Novel forms of copper–metallothionein and anti-oxidant defense enzymes in crustacea that use copper for oxygen transport

Copper is an essential, but potentially toxic, metal. Defense mechanisms against copper-mediated oxidative damage include sequestration of copper by metallothionein (MT) and increased levels of anti-oxidant defense enzymes. The blue crab, Callinectes sapidus, has two copper-inducible (CuMT-I and CuMT-II) and two cadmium-inducible (CdMT-I and CdMT-II) MT isoforms. CuMT-I and CdMT-I are the same protein with different metals. CuMT-II is a unique copper-specific MT that cannot be induced by cadmium. CuMT-I and CuMT-II are structurally and functionally distinct proteins as evidenced by: (1) amino acid sequence; (2) modes of Cu(I)-thiolate coordination; (3) metal binding properties; (4) mechanism of Cu(I)-thiolate cluster formation; (5) interaction with glutathione; and (6) response to copper exposure. In addition to a novel form of CuMT, blue crabs have two novel anti-oxidant defense enzymes. They have replaced cytosolic CuZn-superoxide dismutase (CuZnSOD) with a mitochondrial MnSOD that is retained in the cytosol by having an abnormal mitochondrial-targeting sequence. This phenomenon occurs in all crustacea that use copper for oxygen transport. Finally, we have characterized a novel form of extracellular SOD, which has replaced the commonly found extracellular CuZnSOD. A possible explanation for these intriguing observations will be discussed.