Abstract:
Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. In
this study, caspase 3 was identified (Rbcasp3) and characterized from rock bream (Oplegnathus fasciatus).
The full-length cDNA of Rbcasp3 is 2683 bp and contains an open reading frame of 849 bp, which encodes
a 283 amino acid protein with a calculated molecular mass of 31.2 kDa and isoelectric point of 6.31. The
amino acid sequence resembles the conventional caspase 3 domain architecture, including crucial amino
acid residues in the catalytic site and binding pocket. The genomic length of Rbcasp3 is 7529 bp, and
encompasses six exons interrupted by five introns. Phylogenetic analysis affirmed that Rbcasp3 represents a complex group in fish that has been shaped by gene duplication and diversification. Many
putative transcription factor binding sites were identified in the predicted promoter region of Rbcasp3,
including immune factor- and cancer signal-inducible sites. Rbcasp3, excluding the pro-domain, was
expressed in Escherichia coli. The recombinant protein showed a detectable activity against the
mammalian caspase 3/7-specific substrate DEVD-pNA, indicating a functional role in physiology. Quantitative real time PCR assay detected Rbcasp3 expression in all examined tissues, but with high abundance in blood, liver and brain. Transcriptional profiling of rock bream liver tissue revealed that challenge
with lipopolysaccharides (LPS) caused prolonged up-regulation of Rbcasp3 mRNA whereas, Edwardsiella
tarda (E. tarda) stimulated a late-phase significant transcriptional response. Rock bream iridovirus (RBIV)
up-regulated Rbcasp3 transcription significantly at late-phase, however polyinosinic-polycytidylic acid
(poly(I:C)) induced Rbcasp3 significantly at early-phase. Our findings suggest that Rbcasp3 functions as
a cysteine-aspartate-specific protease and contributes to immune responses against bacterial and viral
infections.
