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 cystatinlike 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 lmol/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.
