Key Laboratory of Marine Biogenetics and Resources, Third Institute of Oceanography, State Oceanic Administration, 184 Daxue Road, Xiamen 361005, China.
Key Laboratory of Marine Biogenetics and Resources, Third Institute of Oceanography, State Oceanic Administration, 184 Daxue Road, Xiamen 361005, China.
Dev Comp Immunol. 2014 Aug;45(2):313-20. doi: 10.1016/j.dci.2014.03.019. Epub 2014 Apr 3.
In teleost, cathepsin B has been identified from several species and shown to play roles in the host immune response during pathogen challenge. However, the mechanism of how cathepsin B modulates the immune response in teleosts remains poorly understood. In this study, we identified and characterized cathepsin B (LycCatB) and invariant chain (LycIi) from the large yellow croaker (Pseudosciaena crocea). Sequence comparison and phylogenetic analysis indicated that LycCatB and LycIi are highly conserved within teleosts. Quantitative RT-PCR analysis showed that LycCatB mRNA was widely expressed in all examined tissues. We then recombinantly expressed LycCatB and Lyc-TR-Ii (transmembrane domain removed Ii chain) in Pichia pastoris and Escherichiacoli, respectively. The recombinant LycCatB (rLycCatB) can hydrolyze the substrate Z-FR-AMC with a Km value of 40.68μM. Furthermore, co-incubation of rLycCatB with rLyc-TR-Ii led to an efficient cleavage of rLyc-TR-Ii in a time-dependant manner. These results indicated that cathepsin B may be involved in MHC class II-associated Ii processing in large yellow croaker, and provide new information helping to elucidate the immunological functions of teleost cathepsin B.
在硬骨鱼中，已经从多个物种中鉴定出组织蛋白酶 B，并表明其在病原体挑战期间参与宿主免疫反应。然而，组织蛋白酶 B 如何调节硬骨鱼的免疫反应的机制仍知之甚少。在这项研究中，我们从大黄鱼（Pseudosciaena crocea）中鉴定和表征了组织蛋白酶 B（LycCatB）和不变链（LycIi）。序列比较和系统发育分析表明，LycCatB 和 LycIi 在硬骨鱼中高度保守。定量 RT-PCR 分析显示 LycCatB mRNA 在所有检测组织中广泛表达。然后，我们分别在毕赤酵母和大肠杆菌中重组表达了 LycCatB 和 Lyc-TR-Ii（跨膜结构域去除的 Ii 链）。重组 LycCatB（rLycCatB）可以水解底物 Z-FR-AMC，Km 值为 40.68μM。此外，rLycCatB 与 rLyc-TR-Ii 共孵育可导致 rLyc-TR-Ii 在时间依赖性方式下有效切割。这些结果表明，组织蛋白酶 B 可能参与大黄鱼 MHC Ⅱ类相关 Ii 加工，并提供有助于阐明硬骨鱼组织蛋白酶 B 免疫功能的新信息。
