Caspase 3 from rock bream (Oplegnathus fasciatus): Genomic characterization and transcriptional profiling upon bacterial and viral inductions

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• 作者：Don Anushka Sandaruwan Elvitigala^a ; ¹ ; Ilson Whang^a ; ¹ ; H.K.A. Premachandra^a ; Navaneethaiyer Umasuthan^a ; Myung-Joo Oh^b ; Sung-Ju Jung^b ; Sang-Yeob Yeo^c ; Bong-Soo Lim^d ; Jeong-Ho Lee^e ; Hae-Chul Park^f ; ^{hcpark67@korea.ac.kr} ; Jehee Lee^a ; ^d ; ^{jehee@jejunu.ac.kr} ; ^{jeheedaum@hanmail.net}
• 关键词：Caspase 3 ; Rock bream ; Recombinant protein ; Hydrolyzing activity ; Transcriptional analysis
• 刊名：Fish & Shellfish Immunology
• 出版年：2012
• 期刊代码：115_10504648
• 类别：abs
• 出版时间：July, 2012
• 卷：33
• 期：1
• 页码：99-110
• 文件大小：2462 K

摘要

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聽bp and contains an open reading frame of 849聽bp, which encodes a 283 amino acid protein with a calculated molecular mass of 31.2聽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聽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.