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.
Highlights
Thrombin, the key enzyme responsible for converting fibrinogen to fibrin in the mammalian blood clotting cascade, is mainly regulated by the target serpin human antithrombin [1, 6]
Wild-type SPN48 was resistant to proteolysis by thrombin and was degraded by trypsin without forming a complex, which is distinct from the complex formation of SPN48 and Spatzle-processing enzyme (SPE)
Native full-length heparin accelerated the SPN48 inhibition of SPE (Fig. 5A), whereas a commercially available heparin pentasaccharide, which has anti-coagulation activity in mammalian blood [37], did not alter the reactivity of SPN48. Another low molecular weight heparin (15-saccharide with molecular mass of ϳ5000 Da) did not affect the SPN48 activity like the heparin pentasaccharide. These results show that heparin enhances the reactivity of SPN48 with SPE depending on the length of the heparin chain
Summary
Overexpression, Purification, and Site-directed Mutagenesis— Overexpression and purification of recombinant SPN48 (residues 17–389) using a bacterial expression system were reported previously [22]. To construct wild-type (residues 336 –361) and mutant RCL-linker (E353K), DNA fragments encoding SPN48 RCL (residues 336 –361) were amplified by PCR. The resulting PCR DNA was digested with BamHI and EcoRI and ligated into the same sites of the pGEXTEV-MliC vector, which expresses the Nterminal GST protein, C-terminal Pseudomonas aeruginosa MliC protein, and the tobacco etch virus protease cleavage site between the two proteins [23]. The structure was refined in the program CNS [27] and was further refined using PHENIX.REFINE [28] Another crystal grown in a reservoir solution (13% polyethylene glycol 8000 and 0.11 M MES (pH 6.0)) was obtained, from which a 1.9-Å resolution dataset with similar cell dimensions was collected. To measure the protease activity of SPE and thrombin toward wild-type or the mutant RCL linker, 5 g of the RCL linker protein was incubated with 0.15 g of SPE or 25 units of thrombin at 30 °C for 2 h in buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl, and resolved by SDS-PAGE
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