State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China; Chongqing Key Laboratory of Sericultural Science, Chongqing engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, 400715, China.
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China.
Insect Biochem Mol Biol. 2019 Mar;106:19-27. doi: 10.1016/j.ibmb.2019.01.003. Epub 2019 Jan 10.
Fibroinase, a cathepsin L-like cysteine protease, was previously identified in the silk gland of the silkworm, Bombyx mori. It shows high degradation activity during the pre-pupa period, when the silk gland undergoes apoptosis and remodeling. Here, we recombinantly expressed pro-fibroinase and activated it in vitro. Fibroinase showed optimal hydrolytic activity at pH 4.0 and its optimum temperature was about 42 °C. One physiological inhibitor, B. mori cysteine protease inhibitor (BCPI) was found, which showed strong inhibitory activity against fibroinase. The inhibitory reaction was caused by the formation of a non-covalent complex; this is in contrast to a previously reported mode of fibroinase inhibition by Serpin18. Expression profiles and immunolocalization analysis demonstrated that fibroinase was involved in silk gland development by degrading silk proteins and apoptosis/remodeling of silk glands at specific points. Furthermore, the comparison of the temporal expression of fibroinase and its inhibitors, BCPI and Serpin18, indicated that these inhibitors were involved in the silk gland development by regulating the activity of fibroinase from the fifth instar until the early spinning stage. These findings improve our understanding of the mechanism of protease regulation and its inhibitors in silk gland development.
丝胶酶是一种组织蛋白酶 L 样半胱氨酸蛋白酶，先前在桑蚕的丝腺中被鉴定出来。它在丝腺经历细胞凋亡和重塑的预蛹期显示出很高的降解活性。在这里，我们重组表达了 pro-fibroinase 并在体外激活了它。丝胶酶在 pH 值为 4.0 时表现出最佳的水解活性，其最适温度约为 42°C。发现了一种生理抑制剂，即家蚕半胱氨酸蛋白酶抑制剂（BCPI），它对丝胶酶表现出很强的抑制活性。抑制反应是由形成非共价复合物引起的；这与先前报道的丝氨酸蛋白酶抑制剂 18 抑制丝胶酶的模式不同。表达谱和免疫定位分析表明，丝胶酶通过降解丝蛋白和丝腺的细胞凋亡/重塑参与丝腺发育，在特定时间点发挥作用。此外，丝胶酶与其抑制剂 BCPI 和 Serpin18 的时间表达比较表明，这些抑制剂通过调节五龄期到早期吐丝期丝胶酶的活性参与丝腺发育。这些发现提高了我们对蛋白酶调节及其抑制剂在丝腺发育中的机制的理解。
