold the catalytic rate. AtpH < 6, in line with all previous reports, chloride strongly inhibited the activity. At intermediate pH values, i.e., around 6, the effect was composite, with an activating effect at low concentration and an inhibitory effect at higher concentration. Since chloride is present at very high concentrations in the plasma, these results suggest that human ceruloplasmin is, in the plasma, under control of this anion.