ESI-MS and MALLS analysis of quaternary structure of molluscan hemocyanins.

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences.

Role of the cellular prion protein in the neuron adaptation strategy to copper deficiency.

Copper transporter 1 (CTR1), cellular prion protein (PrP(C)), natural resistance-associated macrophage protein 2 (NRAMP2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In this study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn superoxide dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrP(C) transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expressions were unvaried. The up-regulated PrP(C) has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrP(C) in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrP(C) accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances.

Laboratory evolution of copper tolerant yeast strains.

BACKGROUND: Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts. RESULTS: We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts. CONCLUSIONS: Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper-binding proteome. However, copper elicits different physiological and molecular reactions in yeasts with different backgrounds.

Cuprizone neurotoxicity, copper deficiency and neurodegeneration.

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.

Fluorimetric analysis of copper transport mechanisms in the b104 neuroblastoma cell model: a contribution from cellular prion protein to copper supplying.

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP(C)) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP(C) proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP(C)-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP(C) in mediating copper uptake by clathrin-dependent endocytosis.

Structural characterization of the ceruloplasmin: lactoferrin complex in solution.

Ceruloplasmin is a copper protein found in vertebrate plasma, which belongs to the family of multicopper oxidases. Like transferrin of the blood plasma, lactoferrin, the iron-containing protein of human milk, saliva, tears, seminal plasma and of neutrophilic leukocytes tightly binds two ferric ions. Human lactoferrin and ceruloplasmin have been previously shown to interact both in vivo and in vitro forming a complex. Here we describe a study of the conformation of the human lactoferrin/ceruloplasmin complex in solution using small angle X-ray scattering. Our ab initio structural analysis shows that the complex has a 1:1 stoichiometry and suggests that complex formation occurs without major conformational rearrangements of either protein. Rigid-body modeling of the mutual arrangement of proteins in the complex essentially yields two families of solutions. Final discrimination is possible when integrating in the modeling process extra information translating into structural constraints on the interaction between the two partners.

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry.

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin.

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.

Molecular evolution and phylogeny of sipunculan hemerythrins.

We sequenced seven new hemerythrin (Hr) and myohemerythrin (myoHr) cDNAs from Sipunculus nudus and Golfingia vulgaris vulgaris, thus providing new comparative data that significantly increase the set of the known Hr and myoHr sequences. Bayesian inference, maximum likelihood, and maximum parsimony phylogenetic analyses were performed to investigate the evolutionary relationships among the sipunculan and annelid Hr and myoHr sequences. Annelid myoHrs and sipunculan Hrs were resolved as monophyletic groups. Conversely sipunculan myoHrs did not form a clade. The Hrs having an octameric quaternary structure were resolved as a monophyletic group. The octameric cluster includes the Hr sequences of G. v. vulgaris, Themiste zostericola, Themiste discriptum, and Phascolopsis gouldii. Siphonosoma cumanense Hr, which has a trimeric quaternary structure, assumes a sister group position of the octameric clade. The S. nudus Hrs, having a quaternary structure that is not well resolved, assume an isolate position within the Hrs clade. Likelihood-based analyses reveal that purifying selection mainly characterized the evolution of Hr and myoHr. We suggest that starting from a common gene ancestor, two distinct quaternary structures evolved in the sipunculan Hrs and this differentiation was probably favored by the acquisition of distinct physiological advantages.

Optimization of two-joint arm movements: a model technique or a result of natural selection?

The fossil record of early hominids suggests that their Arm length, and presumably stature and weight, had a tendency to increase. Using the minimum jerk principle and a related formulation of averaged specific power, ASP, with regard to selected two-joint Arm movements, the current paper explores relationships between ASP, hand trajectory length (or Arm length, or body mass) and mean movement speed, deriving relationships which indicate that ASP is proportional to cubic mean movement speed, but inversely proportional to hand trajectory length (or Arm length, or 1/3 power of body mass). Accordingly, an ;ecological niche' is modeled in a three-parameter space. Either ASP maximization for fixed movement time, or ASP minimization for fixed mean movement speed, taken as selective optimization criterion, allows the increasing of human Arm length during evolution, regardless of the arm-to-forearm length ratio.

Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii.

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.

Comparative structural analysis of low-molecular mass fragments of Rapana venosa hemocyanin obtained using two different procedures.

Different fragments of the hemocyanin (Hc) isolated from the gastropod Rapana venosa containing a single functional unit (50 kDa), two functional units (100 kDa) and three functional units (150 kDa) were obtained in a dissociating buffer in the presence of Zn2+ and purified to homogeneity. Their conformations in solution were studied by means of small angle X-ray scattering (SAXS) and compared with those of the corresponding fragments previously obtained by limited proteolysis [Arch. Biochem. Biophys., 2000, 373, 154]. The overall shape of each fragment was determined using an ab initio approach. The crystal structures of the functional unit e from the same Hc and from another molluscan Hc (Octopus dofleini) were used to model 100 and 150 kDa fragments using rigid body movements to fit the corresponding SAXS patterns. Interesting differences were observed between the functional unit organization in the low-molecular mass fragments according to the two preparation methods, suggesting different localizations within the 11S functional subunit.

Extracellular copper ions regulate cellular prion protein (PrPC) expression and metabolism in neuronal cells.

The physiological functions of cellular prion protein (PrP(C)) remain unclear. It has been demonstrated that PrP(C) is a copper binding protein and proposed that its functions could be strictly linked to copper metabolism and neuroprotection. The aim of this study was to clarify how extracellular copper modifies PrP(C) expression and metabolism in cultured neurones. We reported here that copper delivered at physiological concentrations significantly decreases PrP(C) mRNA expression in GN11 neurones. Moreover, copper increases the release of PrP(C) into the culture medium. These results indicate that extracellular copper strongly affects the amount of cellular PrP and might represent an interesting strategy to decrease the expression of PrP(C) in neurones and its conversion in the pathological isoform PrP(Sc).

Model building of a molluscan hemocyanin from X-ray solution scattering.

Molluscan hemocyanins are proteins of a truly enormous size. Because of this, determination of their quaternary structure at high resolution cannot easily be obtained by standard methods such as X-ray crystallography and NMR. Therefore, different approaches, using several low-resolution techniques are currently necessary to understand hemocyanin structure. In this work a model of the Rapana venosa hemocyanin has been obtained from a template model and small angle X-ray scattering (SAXS) data. The template model was built from the electron density of the closely related Haliotis tuberculata hemocyanin and a computer program was written to fit this model to the SAXS data using the simulated annealing algorithm.

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution.

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.

Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion.

The primary structures of two biantennary N -glycans of the glycoprotein Rapana venosa (marine snail) haemocyanin were determined. Two different structural subunits have been found in R. venosa haemocyanin: RvH1 and RvH2. The carbohydrate content of the N-terminal functional unit RvH1-a of RvH1 was studied and compared with the N-terminal functional unit RvH2-a of RvH2. Oligosaccharide fragments were released from the glycoprotein by Smith degradation of a haemocyanin pronase digest and separated on a Superdex 300 column. The glycopeptide fragments, giving a positive reaction for the orcinol/H2SO4 method, were separated by HPLC. In order to determine the linked sugar chains to the hinge glycopeptides isolated from the functional unit RvH1-a, several techniques were applied, including capillary electrophoresis, matrix-assisted laser desorption ionization-MS and electrospray ionization-MS in combination with glycosidase digestion. On the basis of these results and amino acid sequence analysis, we concluded that the functional unit RvH1-a contains 7% oligosaccharides N-glycosidically attached to Asn262 and Asn401, and the following structures were suggested:[structure: see text]

Structure and function of the globin and globin gene from the Antarctic mollusc Yoldia eightsi.

The mechanism of adaptation of haemoglobin from the Antarctic mollusc Yoldia eightsi to its low-temperature environment is a decrease in the oxygen affinity via an increased ligand-dissociation rate. At 2 degrees C this haemoglobin has an oxygen affinity similar to other haemoglobins at 25 degrees C. At 25 degrees C, Yoldia haemoglobin shows a low oxygen affinity, resembling that of human deoxyhaemoglobin. The mechanism involves a lower binding energy to oxygen, suggesting a loss or weakening of the usual hydrogen bond, leading to a higher oxygen-dissociation rate. However, Yoldia haemoglobin has the usual distal and proximal histidines, so the primary structure alone does not provide an obvious explanation for the low affinity. The CO-binding kinetics are biphasic, with the fraction of slow phase increasing at higher protein concentrations, indicating the formation of dimers or a higher level of polymerization. The protein-protein interaction appears to be of hydrophobic nature, since it can be partially reversed by addition of ethylene glycol as co-solvent. While the CO-association rates differ by a factor of 10, the oxygen equilibrium data could be simulated with a single affinity. The Yoldia haemoglobin gene contains three introns, interrupting the coding region at position NA1.2, B12.2 and G7.0. The conservation of the B12.2 and G7.0 introns is in contrast with the unprecedented NA1.2 intron. Phylogenetic analyses reveal a gene tree where the Yoldia haemoglobin gene is separated from other mollusc globin genes, confirming the specific adaptation of the Yoldia haemoglobin.

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.

Oxidized derivatives of Octopus vulgaris and Carcinus aestuarii hemocyanins at pH 7.5 and related models by x-ray absorption spectroscopy.

The binuclear copper sites of the met and met-azido derivatives of Octopus vulgaris and Carcinus aestuarii hemocyanins at pH 7.5 were characterized by high-resolution x-ray absorption spectroscopy in the low energy region (XANES) and in the higher region (EXAFS). The accuracy of the analysis of the data was tested with two mononuclear and six binuclear copper(II) complexes of the poly(benzimidazole) ligand systems 2-BB, L-5,5 and L-6,6 (Casella et al., 1993, Inorg. Chem. 32:2056-2067; 1996, Inorg. Chem. 35:1101-1113). Their structural and reactivity properties are related to those of the protein's derivatives. The results obtained for those models with resolved x-ray structure (the 2-BB-aquo and azido mononuclear complexes, and the binuclear L-5,5 Cu(II)-bis(hydroxo) (Casella et al., unpublished)), extends the validity of our approach to the other poly(benzimidazole)-containing complexes and to the hemocyanin derivatives. Comparison between the protein's and the complexes' data, support a description of the met-derivatives as a five-coordinated O-bridged binuclear copper(II) center and favors, for both species, a bis(hydroxo) structure with a 3-A Cu-Cu distance. For O. vulgaris met-azido derivative a mu-1,3 bridging mode for the ligand appears the most likely. The structural situation of C. aestuarii met-azido-derivative is less clear: a mu-1,1 mode is favored, but a terminal mode cannot be excluded.