Molecular mechanism of the transport and reduction pathway of vanadium in ascidians

Metal ions are required for physiologically essential functions such as metalloenzymatic reactions, redox reactions, electron transfer, regulation of transcription factor activity, and respiration in living cells. Generally, metal ions are homeostatically maintained at very low concentrations in the sub-micromolar to micromolar range in living cells. However, some organisms, called hyperaccumulators, collect extremely high levels of metal ions, thereby providing experimental systems in which to study the mechanisms underlying the selective accumulation of metal ions. Typical of such organisms are the ascidians, more commonly known as sea squirts or tunicates. They are sessile marine animals belonging to the chordates. Several species of ascidians are known to accumulate extremely high levels of vanadium ions in their blood cells. Vanadium is usually in the V-V state in the natural environment, but in ascidians, most of vanadium is reduced to V-III via V-IV during the assimilation process. In this review, we first summarize the history of the studies on vanadium accumulation in ascidians and then focus on the recent progress of molecular studies, especially on the transport and reduction of vanadium, using primarily two ascidian species, Ascidia sydneiensis samea and Ciona intestinalis. Several candidate genes for V-IV transporters and enzymes catalyzing the redox reactions of V-V/V-IV are addressed in detail. The function of accumulated V-III is discussed in relation to redox reactions. (C) 2011 Elsevier B.V. All rights reserved

Mechanism of vanadium accumulation and possible function of vanadium in underwater adhesion in ascidians

Ascidians are marine animals that belong to the same phylogenetic group (Phylum Chordata) as human beings do. One of the three suborders in ascidians can accumulate a high level of vanadium ions in blood cells. Ascidia gemmata has been reported to accumulate the highest levels of vanadium at 350 mM, which is 107 -fold higher than the vanadium concentration in seawater. In the last two decades, many genes and proteins related to vanadium accumulation and reduction have been revealed by molecular biological and biochemical methods. Modern omics approach enhanced the comprehensive identification of factors related to this phenomenon. In this review article, first, we would like to summarize the history of studies on vanadium accumulation in ascidians briefly. Then, we would like to overview recent advances by omics studies. How ascidians selectively accumulate vanadium is discussed from biochemical properties of proteins responsible for each step, and why ascidians accumulate vanadium is discussed in relation to the underwater adhesion

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 A. gemmata were determined to be 32.4 mM 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, 2 genes, 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.Financial support was provided in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#20570070 and #21570077)

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 VO2+ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (Km and Vmax) of AsNramp-mediated VO2+ transport at pH 8.5 were 90 nM and 9.1 pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO2+ under the same conditions. Excess Fe2+, Cu2+, Mn2+ or Zn2+ inhibited the transport of VO2+. Conclusions: AsNramp was revealed to be a novel VO2+/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.This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#17370026, #18570070, #20570070, and #21570077)

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 VIV, but the two constructs, VBP-N52 and VBP-Int55, added with additional 11 or 14 neighboring amino acids bound to VIV. Mutations for cysteine-47 and lysine-50 in VBP-Int55 diminished VIV-binding in VBP-Int55, suggesting that these amino acid residues play important roles in binding VIV. ESR titration analysis revealed that VBP-129, VBP-N52 and VBP-Int55 could bind to 6, 3 and 2 VIV ions, respectively. ESR spectrum analysis indicated a N2O2 coordination geometry, which is similar to Vanabins. The cysteines may contribute to the maintenance of the three-dimensional structure that is necessary for binding VIV ions. VBP-129 did not have a VV-reductase activity, as expected from its tissue localization in blood plasma. This study provided the evidences that VBP-129 possesses VIV-binding domains that make a similar coordination to VIV as those by Vanabins but VBP-129 acts solely as VIV-chaperon in blood plasma.This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (nos. 20570070 and 21570077)

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 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

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

Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (</= 350mM). 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

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 A1, neutralized the contents of the vacuoles. To understand the function of V ATPase in vanadocytes, we isolated cDNA encoding subunit C of V ATPase from vanadocytes since 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 1,443 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

Novel vanadium-binding proteins (Vanabins) identified in cDNA libraries and the genome of the ascidian Ciona intestinalis

Ascidians, especially those belonging to the suborder Phlebobranchia, can accumulate high levels of vanadium. Vanadium-binding proteins (vanabins) were first isolated from a vanadium-accumulating ascidian, Ascidia sydneiensis samea, and then the vanabins were cloned, their expression was studied, and metal-binding assays were conducted. In order to unravel the mechanism of vanadium accumulation, we searched for vanabin-like genes in other animals, including other ascidians. A database search revealed five groups of cDNAs that encoded vanabin-like proteins in another ascidian, Ciona intestinalis. The genes encoding C. intestinalis vanabins, CiVanabin1 to CiVanabin5, were clustered in an 8.4-kb genomic region. The direction of transcription of each gene was identical and each gene had a single intron. All the C. intestinalis vanabins were cysteine rich, and the repetitive pattern of cysteines closely resembled that of A. sydneiensis samea vanabins. Using immobilized metal ion affinity chromatography, we found that a recombinant protein of at least one of the C. intestinalis vanabins (CiVanabin5) bound to vanadium(IV) ions

Vanadocytes, Cells hold the Key to Resolving the Highly Selective Accumulation and Reduction of Vanadium in Ascidians

Since Henze discovered vanadium in the blood (or coelomic) cells of an ascidian in 1911, this unusual phenomenon has attracted the interest of many investigators. The highest concentration of vanadium (350 mM) in the blood cells of Ascidia gemmata, which belongs to the suborder Phlebobranchia, is 107 times higher than that in sea water. Of the approximately ten types of blood cells, a combination of cell fractionation and neutron-activation analysis revealed that the signet ring cells were the true vanadocytes. In the vanadocytes, 97.60f the vanadium is in the +3 oxidation state (III). The extremely low pH of 1.9 found in vanadocytes suggests that protons, concentrated by an H+-ATPase, might be linked to the accumulation of vanadium energetically. The antigen recognized by a monoclonal antibody, S4D5, prepared to identify vanadocytes, was determined to be 6-PGDH in the pentose phosphate pathway. NADPH produced in the pentose phosphate pathway in vanadocytes is thought to participate in the reduction of vanadium(V) to vanadium(IV). During embryogenesis, a vanadocyte-specific antigen first appears in the body wall at the same time as significant accumulations of vanadium become apparent. Three different vanadium-associated proteins (VAPs) were extracted from the blood cells of vanadium-rich ascidians. These are 12.5, 15, and 16 kDa in size and are associated with vanadium in an approximate ratio of 1:16. The cDNA encoding the 12.5 and 15 kDa VAPs was isolated and the proteins encoded were found to be novel. Further biochemical and biophysical characterization of the VAPs is in progress