Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

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.

Hemolymph Melanization in the Silkmoth Bombyx mori Involves Formation of a High Molecular Mass Complex That Metabolizes Tyrosine

“…4D). As noted in other studies (16,22,27,28,38), these results showed that PPO and PO did not elute from the column at their predicted masses due to binding to other proteins and/or the column matrix. However, these data did show that melanization activity localized primarily to fraction 1, which contained little monomeric PO.…”

“…5B showed that processing of PPO to PO is associated with the rapid formation of many PO-containing bands in plasma that are chemically cross-linked and larger than monomeric PO, whereas our gel filtration data indicated that one of these complexes elutes in fraction 1, which also exhibits most of the melanization activity. Earlier studies with B. mori, T. molitor, and H. diomphalia (16,22,27,28,38) had already noted that processing of PPO to PO results in formation of PO-containing complexes that are chemically cross-linked. Adding a catecholamine substrate to a complex containing PO and SPH1 from T. molitor further increased its mass (27), whereas adding PTU to B. mori plasma inhibited the formation of most POcontaining bands (22).…”

“…Thus, it is unclear how the substrate preferences of in vitro activated PPO relate to the substrates actually available in hemolymph. Second, cleavage of PPO may not be the terminal step of the PO cascade because studies with the moths Bombyx mori and Manduca sexta plus the beetles Tenebrio molitor and Holotrichia diomphalia indicate that POs form chemically cross-linked high molecular mass complexes (16,(22)(23)(24)(25)(26)(27)(28). In vitro studies further show that mixing purified components of the PO cascade from T. molitor and H. diomphalia resulted in the formation of complexes that contained PO and a serine protease homolog (SPH).…”

“…In vitro studies further show that mixing purified components of the PO cascade from T. molitor and H. diomphalia resulted in the formation of complexes that contained PO and a serine protease homolog (SPH). These complexes also bound the surface of bacteria and oxidized a catecholamine, which suggests that complex formation is important in localizing melanin deposition to the surface of pathogens (27,28).…”

Structural and Inhibitory Effects of Hinge Loop Mutagenesis in Serpin-2 from the Malaria Vector Anopheles gambiae

“…Melanization is employed to encapsulate and kill invading pathogens and is initiated upon pathogen detection (16). Recognition proteins in the insect hemolymph recognize non-self biomolecules and activate a clip domain serine proteinase cascade (17)(18)(19)(20). This cascade culminates in the activation of prophenoloxidase-activating proteinases, which convert prophenoloxidase to phenoloxidase (21)(22)(23).…”