A New Insight into Metallothionein (MT) Classification and Evolution

Abstract

We report the synthesis and characterization of a <i>Homarus americanus</i> MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous <i>Escherichia coli</i> expression in zinc- and copper-supplemented medium was achieved for MTH, the two domains ββMTH and βαMTH and three site-directed mutants, ββC9H, βαC37H, and βαE31C/T34C. The <i>in vivo</i> conformed metal complexes and the <i>in vitro</i> substituted cadmium aggregates were characterized. Major stoichiometries of M^II₆-MTH for the entire MTH and M^II₃-ββMTH and M^II₃-βαMTH for the independent domains fully validated our expression system. A low affinity binding site for a seventh Zn(II) in the <i>in vivo</i> synthesized MTH was located in the βα domain. Additionally, minor M^II₄species were found for each domain. Both single Cys to His mutations exhibited a similar reduction of their <i>in vivo</i> zinc binding ability but differed in their cadmium binding behavior when compared with the wild-type forms. Conversely, the double mutant showed an enhanced zinc and cadmium binding capacity. <i>In vivo</i>synthesis of MTH and of its independent domains in the presence of copper only afforded heterometallic copper-zinc species. These findings allow consideration of MTH as a zinc thionein and question the view of all crustacea MT structures as copper thioneins. Furthermore, a new approach for the evolutionary and functional classification of MT is proposed, based on the stoichiometry of metal-MT species and molecular phylogenetic analysis.