Metal binding characterization of heterologously expressed metallothionein of the freshwater crab Sinopotamon henanense

Abstract

We characterized the metal tolerance of recombinant strains harboring metallothionein from the freshwater crab Sinopotamon henanense (ShMT) in vivo and metal binding properties of ShMT purified in vitro. The recombinant strains harboring ShMT were exposed to 0.1 mM Cd²⁺, 0.3 mM Cu²⁺, 0.5 mM Pb²⁺, and 0.8 mM Zn²⁺. The growth curves and spot assays of recombinant strains and the contents of heavy metal ions were analysed in the media supplemented with above metal ions provided to recombinant E. coli synthesis. The structural characteristics of the Cd-, Cu-, Pb-, and Zn-ShMT were determined through ultraviolet spectroscopy (UV-vis), circular dichroism (CD), and isothermal titration calorimetry (ITC). The in vivo results showed that, compared to control strains, recombinant strains tolerated Cd²⁺, Cu²⁺, Pb²⁺, and Zn²⁺. Furthermore, the contents of Cd²⁺ and Pb²⁺ in media decreased substantially. In vitro and the Cd-ShMT had a higher degree of folding compactness in solution. 5,5'-Dithiobis-(2-nitrobenzoic) acid (DTNB) reaction and ITC results demonstrated that ShMT yielded Cd₆-, Cu₇-, and Pb₆-ShMT. The binding stability order was Cu-ShMT > Cd-ShMT > Pb-ShMT > Zn-ShMT. Overall, ShMT is a canonical crustacean MT and is defined as a Cd-specific MT isoform that functions mainly in a detoxifying Cd²⁺ and Pb²⁺ and in regulating Zn²⁺ homeostasis in S. henanense. This research on the metal binding properties of ShMT provides a better understanding of the physiological function of ShMT reducing heavy metal bioavailability and by regulating essential trace metals.

Keywords: In vivo and in vitro; Metal binding characterization; Metal ion; Sinopotamon henanense metallothionein; Spectroscopy; Thermodynamic.