Trypsin-type serine protease p37k hydrolyzes CPAP3-type cuticle proteins in the molting fluid of the silkworm Bombyx mori
Trypsin-type serine protease p37k hydrolyzes CPAP3-type cuticle proteins in the molting fluid of the silkworm Bombyx mori
复制标题
胰蛋白酶型丝氨酸蛋白酶 p37k 水解家蚕蜕皮液中的 CPAP3 型角质层蛋白
DOI：
10.1016/j.ibmb.2021.103610
复制
发表时间：
2021-08-18
影响因子：
3.8
通讯作者：
Xia, Qingyou
中科院分区：
文献类型：
--
作者：
Hou, Yong;Yang, Lingzhen;Xia, Qingyou
类似于围营养蛋白 3 (CPAP3) 型角质层蛋白的角质层蛋白构成了具有三个几丁质结合域 (CBD) 的蛋白质家族，它们通过与几丁质结合在角质层形成中发挥重要作用。在我们之前的研究中，我们鉴定了家蚕基因组中的CPAP3型角质层蛋白，其中我们鉴定了CPAP3-A2（BmCBP1），这是一种在表皮中高表达的蛋白。在本研究中，为了阐明CPAP3型角质层蛋白的消化机制，我们在体外将CPAP3-A2与蜕皮液一起孵育，发现其水解被丝氨酸和半胱氨酸蛋白酶抑制剂抑制，产生两条主要条带，分子量约为22 kD和11 kD。根据对自然纯化蜕皮液的液相色谱-串联质谱分析，推测胰蛋白酶型丝氨酸蛋白酶 p37k 负责水解 CPAP3-A2。为了验证这一点，随后使用 Bac-to-Bac 杆状病毒表达系统在 Sf9 细胞中表达 p37k。在其活性形式下，重组蛋白酶可以成功水解 CPAP3-A2。最后，我们分析了 CPAP3-A2 蜕皮液消化位点。当CPAP3-A2的精氨酸169突变为丙氨酸时，与正常CPAP3-A2相比，突变体CPAP3-A2的水解作用较弱。总的来说，我们鉴定了一种胰蛋白酶型丝氨酸蛋白酶，它参与 CPAP3 型角质层蛋白（包括 CPAP3-A2）的降解，表明这种蛋白酶在家蚕蜕皮过程中发挥着重要作用。这些发现为进一步阐明昆虫蜕皮和变态的机制提供了基础。
Cuticular proteins analogous to peritrophin 3 (CPAP3)-type cuticle proteins constitute a family of proteins with three chitin-binding domains (CBDs) that play an important role in cuticle formation by associating with chitin. In our previous study, we identified CPAP3-type cuticle proteins in the silkworm genome, of which we characterized CPAP3-A2 (BmCBP1), a protein highly expressed in the epidermis. In this study, to elucidate the digestion mechanism of CPAP3-type cuticle proteins, we incubated CPAP3-A2 with molting fluid in vitro and found that its hydrolysis, which was inhibited by serine and cysteine protease inhibitors, produced two major bands with a molecular weight of approximately 22 kD and 11 kD. A trypsin-type serine protease, p37k, was presumed to be responsible for hydrolyzing CPAP3-A2 based on liquid chromatography-tandem mass spectrometry analysis of naturally purified molting fluid. To verify this, p37k was subsequently expressed in Sf9 cells using the Bac-to-Bac baculovirus expression system. In its active form, the recombinant protease could successfully hydrolyze CPAP3-A2. Finally, we analyzed the CPAP3-A2 molting fluid digestion site. When arginine 169 of CPAP3-A2 was mutated to alanine, a weaker hydrolysis of mutant CPAP3-A2 was observed compared to that of normal CPAP3-A2. Collectively, we identified a trypsin-type serine protease that is involved in the degradation of CPAP3-type cuticle proteins, including CPAP3-A2, suggesting that this protease plays an important role during molting in Bombyx mori. These findings provide the basis for further elucidation of the mechanisms underlying insect molting and metamorphosis.