单结构域血管性血友病因子C型蛋白(SVWCs),主要在节肢动物中发现,对各种病原体引起的感染有反应。已在家蚕中鉴定出三种SVWCs,BmSVWC2可能在免疫系统中起关键作用。然而,BmSVWC2的调控机制仍不清楚。本研究旨在全面探讨BmSVWC2在家蚕免疫系统中的生化功能。系统发育分析显示BmSVWC1、BmSVWC3和BmSVWC2分布在不同的群体中,暗示不同的生化功能。响应细菌感染,BmSVWC2的mRNA和蛋白水平显着增加。BmSVWC2对细菌和真菌的多糖病原体相关分子模式表现出明确的结合活性,增强体内细菌清除,但不在体外。脂肪体和血细胞的RNA测序分析显示,许多免疫基因明显上调,BmSVWC2水平较高,主要影响识别,信令,以及Toll和免疫缺陷(IMD)信号通路的反应产生。这导致在血淋巴中产生各种抗微生物肽和显著的抗菌活性。BmSVWC2在脂肪体和血细胞中上调吞噬相关基因,和吞噬试验证实BmSVWC2提高了血细胞对细菌的吞噬能力。此外,BmSVWC2诱导脂肪体内一氧化氮合成酶(NOS)的表达,和生物测定证实,BmSVWC2增加了脂肪体和血淋巴中的NOS活性,导致一氧化氮积累。然而,BmSVWC2不影响酚氧化酶活性,尽管它引起了一些丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的差异表达。免疫共沉淀和质谱分析显示,BmSVWC2与30K蛋白相互作用,如30K蛋白2,30KpBmHPC-19,30K19G1样,30K蛋白8、30K蛋白7、30KpBmHPC-23和低分子质量脂蛋白4样。我们的研究提供了BmSVWC2的全面表征,并阐明了其调节免疫反应激活的潜在机制。
Single-domain von Willebrand factor type C proteins (SVWCs), primarily found in arthropods, responds to infections caused by various pathogens. Three SVWCs have been identified in the
silkworm and BmSVWC2 might play a crucial role in the immune system. However, the regulatory mechanism of BmSVWC2 remains largely unknown. This study aimed to investigate the biochemical functions of BmSVWC2 in the immune system of B. mori comprehensively. Phylogenetic analysis revealed that BmSVWC1, BmSVWC3, and BmSVWC2 were distributed in diverse groups, suggesting distinct biochemical functions. The mRNA and protein levels of BmSVWC2 increased significantly in response to bacterial infection. BmSVWC2 exhibited clear binding activity to the polysaccharide pathogen-associated molecular patterns of bacteria and fungi, enhancing bacterial clearance in vivo but not in vitro. RNA-sequencing assays of the fat body and hemocytes showed that numerous immune genes were markedly up-regulated with higher level of BmSVWC2, primarily affecting recognition, signaling, and response production of the Toll and immune deficiency (IMD) signaling pathways. This led to the production of various antimicrobial peptides and significant antibacterial activities in the hemolymph. BmSVWC2 up-regulated phagocytosis-related genes in the fat body and hemocytes, and phagocytosis assays confirmed that BmSVWC2 improved the phagocytic ability of hemocytes against bacteria. Additionally, BmSVWC2 induced the expression of nitric oxide synthetase (NOS) in the fat body, and bioassays confirmed that BmSVWC2 increased NOS activity in the fat body and hemolymph, resulting in nitric oxide accumulation. However, BmSVWC2 did not affect phenoloxidase activity, despite it caused differential expression of a few serine proteases and serine protease inhibitors. Co-immunoprecipitation and mass spectrometry assays showed that BmSVWC2 interacted with 30 K proteins, such as 30 K protein 2, 30 K pBmHPC-19, 30 K 19G1-like, 30 K protein 8, 30 K protein 7, 30 K pBmHPC-23, and low molecular mass lipoprotein 4-like. Our study provides a comprehensive characterization of BmSVWC2 and elucidates the mechanism underlying its regulation of immune responses activation.