Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
J Inorg Biochem. 2012 Nov;116:70-6. doi: 10.1016/j.jinorgbio.2012.08.003. Epub 2012 Aug 18.
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.
海鞘在其血细胞中积累极高水平的钒是众所周知的。已经从富含钒的海鞘中分离出与钒积累和生理功能相关的几种关键蛋白质。其中，钒（IV）结合蛋白-129（VBP-129）是一种独特的蛋白质，已从海鞘 Ascidia sydneiensis samea 的血浆中鉴定出来，但它的金属结合结构域尚不清楚。在这项研究中，生成了 VBP-129 的几个缺失和点突变体，并通过固定化金属离子亲和层析（IMAC）和电子自旋共振光谱（ESR）评估了它们的金属结合能力。内部部分蛋白 VBP-Int41 不与 V(IV)结合，但两个构建体 VBP-N52 和 VBP-Int55 添加了另外 11 或 14 个相邻氨基酸与 V(IV)结合。VBP-Int55 中半胱氨酸 47 和赖氨酸 50 的突变降低了 VBP-Int55 中的 V(IV)-结合，表明这些氨基酸残基在结合 V(IV)中起重要作用。ESR 滴定分析表明，VBP-129、VBP-N52 和 VBP-Int55 分别可以结合 6、3 和 2 个 V(IV)离子。ESR 光谱分析表明，N(2)O(2)配位几何形状与 vanabins 相似。半胱氨酸可能有助于维持结合 V(IV)离子所必需的三维结构。VBP-129 没有 V(V)-还原剂活性，这与其在血浆中的组织定位相符。这项研究提供了证据表明，VBP-129 具有钒（IV）结合结构域，与 vanabins 结合钒（IV）的结构域相似，但 VBP-129 在血浆中仅作为钒（IV）伴侣发挥作用。
