0.05. HP5 Stimulates proPO Activation. (trimming after His) that coordinates immune responses in the caterpillar. Serine protease cascades have developed in vertebrates and invertebrates to mediate defense responses to pathogen attack and tissue damage (1, 2). Once microbial surfaces or aberrant tissues are sensed by host factors in the blood circulation, specific proteolysis occurs to activate PP58 sequentially the protease cascade users within minutes. In insects, the cascades include serine proteases with multiple modular regulatory domains (3) and proteases with amino-terminal clip domains (2). Terminal clip proteases in the pathways generate active phenoloxidases (POs) and the Toll ligand Sp?tzle by cleaving their inactive precursors. POs catalyze the formation of reactive PP58 compounds and melanin to kill and sequester pathogens (4). Sp?tzle binds the Toll receptor to induce synthesis of antimicrobial peptides (5). Noncatalytic serine protease homologs (SPHs) are also activated by limited proteolysis and become PP58 a cofactor for proPO activating proteases (PAPs) (6, 7). The proteases in these pathways are regulated by protease inhibitors from your serpin superfamily (8). The immune serine protease-SPH systems and their regulation by serpins have been studied in that includes an initiating modular serine protease (ModSP) and terminal Sp?tzle processing enzyme (SPE), with the clip protease Grass between these two proteases, but the substrates of ModSP and Grass have not yet been determined experimentally (11). An alternative pathway that leads to Sp?tzle activation involves the clip protease Persephone but does not require ModSP (11). Persephone can be activated by a combination of microbial proteases and a hemolymph cathepsin (12), which cleaves after an unusual His residue at the activation site of Persephone. The melanization cascade in includes the clip proteases MP1, MP2, and Hayan (13, 14). MP2 is usually a PAP for proPO1 (15). There is not a well-characterized linkage between the protease cascades that activate proSp?tzle and proPO in immune responses. However, the serpins Necrotic and Spn28Da impede both Toll and proPO activation in (16, 17). Biochemical studies in a beetle, has additional complexity. In a lepidopteran insect, Persephone, cleaves proPAP1 for proPO activation and also cleaves hemolymph protease 8 (HP8), which activates proSp?tzle-1 (22, 23) (Fig. 1). Like Persephone, proHP6 has a His residue at its activation cleavage site. Some evidence indicates that hemolymph protease 1 (HP1) may activate proHP6 to form HP6 (24, 25), but it is still unclear whether proHP6 activation may be linked to HP14 or HP21. Here we statement our results indicating that HP5 acts as a previously unknown link between HP21 and HP6, cleaving proHP6 at its activation site His residue. We also found that HP5 is usually inhibited by multiple serpins present in hemolymph. Open in a separate windows Fig. 1. A simplified model of the serine protease network in HP5. Results Structural Features, Expression Pattern, and Production of Recombinant proHP5. HP5 is a member of the CLIP family of proteases in subfamily B (2). The proHP5 cDNA sequence encodes a signal peptide and a group 2 clip domain, followed by a 96-residue linker region rich in Ser (11), Asp (10), Thr (9), Asn (9), Pro (9), and Gly (8) (proHP5 in a baculovirusCinsect cell system. The purified proHP5 migrated as a 52-kDa band around the Coomassie-stained gel, which was recognized by HP5 antiserum produced using HP5 protein synthesized in as antigen (Fig. 2HP5 in melanization and antimicrobial peptide induction. (= 3). ((0.1 g), proHP5 (0.1 g), or both for 45 min at 25 C. PO activities were assayed and plotted as the bar graph (mean SEM, PP58 = 3). Interactions of plasma with and proHP5 both led to proPO activation. (test is run to reveal statistical significance of the mRNA level changes. * 0.05; n.s. 0.05. HP5 Stimulates proPO Activation. To test a possible role for HP5 in melanization, we incubated 100C500 ng proHP5 with diluted plasma from bacteria-induced larvae and detected increased PO activity that depended around the concentration of added proHP5 (Fig. 2(Fig. 2that is brought on by acknowledgement of bacteria. ProHP5 Is usually Cleaved at Its Activation Site by PAP3 and HP21. ProHP5 must be cleaved by another protease at its zymogen activation site to elicit its activity during an immune response. We tested several hemolymph clip domain name proteases (PAP1, PAP2, PAP3, HP2, and HP21) as proHP5 activators, based on their known specificity PP58 for cleavage after a basic residue, with the prediction that a proHP5 activator will cleave after Arg153 in proHP5. After incubation with active PAP3, the 52-kDa proHP5 was converted to a 30-kDa band visible with Coomassie staining and with detection by HP5 antibodies, consistent with the size of the C-terminal protease domain name of HP5 (Fig. 3proHP5 by PAP3 and HP21. XLKD1 (gel; lane 1, 0.8 g proHP5; lane 2, 50 ng PAP3; and lane 3, both proHP5 and.
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