93-kDa Twin-domain Serine Protease Inhibitor (Serpin) Has a Regulatory Function on the Beetle Toll Proteolytic Signaling Cascade

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 Spatzle-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 Spatzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited beta-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 beta-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.