Single von Willebrand factor C-domain protein-2 confers immune defense against bacterial infections in the silkworm, Bombyx mori.

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.