Structural Characterization and Physiological Role of Bombyx mori Fibroinase in the Silk Gland Development

Silkworm is a highly valuable insect that produces silk through secretion by a silk gland. Within this gland, a type of cathepsin L protease called Fibroinase was identified as an enzyme for hydrolyzing the primary components of silk, including fibroin and sericin. Here, we determined the crystal structure of Fibroinase fromBombyx mori at a resolution of 1.56 Å. Comparative structural analysis revealed that Fibroinase adopted a similar structural pattern with papain-type cathepsin, consisting of an N-terminal domain and a C-terminal domain. The interface between the domains forms a substrate-binding cleft, where the E64 inhibitor noncovalently binds in a novel manner. Additionally, computational simulations combined with biochemical analysis allowed us to define the binding mode and inhibition mechanism of physiological inhibitor Bombyx cysteine protease inhibitor (BCPI) with Fibroinase. Moreover, the expression profiles and RNA interference of Fibroinase indicated its critical role in removing silk proteins in the silk gland lumen and the destruction of silk gland tissue during the larval–pupal metamorphosis. These findings enhance our understanding of the structural and biochemical features of Fibroinase and its inhibitors, while also providing evidence for the physiological role of Fibroinase in silk gland development.家蚕（Silkworm）是一类极具经济价值的昆虫，可通过丝腺分泌蚕丝。在该丝腺中，研究人员鉴定出一种名为丝纤酶（Fibroinase）的组织蛋白酶L（cathepsin L）蛋白酶，其可作为水解蚕丝主要成分——丝素蛋白（fibroin）与丝胶蛋白（sericin）——的酶类。本研究解析了家蚕（Bombyx mori）来源的丝纤酶的晶体结构，分辨率达1.56埃（Å）。比较结构分析显示，丝纤酶与木瓜蛋白酶型组织蛋白酶（papain-type cathepsin）具有相似的结构模式，由N端结构域与C端结构域组成。两个结构域的界面形成底物结合裂隙，E64抑制剂以全新的非共价结合方式结合于该裂隙中。此外，本研究结合计算模拟与生化实验分析，明确了生理抑制剂家蚕半胱氨酸蛋白酶抑制剂（Bombyx cysteine protease inhibitor, BCPI）与丝纤酶的结合模式及抑制机制。进一步的表达谱分析与RNA干扰（RNA interference）实验结果表明，丝纤酶在幼虫-蛹变态发育过程中，对丝腺腔中的蚕丝蛋白清除以及丝腺组织的降解发挥关键作用。本研究成果不仅加深了人们对丝纤酶及其抑制剂的结构与生化特性的理解，同时为丝纤酶在丝腺发育中的生理功能提供了坚实的实验证据。