Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea.
Fish Shellfish Immunol. 2012 Jul;33(1):99-110. doi: 10.1016/j.fsi.2012.04.008. Epub 2012 Apr 21.
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.
半胱天冬酶 3 是细胞凋亡的主要介质，参与细胞死亡信号级联反应。在这项研究中，从石斑鱼（Oplegnathus fasciatus）中鉴定并表征了半胱天冬酶 3（Rbcasp3）。Rbcasp3 的全长 cDNA 为 2683bp，包含一个 849bp 的开放阅读框，编码一个 283 个氨基酸的蛋白质，预测分子量为 31.2kDa，等电点为 6.31。氨基酸序列类似于常规的半胱天冬酶 3 结构域架构，包括催化位点和结合口袋中的关键氨基酸残基。Rbcasp3 的基因组长度为 7529bp，包含六个外显子和五个内含子。系统发育分析证实，Rbcasp3 是鱼类中一个由基因复制和多样化形成的复杂群体。在 Rbcasp3 的预测启动子区域中鉴定出许多假定的转录因子结合位点，包括免疫因子和癌症信号诱导的位点。Rbcasp3 （不包括前导肽）在大肠杆菌中表达。重组蛋白对哺乳动物半胱天冬酶 3/7 特异性底物 DEVD-pNA 表现出可检测的活性，表明其在生理机能中具有功能作用。定量实时 PCR 检测显示 Rbcasp3 在所有检测组织中均有表达，但在血液、肝脏和大脑中丰度较高。石斑鱼肝脏组织的转录谱分析显示，脂多糖（LPS）刺激导致 Rbcasp3 mRNA 长时间上调，而爱德华氏菌（E. tarda）刺激则在后期产生显著的转录反应。石斑鱼虹彩病毒（RBIV）在晚期显著上调 Rbcasp3 转录，而聚肌胞苷酸（poly(I:C)) 在早期显著诱导 Rbcasp3 转录。我们的研究结果表明，Rbcasp3 作为半胱氨酸天冬氨酸特异性蛋白酶发挥作用，有助于对细菌和病毒感染的免疫反应。
