Identification of the extracellular copper–zinc superoxide dismutase (ecCuZnSOD) gene of the mud crab Scylla serrata and its expression following β-glucan and peptidoglycan injections

An extracellular copper–zinc superoxide dismutase (ecCuZnSOD, or EC-SOD) gene was cloned from haemocytes of mud crab Scylla serrata. The ecCuZnSOD cDNA consists of 939 bp with an open reading frame (ORF) of 585 bp that encodes 195 amino acids (aa) with 31 residues of a signal peptide sequence. The predicted molecular mass of the mature protein (164 aa) is 17.1 kDa with an estimated pI of 6.89. Amino acids responsible for binding Cu (His84, 86, 101, and 163) and Zn (His101, 109, and 121, and Asp 124), two cysteines (95 and 189) that form a disulfide bond, two CuZnSOD signatures (GFHVHAEGDLS) from 82 to 92 and (GNAGGRAGCGLI) from 181 to 192, as well as a N-linked glycosylation site (NVSG) were conserved. The deduced amino acid sequence of S. serrata ecCuZnSOD showed similarity of 84% and 54% with the ecCuZnSOD of blue crab Callinectes sapidus, and crayfish Pacifastacus leniusculus, respectively. The phylogenetic analysis revealed that the ecCuZnSOD of S. serrata grouped together with ecCuZnSODs of crustaceans and insects, but was far way from the intracellular (ic)CuZnSODs of invertebrates and vertebrates. The ecCuZnSOD transcript in haemocytes significantly increased in 6–48 h, and had returned to the original value by 72 h after the β-glucan (βG) injection, whereas the ecCuZnSOD transcript significantly increased 6–72 h after the peptidoglycan (PG) injection indicating induction of the immune system within a short time.