The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab.
Abbreviations used: DTPA, diethylenetriaminepenta-acetic acid; MT, metallothionein; SOD, superoxide dismutase; Cu,ZnSOD, copper/zinc SOD; cytCu,ZnSOD, cytosolic Cu,ZnSOD; ecCu,ZnSOD, extracellular Cu,ZnSOD; MnSOD, manganese SOD; mtMnSOD, mitochondrial MnSOD; cytMnSOD, cytosolic MnSOD; RACE, rapid amplification of cDNA ends; UAP, universal amplification primer; UDG, uracil DNA glycosylase; UTR, untranslated region.
The nucleotide sequence data reported for the blue crab cytMnSOD, mtMnSOD and ecCu,ZnSOD, brown shrimp cytMnSOD and mtMnSOD, and grass shrimp cytMnSOD will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AF264030, AF264029, AF264031, AY211085, AY211086 and AY211084 respectively.