Purification And Biological Function Study Of Three Novel Serpins Involved In Regulation Of Insect Immune Responses

Analogous to blood coagulation and complement system in human, many insect defense responses such as melanin biosynthesis and Toll pathway signaling are mediated by serine proteinase (SP) cascades, however, excessive activation of the immune response is harmful to host tissues and cells, thus, a precise negative regulation of the upstream signal is extremely critical for host surviving. Serpins (serine protease inhibitors) are the largest and most important superfamily of protease inhibitors, they act as suicide substrates by binding covalently to their target proteases, leading inactivation of activity. As we known, SP cascades are usually regulated by the serpins in vertebrates, 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. By using these SPs, we detected and purified three novel serpins from the hemolymph of T. molitor, according their molecule sizes on SDS-PAGE gel, we named them SPN40, SPN55 and SPN48, respectively.We then cloned the serpin genes. The nucleotide sequence of serpins has been submitted to Genbank with accession numbers of AB514563, AB514564 and AB514565. These serpins made specific serpin-SP pairs with three Toll cascade-activating serine proteases:such as SPN40-MSP (Modular Serine Protease), SPN55-SAE (Spatzle-processing enzyme-Activating Enzyme), and SPN48-SPE (Spatzle-Processing Enzyme). we determined cleavage sites of each serpin on their reactive center loops (RCL). Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in RCL, respectively, despite being targeted by trypsin-like SAE and SPE。The levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll-ligand, processed Spatzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors.Results of in vitro reconstitution experiment suggest three serpins cooperatively blocked the Toll signaling cascade, furthermore, injection of three serpins into Tenebrio larvae inhibitedβ-1, 3-glucan mediated melanin biosynthesis in vivo.Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins, This is the first determination and functional study of specific SP-serpin pairs that are directly involved in the regulation of the pattern recognition protein-dependent Toll signaling cascade.