[Show abstract][Hide abstract] ABSTRACT: Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35-50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.
[Show abstract][Hide abstract] ABSTRACT: In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.
[Show abstract][Hide abstract] ABSTRACT: Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and beta-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.
[Show abstract][Hide abstract] 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.