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.
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The acidic amino acid-rich C-terminal domain of VanabinX enhances reductase activity, attaining 1.3- to 1.7-fold vanadium reduction.
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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 transcriptome analysis of blood cells from a V-rich ascidian, , and constructed a database containing 8532 predicted proteins.
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Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan; Hiroshima University, Japan.
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Dalton Trans
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A novel vanadium reductase, Vanabin2, forms a possible cascade involved in electron transfer.
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The unusual ascidian ability to accumulate high levels of vanadium ions at concentrations of up to 350 mM, a 10(7)-fold increase over that found in seawater, has been attracting interdisciplinary attention for a century. Accumulated V(V) is finally reduced to V(III) via V(IV) in ascidian vanadocytes. Reducing agents must therefore participate in the reduction.
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