Participation of thioredoxin in the V(V)-reduction reaction by Vanabin2

Ueki Tatsuya

Uwagaki Masayuki

Yamamoto Sohei

Michibata Hitoshi

Biochimica et Biophysica Acta (BBA) - General Subjects

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 0.066 mol-V(IV)/min/mol-Vanabin2 and 0.19 mM, 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.

Ascidian

Vanadium

Reductase

Metal

Redox

Biology [ 460 ]

eng

Elsevier B.V.

Copyright (c) 2014 Elsevier B.V. All rights reserved.

This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

[ISSN] 0304-4165

[NCID] AA00564679

[DOI] 10.1016/j.bbagen.2014.07.023

[DOI] http://doi.org/10.1016/j.bbagen.2014.07.023 isVersionOf