Molecular and functional characterization of a type-1 cystatin in amphioxus (Branchiostoma japonicum)
Molecular and functional characterization of a type-1 cystatin in amphioxus (Branchiostoma japonicum)
复制标题
DOI：
10.1016/j.fsi.2024.109423
复制
发表时间：
2024-02-14
影响因子：
4.7
通讯作者：
Wang，Peng
中科院分区：
文献类型：
--
作者：
Du，Ronghuan;Zheng，Xian;Wang，Peng
胱氨酸蛋白酶抑制剂包括进化保守蛋白质的庞大超家族，主要被认为是通过调节半胱氨酸蛋白酶的活性而作为内源性抑制剂的作用。新兴的研究证据也提供了对它们在一系列生物和病理过程中的替代作用的见解，包括神经退行性疾病、肿瘤进展、炎症性疾病和免疫反应。目前，在脊椎动物中发现了多种1型半胱氨酸蛋白酶抑制剂（stefins），而在头索动物文昌鱼中发现的1型半胱氨酸蛋白酶抑制剂（stefins）却知之甚少。本研究首次成功克隆了日本文昌鱼1型半胱氨酸蛋白酶抑制剂同源物Bjcystatin-1。推导的Bjcystatin-1蛋白的结构特征在于存在典型的楔形半胱氨酸蛋白酶抑制剂特征，包括'QxbaseG'和'Px'基序，以及保守的N-末端甘氨酸残基。利用不同的半胱氨酸蛋白酶抑制剂对应物的系统基因组分析证实了Bjcystatin-1和1型半胱氨酸蛋白酶抑制剂同源物的密切进化关系。Bjcystatin-1主要在鳃和后肠中以组织特异性模式表达，并且其表达响应于细菌或其特征分子LPS和LTA的攻击而显著上调，表明其参与免疫应答。此外，重组Bjcystatin-1（rBjcystatin-1）蛋白对木瓜蛋白酶具有明显的抑制活性，并能与LPS和LTA结合，表明其可能作为模式识别受体参与免疫应答。亚细胞定位结果显示Bjcystatin-1定位于细胞质和细胞核，其过表达可减弱LPS诱导的核转录因子NF-κB的激活。综上所述，我们的研究表明，文昌鱼Bjcystatin-1具有蛋白酶抑制剂和免疫活性因子的双重作用，为研究1型半胱氨酸蛋白酶抑制剂在文昌鱼免疫防御中的作用提供了新的思路。
Cystatins comprise a vast superfamily of evolutionary conserved proteins, predominantly recognized for their roles as endogenous inhibitors by regulating the activity of cysteine proteases. Emerging lines of research evidence also provides insight into their alternative roles in a spectrum of biological and pathological processes, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. Nowadays, various type-1 cystatins (stefins) have been demonstrated among a variety of discovered vertebrate groups, while little is known about the related homologue in cephalochordate amphioxus, which are repositioned at the base of the chordate phylum. In the present study, a single type-1 cystatin homologue inBranchiostoma japonicumwas first successfully cloned and designated as Bjcystatin-1. The deduced Bjcystatin-1 protein is structurally characterized by the presence of typical wedge-shaped cystatin features, including the ‘QxVxG’ and ‘Px’ motif, as well as the conserved N-terminal glycine residue. Phylogenomic analyses utilizing different cystatin counterparts affirmed the close evolutionary relationship of Bjcystatin-1 and type-1 cystatin homologue. Bjcystatin-1 was predominantly expressed in the gills and hind-gut in a tissue-specific pattern, and its expression was remarkably up-regulated in response to challenge with bacteria or their signature molecules LPS and LTA, suggesting the involvement in immune response. Additionally, the recombinant Bjcystatin-1 (rBjcystatin-1) protein showed significant inhibitory activity towards papain and binding ability to LPS and LTA, indicating its hypothesized role as a pattern recognition receptor in immune response. Subcellular localization results also showed that Bjcystatin-1 was located in the cytoplasm and nucleus, and its overexpression could attenuate the activation of LPS-induced nuclear transcription factors NF-κB. Taken together, our study suggests that amphioxus Bjcystatin-1 acts as a dual role in protease inhibitor and an immunocompetent factor, providing new insights into the immune defense effect of type-1 cystatin in amphioxus.