title: Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus).
abstract: 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.
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Science, Jeju National University, Jeju Special Self-Governing Province, Republic of Korea.
authors.forename: H K A
authors.initials: HK
authors.lastname: Premachandra
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wan
authors.forename: Don Anushka Sandaruwan
authors.initials: DA
authors.lastname: Elvitigala
authors.forename: Mahanama
authors.initials: M
authors.lastname: De Zoysa
authors.forename: Cheol Young
authors.initials: CY
authors.lastname: Choi
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003198:Computer Simulation; D055313:Cystatin B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049851:Gastropoda / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D005786:Gene Expression Regulation; D016678:Genome; D006434:Hemocytes / Q000378:metabolism; D054884:Host-Pathogen Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry; D010802:Phylogeny; D059748:Proteolysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D014736:Vibrio parahaemolyticus / Q000502:physiology
chemical_list: D055313:Cystatin B; D010206:Papain