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

## TL;DR

This study explores how a serine protease called Ap-HP6 regulates immune responses in the silkworm Antheraea pernyi.

## Contribution

The study identifies Ap-HP6 as a key regulator of both PPO activation and AMP synthesis in insect immunity.

## Key findings

- Ap-proHP6 expression is significantly induced in hemocytes and fat body after immune stimulation.
- Reduced Ap-proHP6 levels lead to decreased antimicrobial peptide expression and phenoloxidase activity.
- rAp-HP6 exhibits optimal serine protease activity under specific in vitro conditions.

## Abstract

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 A. pernyi (Ap-HP6). Following immune stimulation, the expression of Ap-proHP6 was significantly induced, primarily observed in hemocytes and the fat body. After suppressing Ap-proHP6 expression via RNA interference (RNAi) and infecting larvae with different microbes, the expression levels of AMPs showed a downward trend. When endogenous Ap-proHP6 content in hemolymph was reduced using RNAi technology or anti-rAp-proHP6-His6 polyclonal antibodies, PAMPs/microbe-mediated phenoloxidase (PO) activity significantly decreased. These results suggest that Ap-HP6 has a positive regulatory effect on PPO activation and AMP synthesis. Additionally, the in vitro hydrolysis of rAp-proHP6-Tb-His6 yielded rAp-HP6 with serine protease activity, which exhibited optimal reaction conditions for S-2288 at pH 8.0, 50 °C, and 15 min.