Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus
Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus
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DOI：
10.1016/j.fsi.2016.11.033
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发表时间：
2017
影响因子：
4.7
通讯作者：
Huahui Chen;Miao Lv;Zhimeng Lv;Chenghua Li;Wei Xu;Weiwei Zhang;Xuelin Zhao;Xuemei Duan;C. Jin
中科院分区：
文献类型：
--
作者：
Huahui Chen;Miao Lv;Zhimeng Lv;Chenghua Li;Wei Xu;Weiwei Zhang;Xuelin Zhao;Xuemei Duan;C. Jin
组织蛋白酶B（CTSB）是溶酶体半胱氨酸蛋白酶的一个成员，参与多层次的生理和生物学过程，在宿主抵抗病原体感染的免疫防御中起着重要作用。然而，CTSB在无脊椎动物，特别是海洋棘皮动物的先天免疫系统中的功能，一直缺乏记录。本研究采用分子生物学和蛋白质生物学相结合的方法，对具有重要经济价值的水产养殖品种仿刺参（Apostichopus sp.）CTSB的免疫功能进行了研究。利用重叠EST和RACE技术克隆了AjCTSB的全长cDNA，其开放阅读框为999 bp，编码332个氨基酸残基，预测分子量为36.8 kDa。AjCTSB推导的氨基酸序列具有典型的活性中心，含有3个保守的氨基酸残基（Cys 108，His 277和Asn 297）。系统进化树分析也支持AjCTSB是CTSB家族的一个新成员，并首次与无脊椎动物CTSB聚类。定量真实的时间PCR分析显示，AjCTSB在所有检测组织中普遍表达，在肠中表达水平最高。在0 ~ 0.24 μg μL−1浓度范围内，纯化的重组AjCTSB具有剂量依赖性的CTSB活性。进一步的功能分析表明，0.16倍的siRNA能显著抑制刺参体腔细胞的凋亡，并且特异性的AjCTSB siRNA能显著降低刺参体腔细胞中Ajcaspase 3的凋亡执行。这些结果表明，AjCTSB是一种重要的免疫因子，可作为病原菌攻击海参的细胞凋亡促进剂。
Cathepsin B (CTSB), a member of lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and also plays crucial roles in host immune defense against pathogen infection in vertebrates. However, the function of CTSB within the innate immune system of invertebrates, particularly in marine echinoderms, has been poorly documented. In this study, the immune function of CTSB inApostichopus japonicus(designated asAjCTSB), a commercially important and disease vulnerable aquaculture specie, was investigated by integrated molecular and protein approaches. A 2153 bp cDNA representing the full-length ofAjCTSBwas cloned via overlapping ESTs and RACE fragments.AjCTSBcontained an open reading frame of 999 bp encoding a secreted protein of 332 amino acid residues with a predicted molecular mass of 36.8 kDa. The deduced amino acid ofAjCTSBshared a typical activity center containing three conserved amino acid residues (Cys108, His277and Asn297). Phylogenetic tree analysis also supported thatAjCTSBwas a new member of CTSB family with clustering firstly with invertebrate CTSBs. Quantitative real time PCR analysis revealed thatAjCTSBwas ubiquitously expressed in all examined tissues with the highest levels in intestine. TheVibrio splendiduschallenged sea cucumber and LPS-exposed coelomocytes could both significantly boost the expression ofAjCTSB.Moreover, the purified recombinantAjCTSBexhibited dose-dependent CTSB activities at the concentration ranged from 0 to 0.24 μg μL−1. Further functional analysis indicated that coelomocytes apoptosis was significantly inhibited by 0.16-foldin vivoand the apoptosis executionAjcaspase 3was extremely reduced inApostichopus japonicuscoelomocytes treated with specificAjCTSBsiRNA. Collectively, all these results suggested thatAjCTSBwas an important immune factor and might be served as apoptosis enhancers in pathogen challenged sea cucumber.