A trypsin‐like serine proteinase interacts with Hemocytin and participates in regulating the function of insect nodulation

Nodulation is an efficient innate immune regulatory mechanism primarily mediated by hemocytes, which aids insects in effectively capturing and eliminating pathogens following their invasion into the hemocoel. Hemocytin is a crucial protein that mediates the nodulation process, synthesized and secreted by granulocytes, facilitating the aggregation of hemocytes. However, the mechanisms underlying the synthesis and release of Hemocytin in granulocytes, as well as the molecular mechanisms regulating nodule formation, remain unclear. In this study, we constructed a yeast library and used Hemocytin as the bait protein, combined with SPR molecular interaction experiments, to identify a trypsin-like serine protease (SP) that interacts with Hemocytin. Through localization studies, we found that SP and Hemocytin co-localize in the granules of granulocytes and are co-released into the hemolymph. Additionally, enzymatic cleavage experiments confirmed that SP can cleave the propeptide portion of Hemocytin. Destruxin A, a Hemocytin-targeting drug, modulates the interaction between SP and Hemocytin, thereby influencing insect immune responses. This, together with functional studies of SP, confirms that SP plays a key role in regulating Hemocytin-mediated hemocyte aggregation and nodulation. These findings will enhance our understanding of the molecular mechanisms by which Hemocytin mediates nodulation and deepen our comprehension of the nodule formation process.