Identification and analysis of a Cu/Zn superoxide dismutase from Haliotis diversicolor supertexta with abalone juvenile detached syndrome.

A partial cDNA sequence of a putative Cu/Zn superoxide dismutase (SOD) from Haliotis diversicolor supertexta with abalone juvenile detached syndrome (AJDS) was isolated by suppression subtractive hybridization library screening. The full 988 base pair (bp) abalone Cu/Zn SOD cDNA representing full cDNA coding sequence was obtained by rapid amplification of cDNA ends (RACE), and included a 462-bp open reading frame encoding 154 amino acids, plus 49 bp of 5'-, and 477 bp of 3'-untranslated region. The coding sequence shared high similarity and identity with known Cu/Zn SODs. The deduced amino acids contained two typical Cu/Zn SOD motifs and the conserved geometry active sites. Moreover, the cysteines involved in dimer formation, and the copper- and zinc-binding sites were conserved. Recombinant abalone Cu/Zn SOD (abSOD) was expressed in Escherichia coli, purified under denaturing conditions, and refolded by direct buffer exchange or gel gradient recovery. The activity of the purified protein was enhanced by 1 microM Cu(2+) or 1 microM Cu(2+) plus 1 microM Zn(2+). The transcription of abSOD was significantly increased in AJDS abalones compared to controls, while protein expression and SOD enzyme activity both decreased significantly, suggesting that SOD may play an important role in AJDS, and that the disease etiology may be related to environmental stress. (c) 2009 Elsevier Inc. All rights reserved.