Vanadium Accumulation and Reduction by Vanadium-Accumulating Bacteria Isolated from the Intestinal Contents of Ciona robusta.

The sea squirt Ciona robusta (formerly Ciona intestinalis type A) has been the subject of many interdisciplinary studies. Known as a vanadium-rich ascidian, C. robusta is an ideal model for exploring microbes associated with the ascidian and the roles of these microbes in vanadium accumulation and reduction. In this study, we discovered two bacterial strains that accumulate large amounts of vanadium, CD2-88 and CD2-102, which belong to the genera Pseudoalteromonas and Vibrio, respectively. The growth medium composition impacted vanadium uptake. Furthermore, pH was also an important factor in the accumulation and localization of vanadium. Most of the vanadium(V) accumulated by these bacteria was converted to less toxic vanadium(IV). Our results provide insights into vanadium accumulation and reduction by bacteria isolated from the ascidian C. robusta to further study the relations between ascidians and microbes and their possible applications for bioremediation or biomineralization.

Concomitant downregulation of neuropeptide genes in a marine snail with consecutive sexual maturation after a nuclear disaster in Japan.

Consecutive sexual maturation (CSM), an abnormal reproductive phenomenon of a marine snail, Reishia clavigera, has occurred since 2017 in the vicinity of the Fukushima Daiichi Nuclear Power Plant after the nuclear disaster there. We hypothesized that alterations in animal physiology mediated through genetic/epigenetic changes could sensitively reflect environmental pollution. Understanding the mechanism of this rapid biological response should enable us to quantitatively evaluate long-lasting effects of the nuclear disaster. To determine the molecular basis for CSM, we conducted transcriptome profiling in the ganglia of normal and CSM snails. We assembled the short-read cDNA sequences obtained by Illumina sequencing, and succeeded in characterizing more than 60,000 gene models that include 88 kinds of neuropeptide precursors by BLAST search and experimental curation. GO-enrichment analysis of the differentially expressed genes demonstrated that severe downregulation of neuropeptide-related genes occurred concomitantly with CSM. In particular, significant decreases of the transcripts of 37 genes among 88 neuropeptide precursor genes, including those for myomodulin, PentaFVamide, maturation-associated peptide-5A and conopressin, were commonly observed in female and male CSM snails. By contrast, microseminoprotein precursor was the only exceptional case where the expression was increased in CSM snails. These results indicate that down-regulation of neuropeptide precursors is a remarkable feature of CSM. We also found that factors involved in epigenetic modification rather than transcription factors showed altered patterns of expression upon CSM. Comprehensive expression panels of snail neuropeptide precursors made in this study will be useful tools for environmental assessment as well as for studying marine reproductive biology.

The acidic amino acid-rich C-terminal domain of VanabinX enhances reductase activity, attaining 1.3- to 1.7-fold vanadium reduction.

Ascidians accumulate extremely high levels of vanadium (V) in their blood cells. Several V-related proteins, including V-binding proteins (vanabins), have been isolated from V-accumulating ascidians. In this study, to obtain a deeper understanding of vanabins, we performed de novo transcriptome analysis of blood cells from a V-rich ascidian, Ascidia sydneiensis samea, and constructed a database containing 8532 predicted proteins. We found a novel vanabin with a unique acidic amino acid-rich C-terminal domain, designated VanabinX, in the database and studied it in detail. Reverse-transcription polymerase chain reaction analysis revealed that VanabinX was detected in all adult tissues examined, and was most prominent in blood cells and muscle tissue. We prepared recombinant proteins and performed immobilized metal ion affinity chromatography and a NADPH-coupled V(V)-reductase assay. VanabinX bound to metal ions, with increasing affinity for Cu(II) > Zn(II) > Co(II), but not to V(IV). VanabinX reduced V(V) to V(IV) at a rate of 0.170 µM per micoromolar protein within 30 min. The C-terminal acidic domain enhanced the reduction of V(V) by Vanabin2 to 1.3-fold and of VanabinX itself to 1.7-fold in trans mode. In summary, we constructed a protein database containing 8532 predicted proteins expressed in blood cells; among them, we discovered a novel vanabin, VanabinX, which enhances V reduction by vanabins.

Characterization of vanadium of biological origin for possible applications in physics experiments.

For the first time, vanadium of biological origin, extracted from centrifugal fraction of vanadium-storing blood cells of the Ascidia sydneiensis samea species, was characterized as regards its isotopic composition and content of natural radioactive elements potassium (K), thorium (Th) and uranium (U). The natural abundance of vanadium isotopes has been confirmed with high accuracy, thus excluding a possible selectivity within bio-chemical reactions of vanadium concentration in blood cells from seawater. A large potassium concentration (up to 5500 × 10-6 g g-1) was found in the blood cell samples. The concentration of thorium was determined to be about 30 × 10-9 g g-1, while the uranium concentration was about 150 × 10-9 g g-1. Hence, a highly efficient two-stage purification approach with a total vanadium recovery of better than 70% was developed and applied. The final concentrations of K < 100 × 10-6 g g-1 and of U/Th < 0.5 × 10-9 g g-1 in the purified vanadium-containing samples were achieved. Vanadium extracted from centrifugal fraction of vanadium-storing blood cells after two-stage purification approach could be utilized in various applications, where a high chemical purity compound is required. However, to be used as a source of radiopure vanadium in ultra-low-background experiment aimed to search for 50V beta decay, it should be further purified by Electron Beam Melting against residual potassium.

A draft nuclear-genome assembly of the acoel flatworm Praesagittifera naikaiensis.

BACKGROUND: Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis. FINDINGS: To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ∼17-kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ∼656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ∼70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ∼99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules. CONCLUSIONS: Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians.

Symbiotic bacteria associated with ascidian vanadium accumulation identified by 16S rRNA amplicon sequencing.

Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life.

Xenacoelomorph-Specific Hox Peptides: Insights into the Phylogeny of Acoels, Nemertodermatids, and Xenoturbellids.

Xenacoelomorpha has recently been proposed as an animal taxon that includes acoels, nemertodermatids, and xenoturbellids. Their flattened bodies are very simple and lack discrete organs. The Acoela and Nemertodermatida (which comprise Acoelomorpha) were traditionally regarded as early-diverged extant orders of the class Turbellaria of the phylum Platyhelminthes. Recent anatomical studies and molecular phylogenetic studies demonstrate that the two groups belong to the phylum Xenacoelomorpha together with Xenoturbellida. However, debate remains in regard to whether Xenacoelomorpha is monophyletic, and whether xenacoelomorphs are sisters to all other bilaterians or have close affinity to ambulacrarians. The present study addresses the first question by examining the presence or absence of diagnostic peptide sequences shared by the three taxa. Hox genes have been used to investigate the phylogenetic relationships of metazoans. It has been shown that lophotrochozoans, rotifers, and chaetognaths share diagnostic peptide sequences in the C-terminal region of the Lox5 (Hox5/6/7) homeodomain proteins, which supports the clustering of these taxa. Examination of the decoded genome of the acoel Praesagittifera naikaiensis and reported xenacoelomorph Hox genes revealed that acoels share a peptide NLK(S/T)MSQ(V/I)D, which starts immediately after the homeodomain sequence of the central Hox4/5/6. In addition, we found another diagnostic peptide, KEGKL, in the C-terminal region of the anterior Hox1, which is shared by all the three groups of xenacoelomorphs, but not other bilaterians. Furthermore, two acoels, Praesagittifera naikaiensis and Symsagittifera roscoffensis, share another peptide SG(A/P)PGM in the posterior Hox9/11/13. These results support the designation of the phylum Xenacoelomorpha, in which Acoela is a discrete group.

Structural and Mass Spectrometric Imaging Analyses of Adhered Tunic and Adhesive Projections of Solitary Ascidians.

Most ascidian species settle on underwater substrates during a short free-swimming tadpole larval period. During this process, "rapid adhesion" occurs on adhesive papillae located at the anterior region of the cephalenteron. Settled and transformed ascidians subsequently expand the attachment area by "slow adhesion" with ampullae. In the present study, we attempted to identify the ultrastructures related to the adhesion process and adhesive materials in the ascidian tunic and to elucidate the biological function of vanadium in adhesion. We focused on an adhesive organ named the adhesive projection, which is newly generated by the adhered tunic to enlarge the bonding area between ascidian and substrate. Based on its structure and the presence of vanadiumcontaining blood cells, the adhesive projection was considered to be a large tunic vessel. At the adhered tunic, eosinophilic regions and migrated tunic cells were observed, but metal deposition was not detected. We speculate that the eosinophilic materials were components of the adhesive glue, and these are likey produced in epithelial cells, tunic cells, or both. Furthermore, using imaging mass spectrometry, we identified eight tunic-specific molecules as glue candidates.

A flow-through column electrolytic cell for supercritical fluid chromatography.

A novel flow-through column electrolytic cell was proposed as a detector to obtain current signals for supercritical fluid chromatography. The electrochemical cell consisted of two electrodes and its holder, and a working and a counter electrode were fabricated from 192 carbon strings, which were composed of 400 carbon fibers of 10 µm in diameter filled into a heat-shrinkable tube. These electrodes were placed in the center of a holder made from polyether ether ketone blocks and they were separated by polytetrafluoroethylene membrane filters. To evaluate the sensitivity of this cell, a standard solution of ferrocene was injected into the supercritical fluid chromatography system connected to the electrolytic cell. The ferrocene was eluted through a silica gel column using a mixture of a mobile phase of supercritical CO2 and a modifier of methanol containing ammonium acetate. The current peak area of ferrocene correlated to the ferrocene concentration in the range of 10-400 µmol/L (r = 0.999). Moreover, the limit of detection on the column estimated from a signal-to-noise ratio of 3 was 9.8  × 10-13  mol.

Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

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 0.67 mM, 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.

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. Vapp and Kapp values for Vanabin2-catalyzed V(V)-reduction in the thioredoxin cascade were 0.066mol-V(IV)/min/mol-Vanabin2 and 0.19mM, respectively. The Kapp 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.

Metal ion selectivity of the vanadium(V)-reductase Vanabin2.

In a previous study, Vanabin2, a member of a family of V(IV)-binding proteins, or Vanabins, was shown to act as a V(V)-reductase. The current study assesses the ability of Vanabin2 to reduce various transition metal ions in vitro. An NADPH-coupled oxidation assay yielded no evidence of reduction activity with the hexavalent transition metal anions, Mo(VI)O4(2-) and W(VI)O4(2-), or with three divalent cations, Mn(II), Ni(II), and Co(II). Although Cu(II) is readily reduced by glutathione and is gradually oxidized in air, this process was not affected by the presence of Vanabin2. In the experiments conducted thus far, Vanabin2 acts only as a V(V)-reductase. This high selectivity may account for the metal ion selectivity of vanadium accumulation in ascidians.

Metal-binding domains and the metal selectivity of the vanadium(IV)-binding protein VBP-129 in blood plasma.

Ascidians are well known to accumulate extremely high levels of vanadium in their blood cells. Several key proteins related to vanadium accumulation and physiological function have been isolated from vanadium-rich ascidians. Of these, vanadium(IV)-binding protein-129 (VBP-129) is a unique protein that has been identified from the blood plasma of an ascidian Ascidia sydneiensis samea, but its metal binding domains are not known. In this study, several deletion and point mutants of VBP-129 were generated, and their metal binding abilities were assessed by immobilized metal ion affinity chromatography (IMAC) and electron spin resonance spectroscopy (ESR). The internal partial protein, VBP-Int41, did not bind to V(IV), but the two constructs, VBP-N52 and VBP-Int55, added with additional 11 or 14 neighboring amino acids bound to V(IV). Mutations for cysteine-47 and lysine-50 in VBP-Int55 diminished V(IV)-binding in VBP-Int55, suggesting that these amino acid residues play important roles in binding V(IV). ESR titration analysis revealed that VBP-129, VBP-N52 and VBP-Int55 could bind to 6, 3 and 2 V(IV) ions, respectively. ESR spectrum analysis indicated a N(2)O(2) coordination geometry, which is similar to vanabins. The cysteines may contribute to the maintenance of the three-dimensional structure that is necessary for binding V(IV) ions. VBP-129 did not have a V(V)-reductase activity, as expected from its tissue localization in blood plasma. This study provided the evidences that VBP-129 possesses V(IV)-binding domains that make a similar coordination to V(IV) as those by vanabins but VBP-129 acts solely as a V(IV)-chaperon in blood plasma.

Differential gene regulation by V(IV) and V (V) ions in the branchial sac, intestine, and blood cells of a vanadium-rich ascidian, Ciona intestinalis.

Ascidians are hyperaccumulators that have been studied in detail. Proteins and genes involved in the accumulation process have been identified, but regulation of gene expression related to vanadium accumulation remains unknown. To gain insights into the regulation of gene expression by vanadium in a genome-wide manner, we performed a comprehensive study on the effect of excess vanadium ions on a vanadium-rich ascidian, Ciona intestinalis, using a microarray. RT-PCR and enzyme activity assay were performed from the perspective of redox and accumulation of metal ions in each tissue. Glutathione metabolism-related proteins were significantly up-regulated by V(IV) treatment. Several genes involved in the transport of vanadium and protons, such as Nramp and V-ATPase, were significantly up-regulated by V(IV) treatment. We observed significant up-regulation of glutathione synthesis and degradation pathways in the intestine and branchial sac. In blood cells, expression of Ci-Vanabin4, glutathione reductase activity, glutathione levels, and vanadium concentration increased after V(IV) treatment. V(IV) treatment induced significant changes related to vanadium exclusion, seclusion, and redox pathways in the intestine and branchial sac. It also induced an enhancement of the vanadium reduction and accumulation cascade in blood cells. These differential responses in each tissue in the presence of excess vanadium ions suggest that vanadium accumulation and reduction may have regulatory functions. This is the first report on the gene regulation by the treatment of vanadium-rich ascidians with excess vanadium ions. It provided much information for the mechanism of regulation of gene expression related to vanadium accumulation.

Identification of a novel vanadium-binding protein by EST analysis on the most vanadium-rich ascidian, Ascidia gemmata.

Ascidians are known to accumulate extremely high levels of vanadium in their blood cells (up to 350 mM). The branchial sac and the intestine are thought to be the first tissues to contact the outer environment and absorb vanadium ions. The concentration of vanadium in the branchial sac and the intestine of the most vanadium-rich ascidian Ascidia gemmata were determined to be 32.4 and 11.9 mM, respectively. Using an expressed sequence tag (EST) analysis of a cDNA library from the intestine of A. gemmata, we determined 960 ESTs and found 55 clones of metal-related gene orthologs, 6 redox-related orthologs, and 18 membrane transporter orthologs. Among them, two genes, which exhibited significant similarity to the vanadium-binding proteins of other vanadium-rich ascidian species, were designated AgVanabin1 and AgVanabin2. Immobilized metal ion affinity chromatography revealed that recombinant AgVanabin1 bound to metal ions with an increasing affinity for Cu(II) > Zn(II) > Co(II) and AgVanabin2 bound to metal ions with an increasing affinity for Cu(II) > Fe(III) > V(IV). To examine the use of AgVanabins for a metal absorption system, we constructed Escherichia coli strains that expressed AgVanabin1 or AgVanabin2 fused to maltose-binding protein and secreted into the periplasmic space. We found that the strain expressing AgVanabin2 accumulated about 13.5 times more Cu(II) ions than the control TB1 strain. Significant accumulation of vanadium was also observed in the AgVanabin2-expressing strain as seen by a 1.5-fold increase.

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea.

BACKGROUND: Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified. METHODS: We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function. RESULTS: Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO(2+) into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K(m) and V(max)) of AsNramp-mediated VO(2+) transport at pH 8.5 were 90nM and 9.1pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO(2+) under the same conditions. Excess Fe(2+), Cu(2+), Mn(2+), or Zn(2+) inhibited the transport of VO(2+). AsNramp was revealed to be a novel VO(2+)/H(+) antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H(+)-ATPase in vanadocytes. GENERAL SIGNIFICANCE: This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.

Advances in research on the accumulation, redox behavior, and function of vanadium in ascidians.

The discovery of high levels of vanadium-containing compounds in ascidian blood cells goes back to 1911. Ascidians, which are also known as tunicates or sea squirts, belong to a subphylum of the Chordata, between the vertebrates and invertebrates. This discovery attracted the attention of an interdisciplinary group of chemists, physiologists, and biochemists, in part because of interest in the possible role of vanadium in oxygen transport as a prosthetic group in respiratory pigments, which was later shown not to be such a role, and in part because of the fact that high levels of vanadium were unknown in other organisms. The intracellular concentration of vanadium in some ascidian species can be as high as 350 mm, which is 107 times that in seawater. Vanadium ions, which are thought to be present in the +5 oxidation state in seawater, are reduced to the +3 oxidation state via the +4 oxidation state and are stored in the vacuoles of vanadium-containing cells called vanadocytes, where high levels of protons and sulfate ions are also found. Recently, many proteins and genes that might be involved in the accumulation and reduction of vanadium have been isolated. In this review, we not only trace the history of vanadium research but also describe recent advances in our understanding of the field from several viewpoints: (i) vanadium-accumulating blood cells, (ii) the energetics of vanadium accumulation, (iii) the redox mechanism of vanadium, (iv) the possible role of sulfate, and (v) the physiological roles of vanadium.

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+.

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.