Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino

• A novel cysteine protease inhibitor(HdCpi) was identified in abalone Haliotis discus hannai Ino.
• The unique symmetric structure of HdCpi consisted of two cystatin-like domains.
• HdCpi may play an important role in the antibacterial response of the abalone.

The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly65 and Gly160), the Gln–X–Val–X–Gly motif (Q106IVSG110 and Q202VVAG206), and the less conserved motif at the C-terminus (S136W137 and A254W255). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT–PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone.