Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Qianran Wang,Manli Wang,Xijia Liu,Mengyi Yin,Zhen Zou,Chuanfei Yuan,Zhihong Hu

doi:10.3389/fimmu.2020.00785

Abstract

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

Highlights

Melanization is a prominent defense mechanism in arthropods that plays an essential role in wound healing, killing of microbes, and parasites encapsulation [1, 2]
The extracellular PPO activation pathway usually consists of a three-step proteolytic cascade initiated by one modular serine protease (SP) followed by clip domain SPs, which has been comprehensively revealed in a lepidopteran species Manduca sexta [4,5,6,7] and a coleopteran species Tenebrio molitor [8, 9]. clip domain serine protease (cSP) and the homologs are classified into four subfamilies (A– D) based on phylogenetic analysis [10, 11]
PPO was purified from the hemolymph of H. armigera larvae and, after a cetylpyridinium chloride (CPC)-induced conformation changes, PO activity was confirmed by production of dopamine chrome from dopamine (Supplementary Figure S1A)

Summary

Introduction

Melanization is a prominent defense mechanism in arthropods that plays an essential role in wound healing, killing of microbes, and parasites encapsulation [1, 2]. The extracellular PPO activation pathway usually consists of a three-step proteolytic cascade initiated by one modular SP followed by clip domain SPs (cSPs), which has been comprehensively revealed in a lepidopteran species Manduca sexta [4,5,6,7] and a coleopteran species Tenebrio molitor [8, 9]. The initiating modular SPs without clip domains that activate CLIPC members are characterized by containing low-density lipoprotein receptor class A (LDLa), Sushi and Wonton domains [14, 15]. They could be autoactivated in the presence of pathogens, cleaved the downstream proteases. M. sexta HP1, a member of CLIPD, was identified as a recognition protein of the melanization cascade which was activated without proteolytic cleavage [3, 16]

Methods

Results

Conclusion