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One of the innate immune reactions in invertebrates is the pro-phenoloxidase (pro-PO) activation system that is involved in the generation of superoxide, melanin synthesis, and the subsequent sequestration of foreign matter entering the hemocoel of the invertebrates. However, the molecular mechanism of this biological reaction is still obscure. To expand our understanding of the biological roles of the pro-PO activation system in invertebrates, we performed a yeast two-hybrid screening by using three regions of pro-PO as bait and a yeast two-hybrid cDNA library from Tenebrio molitor larvae as prey We isolated a novel partial cDNA clone that encodes a glycine-rich protein that interacted with the active phenoloxidase (termed phenoloxidase interacting protein, POIP). POIP consists of two domains: One is an N-terminal unique domain and the other is a C-terminal glycine-rich domain. The C-terminal glycine-rich domain showed sequential homology with those of insect antifungal proteins. Also, the yeast two-hybrid screen in a reverse orientation (using POIP as bait) yielded PO, suggesting that the PO-POIP interaction is specific. By using a 315 bP PCR fragment of the N-terminal unique region of POIP, we cloned the full-length cDNA of POIP from the Tenebruo cDNA library constructed by using E. coli injected larvae. The interaction analysis between PO, and a truncated fragment lacking the N-terminal unique region of POIP, indicated that the N-terminal unique region is necessary for interaction between PO and POIP. The expression level of the POIP mRNA is increased by bacterial injection into T. molitor larvae. This suggests that POIP might be engaged in the humoral defense reaction.
Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitors, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1,2 and 3(DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki=3.6×10-9M), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki=6.2×10-8M). Pre-treatment of the 33mg/㎏ of DI-mixture (active fractions from C18 open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.
<P><B>Abstract</B></P> <P>Recently, our group demonstrated that the bean bug, <I>Riptortus pedestris</I>, is a good experimental symbiosis model to study the molecular cross-talk between the host insect and the gut symbiont. The <I>Burkholderia</I> symbiont is orally acquired by host nymphs from the environment in every generation. However, it is still unclear how <I>Riptortus</I> specifically interacts with entomopathogens that are abundant in the environmental soil. In preliminary experiments, we observed that a potent entomopathogen, <I>Serratia marcescens</I>, can colonize the midgut of <I>Riptortus</I> insects and was recovered from the midgut when <I>Serratia</I> cells were orally administered, suggesting that this pathogenic bacterium can escape host immune defenses in the salivary fluid. We examined how orally fed <I>Serratia</I> cells can survive in the presence of antimicrobial substances of the <I>Riptortus</I> salivary fluid. In this study, a 15 kDa trialysin-like protein from the salivary gland of <I>R. pedestris</I> and a potent virulence factor of <I>Serratia</I> cells, a serralysin metalloprotease, from the culture medium of <I>S. marcescens</I> were successfully purified to homogeneity. When the purified <I>Riptortus</I> trialysin (rip-trialysin) was incubated with purified serralysin, rip-trialysin was specifically hydrolyzed by serralysin, leading to the loss of antimicrobial activity. These results clearly demonstrated that a potent virulent metalloprotease of <I>S. marcescens</I> functions as a key player in the escape from the salivary fluid-mediated host immune response, resulting in successful colonization of <I>S. marcescens</I> in the host midgut.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Salivary glands of insects contain some antimicrobial substances to protect the host. </LI> <LI> Two proteins, host antimicrobial peptides and pathogen virulence factor were purified. </LI> <LI> Antimicrobial peptides were specifically hydrolyzed by virulence factor. </LI> <LI> virulent factor can escape from the salivary fluid-mediated host immune response. </LI> </UL> </P>
One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxdase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca^2+ and -1,3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 and β-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conlusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.