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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 x 10(-4) M and 1.2 x 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.

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.

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.

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 2.1x10(-5) and 2.3x10(-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.

Several families of ascidians accumulate extremely high levels of vanadium in their blood cells. The concentration of vanadium has been determined in each species; the highest concentration, found in Ascidia gemmata , reaches 350 mM, corresponding to 10 7 times that of sea water. How and why ascidians accumulate vanadium in a highly selective manner and at such extremely high levels have yet to be determined. To address these questions, our research group sought to identify the genes and proteins responsible for the accumulation and reduction of vanadium in vanadocytes, a type of blood cell, as well as the process of vanadium transport from sea water to blood cells through the branchial sac, intestine, and blood plasma. Here, we review the accumulation steps as a system, especially those related to the concentration and chemical species of vanadium at each step. A comprehensive analysis on each organ has already revealed several categories of protein families, such as vanadium-binding proteins and vanadium transporters. Herein, we also discuss the mechanisms by which ascidians selectively accumulate vanadium ions from a biochemical viewpoint. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]