Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes. In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Eriocheir sinensis (designated EsCystatin) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5′-terminal untranslated region (UTR) of 92 bp, a 3′ UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 5.48 and the predicted molecular weight of 13.39 kDa. A signal Cystatin-like domain (Gly25 to Lys112) was found in the putative amino acid sequences of EsCystatin. Similar to other Cystatins, the conserved central Q70VVSG74 motif was located in the Cystatin-like domain of EsCystatin. But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystatin were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart. After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0.6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01) at 24 h. Afterwards, EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2.8-fold of that in blank (P < 0.01)). The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain. When the concentration of EsCystatin protein was of 300 μg mL−1, almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms.