Metal Binding Feature of Copper-Induced Metallothionein from Fresh Water Crab Sinopotamon Henanense Reveals its Cu-Thionein Character

Abstract

Sinopotamon Henanense expresses two metal-induced metallothioneins (MTs), cadmium (Cd)-induced MT and copper (Cu)-induced MT. Cd-induced MT of S. Henanense is a Cd-thiolate MT isoform (ShCdMT) that could play a role in Cd detoxification and Zn homeostasis (could be). However, it is unknown whether Cu-induced MT (ShCuMT) is a Cu-thiolate MT isoform. In the present study, the ShCuMT protein sequence was analyzed using ClustalX2 software and the ExPASy database. The metal-binding feature of ShCuMT was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI-TOF-MS), and matrix-assisted laser desorption ionization flight mass spectrometry (MALDI-TOF-MS). Bioinformatics analysis demonstrated that ShCuMT possessed the Cys-triplet motif of a Cu-specific MT. The DTNB reaction and ESI-TOF-MS detection at pH 2.4 verified that the stability order of ShCuMT binding metal ions was Cu (Ⅰ)> Cd (Ⅱ)> Zn (Ⅱ). The CD spectrum illustrated that ShCuMT binding with Cu(I) exhibited a compact three-dimensional structure and an optimal thermodynamic conformation. The ITC results indicated that ShCuMT possessed the highest stoichiometric number of Cu(Ⅰ). The emergence of unique Cu12-ShCuMT species, as evidenced by MALDI-TOF-MS at pH 2.4, demonstrated that Cu(Ⅰ) was a cognate metal ion of ShCuMT. Overall, the results suggested that ShCuMT possessed canonical Cu-thiolate metal-binding characteristics, and Cu(Ⅰ) was considered its cognate metal ion. Thus, ShCuMT could mainly adjust Cu metabolism and homeostasis and detoxify Cu in the freshwater crab S. henanense. The metal binding ability of ShCuMT reported here contributes to elucidating the structure‒function relationship of the MT of S. Henanense.

Keywords: Copper-induced metallothionein, metal-binding characterization, Cu-thiolate character, Sinopotamon Henanense