Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus)

• DOI: 10.1016/j.dci.2012.06.010
• Publication Date: 2012-08-07T02:01:01Z
• Authors: H.K.A. Premachandra; Qiang Wan; Don Anushka Sanda...; Mahanama De Zoysa; Cheol Young Choi; Ilson Whang; Jehee Lee
• Keywords: Models, Molecular; 0301 basic medicine; Genome; Hemocytes; Base Sequence; Sequence Homology, Amino Acid; Gastropoda; Molecular Sequence Data; Sequence Analysis, DNA; 3. Good health; 03 medical and health sciences; Gene Expression Regulation; Organ Specificity; Structural Homology, Protein; Host-Pathogen Interactions; Papain; Proteolysis; Animals; Computer Simulation; Amino Acid Sequence; Cystatin B; Cloning, Molecular; Phylogeny

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306 bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4 Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5 μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6 h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.