Vanadium-rich Ascidian Ascidia Sydneiensis Samea Research Articles

Bioaccumulation of Vanadium by Vanadium-Resistant Bacteria Isolated from the Intestine of Ascidia sydneiensis samea.

Isolation of naturally occurring bacterial strains from metal-rich environments has gained popularity due to the growing need for bioremediation technologies. In this study, we found that the vanadium concentration in the intestine of the vanadium-rich ascidian Ascidia sydneiensis samea could reach 0.67mM, and thus, we isolated vanadium-resistant bacteria from the intestinal contents and determined the ability of each bacterial strain to accumulate vanadium and other heavy metals. Nine strains of vanadium-resistant bacteria were successfully isolated, of which two strains, V-RA-4 and S-RA-6, accumulated vanadium at a higher rate than did the other strains. The maximum vanadium absorption by these bacteria was achieved at pH 3, and intracellular accumulation was the predominant mechanism. Each strain strongly accumulated copper and cobalt ions, but accumulation of nickel and molybdate ions was relatively low. These bacterial strains can be applied to protocols for bioremediation of vanadium and heavy metal toxicity.

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 VO 2+ 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 VO 2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO 2+. 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 VO 2+. General significance These results suggested that the site 1 is a high affinity binding site for VO 2+, while the site 2 composes a moderate affinity site for multiple VO 2+.

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians. Methods We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed. Results The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively. General significance This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.

Sequence variation of Vanabin2-like vanadium-binding proteins in blood cells of the vanadium-accumulating ascidian Ascidia sydneiensis samea

The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1–4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea. In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal-ion selectivity, but the overall affinity of 14MT for VO 2+ is higher than that of Vanabin2. Binding number for VO 2+ ions was the same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high-affinity binding sites for VO 2+ are conserved among Vanabin2 variants.

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 VO2+ but also to Fe3+, Co2+, Cu2+, and Zn2+; on the other hand, a truncated form of VBP-129, designated VBP-88, bound only to Co2+, Cu2+ and Zn2+. In a pull-down assay, an interaction between VanabinP and VBP-129 occurred both in the presence and the absence of VO2+. These results suggest that VBP-129 and VanabinP function cooperatively as metallochaperones in blood plasma.

Metal binding ability of glutathione transferases conserved between two animal species, the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum

Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II) > V(IV) > Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.

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.

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.

Glutathione transferases with vanadium-binding activity isolated from the vanadium-rich ascidian Ascidia sydneiensis samea

Some ascidians accumulate vanadium in vanadocytes, which are vanadium-containing blood cells, at high levels and with high selectivity. However, the mechanism and physiological significance of vanadium accumulation remain unknown. In this study, we isolated novel proteins with a striking homology to glutathione transferases (GSTs), designated AsGST-I and AsGST-II, from the digestive system of the vanadium-accumulating ascidian Ascidia sydneiensis samea, in which the digestive system is thought to be involved in vanadium uptake. Analysis of recombinant AsGST-I confirmed that AsGST-I has GST activity and forms a dimer, as do other GSTs. In addition, AsGST-I was revealed to have vanadium-binding activity, which has never been reported for GSTs isolated from other organisms. AsGST-I bound about 16 vanadium atoms as either V(IV) or V(V) per dimer, and the apparent dissociation constants for V(IV) and V(V) were 1.8 × 10 −4 M and 1.2 × 10 −4 M, respectively. Western blot analysis revealed that AsGSTs were expressed in the digestive system at exceptionally high levels, although they were localized in almost all organs and tissues examined. Considering these results, we postulate that AsGSTs play important roles in vanadium accumulation in the ascidian digestive system.

Vanadium-binding protein in a vanadium-rich ascidian Ascidia sydneiensissamea: CW and pulsed EPR studies.

Some of the ascidians belonging to the suborder Phlebobranchia accumulate vanadium ion efficiently from seawater. Clarification of the mechanism of this surprisingly efficient metal-accumulation system is desirable. Two mutually similar vanadium-binding proteins (vanabin1 and vanabin2) have recently been isolated from a vanadium-rich ascidian Ascidia sydneiensis samea. In this study, the vanadium-binding properties of vanabin2 have been investigated by X-band CW EPR and pulsed EPR spectroscopy. CW EPR spectra of samples containing various ratios of VO2+ and vanabin2 invariably exhibited a usual mononuclear-type VO2+ EPR signal with the intensity dependent on the ratio [vanabin]/[V]. EPR titration has shown that vanabin2 can bind up to approximately 23.9 vanadium ions per one molecule, almost all of which ( approximately 84%) are in a mononuclear VO2+ state as estimated by EPR quantitation. Electron spin-echo envelope modulation (ESEEM) spectra of VO-vanabin2 exhibited reasonably intense peaks attributable to amine nitrogen. This is consistent with the fact that vanabin2 is a lysine-rich protein (14 lysines out of 91 amino acids). The present study reveals the uniqueness of vanabin2, which can bind a large number of metal ions in a mononuclear fashion in contrast to the situation for ferritin and metallothionein.

Vanadium-binding proteins (vanabins) from a vanadium-rich ascidian Ascidia sydneiensis samea

Since the beginning of the last century, it has been known that ascidians accumulate high levels of a transition metal, vanadium, in their blood cells, although the mechanism for this curious biological function remains unknown. Recently, we identified three vanadium-binding proteins (vanabins), previously denoted as vanadium-associated proteins (VAPs) [Zool. Sci. 14 (1997) 37], from the cytoplasm fraction of vanadium-containing blood cells (vanadocytes) of the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we describe the cloning, expression, and analysis of the metal-binding ability of vanabins. Recombinant proteins of two independent but related vanabins, vanabin1 and vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1×10 −5 and 2.3×10 −5 M, respectively. The binding of vanadium(IV) to these vanabins was inhibited by the addition of copper(II) ions, but not by magnesium(II) or molybdate(VI) ions. Vanabins are the first proteins reported to show specific binding to vanadium ions; this should provide a clue to resolving the problem regarding the selective accumulation of vanadium in ascidians.

Subunit C of the vacuolar-type ATPase from the vanadium-rich ascidian Ascidia sydneiensis samea rescued the pH sensitivity of yeast vma5 mutants.

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H(+)-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A(1), neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells.

6-Phosphogluconate dehydrogenase is a 45-kDa antigen recognized by S4D5, a monoclonal antibody specific to vanadocytes in the vanadium-rich ascidian Ascidia sydneiensis samea.

We previously prepared a monoclonal antibody, S4D5, specific to vanadocytes, vanadium-containing blood cells, in the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we demonstrate that a 45-kDa antigen recognized by S4D5 is 6-phosphogluconate dehydrogenase (6-PGDH), an enzyme of the pentose phosphate pathway, based on cDNA isolation of RNA samples from blood cells of the ascidian. Western blot analysis confirmed an abundance of 6-PGDH protein in the vanadocytes and localization of 6-PGDH in the soluble extract of the blood cells. Soluble protein exhibited a correspondingly high level of 6-PGDH enzymatic activity. Ascidians are known to selectively accumulate high levels of vanadium in vanadocytes, and the highest recorded concentration of accumulated vanadium is 350 mM, which is 10(7) times the concentration in sea water. Almost all vanadium ions are reduced to the +3 oxidation state via the +4 oxidation state in vanadocytes, indicating that reducing agents must participate in the accumulation. On the other hand, vanadium ions in the +5 oxidation state are reduced to the +4 oxidation state by the presence of NADPH in vitro. Together, these observations suggest that NADPH produced in the pentose phosphate pathway may conjugate the reduction of vanadium from the +5 oxidation state through the +4 oxidation state in vanadocytes of ascidians.