Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses

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 ␤-1,3-glucanmediated 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.

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

“…The production of PGRP-SC2 might be activated under both the Toll signaling pathway and the unidentified monomeric DAP-PG-activated signaling pathway. Recently, we reported three serpins that specifically inactivate MSP, SAE, and SPE, respectively, and two of the three serpins are induced by Toll pathway activation (39). PGRP-SC2 down-regulates the Toll signaling pathway in collaboration with these serpins.…”

Three-dimensional structure of a schistosome serpin revealing an unusual configuration of the helical subdomain

“…The His-to-Gln substitution, for instance, is consistently found in clade e, which contains PAI-1, glia-derived nexin and a myxoma virus serpin, all three of which are well established inhibitory serpins. Another occurrence is found in SPN48 from the mealworm Tenebrio molitor, which also exhibits antiproteinase activity (Jiang et al, 2009). Finally, PAI-1 also contains the Ser-to-Gly exchange found in ShSPI, while in heparin cofactor II the two serines are replaced by alanine and glycine, respectively, without alterations at the remaining positions.…”

Histological Observation and Expression Patterns of antimicrobial peptides during Fungal Infection in Musca domestica (Diptera: Muscidae) Larvae

“…A great number of immune regulators are involved in these processes, including serine proteinase inhibitors (Serpin). In Tenebrio molitor (Jiang et al 2009) and D. melanogaster (Ligoxygakis et al 2002;Fullaondo et al 2011), serpins can regulate the immune response through the Toll signal pathway and prophenoloxidase-activating system. Li discovered at least 11 serpins in housefly, including a gene homologous to serpin27A of D. melanogaster .…”