Unveiling the Multifaceted Role of HP6: A Critical Regulator of Humoral Immunity in Antheraea pernyi (Lepidoptera: Saturniidae)

Serine proteases are widely distributed in both invertebrates and vertebrates, playing critical roles in the regulation of innate immunity. In the insect innate immune system, two pivotal pathways-the prophenoloxidase (PPO) activation cascade and Toll pathway-mediated antimicrobial peptide (AMP) synthesis-are both tightly regulated by serine protease cascades. This study focuses on serine protease-hemolymph protease 6 of <i>A. pernyi</i> (<i>Ap</i>-HP6). Following immune stimulation, the expression of <i>Ap</i>-proHP6 was significantly induced, primarily observed in hemocytes and the fat body. After suppressing <i>Ap</i>-proHP6 expression via RNA interference (RNAi) and infecting larvae with different microbes, the expression levels of AMPs showed a downward trend. When endogenous <i>Ap</i>-proHP6 content in hemolymph was reduced using RNAi technology or anti-r<i>Ap</i>-proHP6-His₆ polyclonal antibodies, PAMPs/microbe-mediated phenoloxidase (PO) activity significantly decreased. These results suggest that <i>Ap</i>-HP6 has a positive regulatory effect on PPO activation and AMP synthesis. Additionally, the in vitro hydrolysis of r<i>Ap</i>-proHP6-Tb-His₆ yielded r<i>Ap</i>-HP6 with serine protease activity, which exhibited optimal reaction conditions for S-2288 at pH 8.0, 50 °C, and 15 min.