Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

TLDR

Melanization by phenoloxidase must be tightly controlled to prevent quinone toxicity and systemic damage, and the Spätzle‑processing enzyme is a key component of the Toll pathway that activates pro‑Spätzle. The study aims to elucidate the in vivo molecular mechanism regulating phenoloxidase‑induced melanin synthesis. Biochemical assays show that the Tenebrio Spätzle‑processing enzyme activates prophenoloxidase and SPH1 into a melanization complex that rapidly melanizes bacteria with strong bactericidal activity, while prophenoloxidase competitively inhibits complex formation, revealing a shared serine protease that links the Toll pathway to melanization.

Abstract

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.