[Show abstract][Hide abstract] ABSTRACT: The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade.
Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified
three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle,
Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue
of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing
that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into
the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site
that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the
inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism
of how serpins tightly regulate innate immune responses in invertebrates.
Journal of Biological Chemistry 01/2011; 286(2):1567-1575. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.
[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 Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-kappaB-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation.
[Show abstract][Hide abstract] ABSTRACT: The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. Here, we demonstrate the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by gram-positive bacteria and fungi. Our analysis shows that ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spätzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions.
Proceedings of the National Academy of Sciences 08/2009; 106(30):12442-7. DOI:10.1073/pnas.0901924106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-kappaB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component beta-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by beta-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of beta-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal beta-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation.
[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.
[Show abstract][Hide abstract] ABSTRACT: The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway.