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Molecular cloning and characterization of a cytoplasmic manganese superoxide dismutase and a mitochondrial manganese superoxide dismutase from Chinese mitten crab Eriocheir sinensis

Wang, Mengqiang, Wang, Lingling, Yi, Qilin, Gai, Yunchao, Song, Linsheng
Fish & shellfish immunology 2015 v.47 no.1 pp. 407-417
prokaryotic cells, crabs, Komagataella pastoris, phylogeny, heart, polypeptides, Micrococcus luteus, Eriocheir sinensis, open reading frames, Vibrio anguillarum, gene expression, amino acids, mitochondria, messenger RNA, complementary DNA, muscles, superoxide dismutase, eukaryotic cells, amino acid sequences, immune response, molecular cloning, enzyme activity, antioxidants, proteins, innate immunity, microorganisms, gills, hemocytes
Superoxide dismutase (SOD) functions as the first and essential enzyme in the antioxidant system and is ubiquitously existed in both prokaryotes and eukaryotes. In the present study, both cytoplasmic and mitochondrial manganese SOD were identified from Chinese mitten crab Eriocheir sinensis (designed as EscytMnSOD and EsmtMnSOD). The complete nucleotide sequence of EscytMnSOD comprised 1349 bp and consisted of a 5′ untranslated regions (UTR) of 43 bp, a 3′ UTR of 445 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acid residues. The full-length cDNA sequence of EsmtMnSOD comprised 990 bp, containing a 5′ UTR of 55 bp, a 3′ UTR of 278 bp and an ORF of 657 bp encoding a polypeptide of 218 amino acid residues. The deduced amino acid sequences of EscytMnSOD and EsmtMnSOD contained highly conserved MnSOD signature and typical functional domain, and exhibited high similarity with their reported homologues. In the phylogenetic tree, EscytMnSOD and EsmtMnSOD were clustered with their homologues from the land crab Cardisoma armatum. The EscytMnSOD and EsmtMnSOD transcripts were constitutively expressed in haemocytes, muscle, heart, gill, haepatopancreas and gonad, with the highest expression level in gills and haepatopancreas, respectively. The mRNA expression levels of them were all up-regulated in haemocytes with similar profiles after the stimulation of Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The EsmtMnSOD with low basal expression level responded to invading microbes intensely, while the EscytMnSOD with high basal expression level exhibited mild responses against stimulating microbes. The purified rEscytMnSOD and rEsmtMnSOD proteins exhibited specific Mn2+-dependent enzymatic activities, while rEscytMnSOD with lower basic activity displayed higher stability than rEsmtMnSOD. All these results indicated that EscytMnSOD and EsmtMnSOD were efficiently antioxidant enzymes and potentially involved in the innate immune responses of E. sinensis with different roles, the former might play a routine role in the innate immune system in crabs, while the later might be involved in the immune response against invading microbes specifically.