Localization of vanabins, vanadium-binding proteins, in the blood cells of the vanadium-rich ascidian, Ascidia sydneiensis samea

Some species of the family Ascidiidae accumulate vanadium in concentrations in excess of 350 mM, which is about 10 (7)-fold higher than the concentration of vanadium in seawater. In these species, signet ring cells with a single large vacuole in which vanadium ions are contained function as vanadium-accumulating cells. These have been termed vanadocytes. We recently isolated five vanadium-binding proteins, which we named Vanabin1, Vanabin2, Vanabin3, Vanabin4, and VanabinP, from vanadocytes of the vanadium-rich ascidian Ascidia sydneiensis samea. In this study, we analyzed localization of the Vanabins in the blood cells of A. sydneiensis samea using monoclonal antibodies and confocal microscopy. The Vanabin1 and Vanabin2 proteins were found in the cytoplasm and/or in some organelles of vanadocytes. Vanabin3 was also detected in the cytoplasm, while Vanabin4 was found exclusively in the cytoplasmic membrane.     

Identification of Vanabin-interacting protein 1 (VIP1) from blood cells of the vanadium-rich ascidian Ascidia sydneiensis samea

Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes.     

Characterization of a novel vanadium-binding protein (VBP-129) from blood plasma of the vanadium-rich ascidian Ascidia sydneiensis samea

The ascidians, the so-called sea squirts, accumulate high levels of vanadium, a transition metal. Since Henze first observed this physiologically unusual phenomenon about one hundred years ago, it has attracted interdisciplinary attention from chemists, physiologists, and biochemists. The maximum concentration of vanadium in ascidians can reach 350 mM, and most of the vanadium ions are stored in the +3 oxidation state in the vacuoles of vanadium-accumulating blood cells known as vanadocytes. Many proteins involved in the accumulation and reduction of vanadium in the vanadocytes, blood plasma, and digestive tract have been identified. However, the process by which vanadium is taken in prior to its accumulation in vanadocytes has not been elucidated. In the present study, a novel vanadium-binding protein, designated VBP-129, was identified from blood plasma of the vanadium-rich ascidian Ascidia sydneiensis samea. Although VBP-129 mRNA was transcribed in all A. sydneiensis samea tissues examined, the VBP-129 protein was exclusively localized in blood plasma and muscle cells of this ascidian. It bound not only to VO(2+) but also to Fe(3+), Co(2+), Cu(2+), and Zn(2+); on the other hand, a truncated form of VBP-129, designated VBP-88, bound only to Co(2+), Cu(2+) and Zn(2+). In a pull-down assay, an interaction between VanabinP and VBP-129 occurred both in the presence and the absence of VO(2+). These results suggest that VBP-129 and VanabinP function cooperatively as metallochaperones in blood plasma.     

Selective metal binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea

Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation. However, the role of Vanabin2 in vanadium accumulation by the ascidian has not yet been determined. In the present study, the effects of acidic pH on selective metal binding to Vanabin2 and on the secondary structure of Vanabin2 were examined. Vanabin2 selectively bound to V(IV), Fe(III), and Cu(II) ions under acidic conditions. In contrast, Co(II), Ni(II), and Zn(II) ions were bound at pH 6.5 but not at pH 4.5. Changes in pH had no detectable effect on the secondary structure of Vanabin2 under acidic conditions, as determined by circular dichroism spectroscopy, and little variation in the dissociation constant for V(IV) ions was observed in the pH range 4.5-7.5, suggesting that the binding state of the ligands is not affected by acidification. Taken together, these results suggest that the reason for metal ion dissociation upon acidification is attributable not to a change in secondary structure but, rather, that it is caused by protonation of the amino acid ligands that complex with V(IV) ions.     

Identification of Vanabin-interacting protein 1 (VIP1) from blood cells of the vanadium-rich ascidian Ascidia sydneiensis samea

Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes.     

Expressed sequence tag analysis of blood cells in the vanadium-rich ascidian,Ascidia sydneiensis samea--a survey of genes for metal accumulation

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10(7) times that found in seawater. The vanadium ions are stored in vacuoles located within vanadium-containing blood cells, vanadocytes. To investigate the phenomenon, an expressed sequence tag analysis (EST) of a cDNA library of Ascidia sydneiensis samea blood cells was carried out. Three hundred clones were obtained and sequenced by EST analysis. A similarity search revealed that 158 of the clones (52.7%) were known genes, and 142 of the clones (47.3%) did not have any similarity to genes registered in the SwissProt database. According to the functions of their genes the identified EST clones were categorized into eight types of clones; these consisted of genes; metal-related proteins (29 clones), signal transduction (22 clones), protein synthesis (17 clones), nuclear proteins (17 clones), cytoskeleton and motility (14 clones), energy conversion (3 clones), hypothetical proteins (11 clones), and others (45 clones). The ferritin homologue has a high degree of similarity to that of mammals; the iron-binding sites of ferritin are well conserved including His-118 which is important for capturing Fe(2+), also works as a ligand for VO(2+).     

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

Background

Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO²⁺ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined.

Methods

In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method.

Results

Mutation at K10/R60 (site 1) markedly reduced the affinity for VO²⁺. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO²⁺. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO²⁺.

General significance

These results suggested that the site 1 is a high affinity binding site for VO²⁺, while the site 2 composes a moderate affinity site for multiple VO²⁺.     

Selective metal binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea

Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation. However, the role of Vanabin2 in vanadium accumulation by the ascidian has not yet been determined. In the present study, the effects of acidic pH on selective metal binding to Vanabin2 and on the secondary structure of Vanabin2 were examined. Vanabin2 selectively bound to V(IV), Fe(III), and Cu(II) ions under acidic conditions. In contrast, Co(II), Ni(II), and Zn(II) ions were bound at pH 6.5 but not at pH 4.5. Changes in pH had no detectable effect on the secondary structure of Vanabin2 under acidic conditions, as determined by circular dichroism spectroscopy, and little variation in the dissociation constant for V(IV) ions was observed in the pH range 4.5–7.5, suggesting that the binding state of the ligands is not affected by acidification. Taken together, these results suggest that the reason for metal ion dissociation upon acidification is attributable not to a change in secondary structure but, rather, that it is caused by protonation of the amino acid ligands that complex with V(IV) ions.     

Expressed Sequence Tag Analysis of Vanadocytes in a Vanadium-Rich Ascidian, <Emphasis Type="Italic">Ascidia sydneiensis samea</Emphasis>

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10⁷ times higher than that in seawater. In these species signet ring cells, with a single huge vacuole in which vanadium ion is contained, function as vanadium-accumulating cells, vanadocytes. To investigate the mechanism underlying this phenomenon, we performed an expressed sequence tag (EST) analysis of a complementary DNA library from vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea. We determined the nucleotide sequences of 1000 ESTs and performed a BLAST analysis against the SwissProt database. We found 93 clones of metal-related gene homologues, including the ferritin heavy subunit, hemocyanin, and metallothionein. Two ESTs, in particular, exhibited significant similarity to vanabins that have been extracted from A. sydneiensis samea blood cells as low molecular weight vanadium-binding proteins. We have named the genes encoding these ESTs vanabin3 and vanabin4. Immobilized metal ion affinity chromatography revealed that these novel vanabin homologues bind vanadium(IV) ions.     

VanabinP, a novel vanadium-binding protein in the blood plasma of an ascidian, Ascidia sydneiensis samea

Some ascidians accumulate high levels of the transition metal vanadium in their blood cells. The process of vanadium accumulation has not yet been elucidated. In this report, we describe the isolation and cDNA cloning of a novel vanadium-binding protein, designated as VanabinP, from the blood plasma of the vanadium-rich ascidian, Ascidia sydneiensis samea. The predicted amino acid sequence of VanabinP was highly conserved and similar to those of other Vanabins. The N-terminus of the mature form of VanabinP was rich in basic amino acid residues. VanabinP cDNA was originally isolated from blood cells, as were the other four Vanabins. However, Western blot analysis revealed that the VanabinP protein was localized to the blood plasma and was not detectable in blood cells. RT-PCR analysis and in situ hybridization indicated that the VanabinP gene was transcribed in some cell types localized to peripheral connective tissues of the alimentary canal, muscle, blood cells, and a portion of the branchial sac. Recombinant VanabinP bound a maximum of 13 vanadium(IV) ions per molecule with a Kd of 2.8 x 10(-5) M. These results suggest that VanabinP is produced in several types of cell, including blood cells, and is immediately secreted into the blood plasma where it functions as a vanadium(IV) carrier.     

Tyrosine modification enhances metal-ion binding

Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK(8) (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe(3+) binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6-2.8 microM at pH 6.5). Measurement of Ca(2+) binding with a Ca(2+)-selective electrode revealed that phosphorylated CCK(8) bound two Ca(2+) ions. CCK(8) and sulfated CCK(8) each bound only one Ca(2+) ion with lower affinity. Binding of Ca(2+), Zn(2+) or Bi(3+) to phosphorylated CCK(8) did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe(3+). The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.     

Bioaccumulation of copper ions by Escherichia coli expressing vanabin genes from the vanadium-rich ascidian Ascidia sydneiensis samea

The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 micro M copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.     

Surface topography of histidine residues in lysozymes.

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.     

Thermostable Pyrococcus woesei beta-D-galactosidase--high level expression, purification and biochemical properties

The gene encoding beta-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei beta-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei beta-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type beta-D-galactosidase (51100 U/mg). The activity of P. woesei beta-D-galactosidase was enhanced by thiol compounds, Mg(2+) ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca(2+) ions had no effect.     

Participation of thioredoxin in the V(V)-reduction reaction by Vanabin2

Background

It is well-understood that ascidians accumulate high levels of vanadium, a reduced form of V(III), in an extremely acidic vacuole in their blood cells. Vanabins are small cysteine-rich proteins that have been identified only from vanadium-rich ascidians. A previous study revealed that Vanabin2 can act as a V(V)-reductase in the glutathione cascade.

Methods

AsTrx1, a thioredoxin gene, was cloned from the vanadium-rich ascidian, Ascidia sydneiensis samea, by PCR. AsTrx1 and Vanabin2 were prepared as recombinant proteins, and V(V)-reduction by Vanabin2 was assessed by ESR and ion-exchange column chromatography. Site-directed mutagenesis was performed to examine the direct involvement of cysteine residues. Tissue expression of AsTrx1 was also examined by RT-PCR.

Results

When reduced AsTrx1 and Vanabin2 were combined, Vanabin2 adopted an SS/SH intermediate structure while V(V) was reduced to V(IV). The loss of cysteine residues in either Vanabin2 or AsTrx1 caused a significant loss of reductase activity. V_app and K_app values for Vanabin2-catalyzed V(V)-reduction in the thioredoxin cascade were 0.066 mol-V(IV)/min/mol-Vanabin2 and 0.19 mM, respectively. The K_app value was 2.7-fold lower than that observed in the glutathione cascade. The AsTrx1 gene was expressed at a very high level in blood cells, in which Vanabins 1–4 were co-expressed.

Conclusions

AsTrx1 may contribute to a significant part of the redox cascade for V(V)-reduction by Vanabin2 in the cytoplasm of vanadocytes, but prevails only at low V(V) concentrations.

General significance

This study is the first to report the reduction of V(V) in the thioredoxin cascade.     

Real-time detection of Cu2+ sequestration and release by immobilized apo-metallothioneins using SECM combined with SPR

Scanning electrochemical microscopy (SECM) combined with surface plasmon resonance (SPR), SECM-SPR, was applied for real-time detection of the incorporation of Cu(2+) by apo-metallothionein (apo-MT) immobilized on the SPR substrate and release of Cu(2+) from surface-confined metallothionein (MT). Cu(2+) anodically stripped from a Cu-coated SECM Au tip was sequestered by apo-MT upon its diffusion to the SPR substrate, and release of Cu(2+) by MT was accomplished by generating protons via oxidation of hydroquinone at the tip. The high sensitivity of the SPR instrument is capable of following the structural and compositional changes of MT molecules during the metal sequestration and release processes. Due to the enhanced mass transfer rate at the SECM tip, the complication of mass transfer limitation on kinetic measurements, commonly encountered in flow injection SPR, is circumvented. The time-resolved SPR response reveals stepwise changes among three stable MT structures and allows the number of copper ions coordinated in each structure to be determined. The numbers of copper ions incorporated by each MT molecule in the three structures were determined to be 5, 9, and 12. This work expands the SECM-SPR approach to assessments of the dynamics and affinity of binding of small ions to surface-confined proteins and to studies of proteins that do not undergo facile electron transfer reactions.     

Chloride channel in vanadocytes of a vanadium-rich ascidian Ascidia sydneiensis samea

Ascidians, so-called sea squirts, can accumulate high levels of vanadium in the vacuoles of signet ring cells, which are one type of ascidian blood cell and are also called vanadocytes. In addition to containing high concentrations of vanadium in the +3 oxidation state, the proton concentrations in vanadocyte vacuoles are extremely high. In order to elucidate the entire mechanism of the accumulation and reduction of vanadium by ascidian vanadocytes, it is necessary to clarify the participation of anions, which might be involved as counter ions in the active accumulation of both vanadium and protons. We examined the chloride channel, since chloride ions are necessary for the acidification of intracellular vesicles and coexist with H(+)-ATPase. We cloned a cDNA encoding a chloride channel from blood cells of a vanadium-rich ascidian, Ascidia sydneiensis samea. It encoded a 787-amino-acid protein, which showed striking similarity to mammalian ClC3/4/5-type chloride channels. Using a whole-mount in situ hybridization method that we developed for ascidian blood cells, the chloride channel was revealed to be transcribed in vanadocytes, suggesting its participation in the process of vanadium accumulation.     

Reversible folding of cysteine-rich metallothionein by an overcritical reaction path

A first-order-like state transition is considered to be involved in the restoration of the activities of a few proteins by correctly folding the protein [Phys. Rev. E 66 (2002) 021903]. In order to understand the general applicability of this mechanism, we studied a metallothionein (MT) protein with an unconventional structure, i.e., without any alpha-helix or beta-sheet. MT is a 61 amino-acid peptide. There are 6-7 Zn(2+) ions, which bind avidly to 20 conserved cysteines (Cys) of MT. These properties indicate that the structure of MT is quite different from those of the other proteins. Similar to our previous findings, the denatured MT can be folded without any aggregation via a designated stepwise quasi-static process (an over-critical reaction path). The particle size of folded MT intermediates, determined by dynamic light scattering, shrank right after the first folding stage. It is consistent with a collapse-model. In addition, results from both atomic absorption and circular dichroism (CD) indicate that the stable intermediates may fold to the native conformation but with only partial Zn(2+) binding, which in turn implies that those folding intermediates are in a molten globular state. These reversible unfolding and folding processes indicate that Cys-rich protein, MT, may also be folded by way of a first-order-like state transition mechanism. We suspect that this process may likely be involved in the reaction of the metal substitution process in metal containing enzymes.     

Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu

The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg(2+)-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn(2+) (0.069 min(-1)) > Mg(2+) (0.037 min(-1)) approximately no Me(2+) (0.034 min(-1)) > VO(2+) (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg(2+) retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange. In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (K(d) approximately 10(-7) m). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me(2+) < VO(2+) < Mn(2+) approximately Mg(2+) whereas the binding affinity of GTP increased by one order of magnitude: no Me(2+) < Mg(2+) < VO(2+) < Mn(2+). Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plot analysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R. C., George, P., and Rutman, R. J. (1969) J. Biol. Chem. 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu.Me(2+).GDP complex over the protein-free Me(2+).GDP complex in solution, with the effect greatest in the presence of Mg(2+) by approximately 10 kJ/mol. These combined results show that Mg(2+) is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg(2+) in EF-Tu function.