Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R³⁶⁶ at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R⁴¹⁰ as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg³⁶⁶ by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg⁴¹⁰ for trypsin-like proteases, Gly⁴⁰⁶ and Ala⁴⁰⁹ for the elastase and Thr⁴⁰⁴ for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0–50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.     

Purification and characterization of a small cationic protein from the tobacco hornworm Manduca sexta

The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 μg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 μg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.     

Oenocytoid cell lysis to release prophenoloxidase is induced by eicosanoid via protein kinase C

Eicosanoids mediate insect cellular immune responses, which depend largely on phenoloxidase (PO) activity. In plasma, PO is activated by the proteolytic cleavage of proPO, which is stored in oenocytoids, a specific hemocyte type, of the beet armyworm, Spodoptera exigua. Eicosanoids induce an acute cell lysis of oenocytoids, which releases proPO into the plasma. We investigated an intracellular signal pathway following a functional interaction of eicosanoid(s) to a putative membrane receptor. U-73122 (a specific inhibitor of phospholipase C) inhibited oenocytoid lysis of S. exigua significantly after bacterial infection. We concluded that oenocytoid lysis required a certain level of calcium ion because EGTA (a calcium chelator) treatment inhibited cell lysis. Two protein kinase C (PKC) inhibitors (staurosporine and calphostin C) significantly inhibited the oenocytoid lysis. Oenocytoid lysis was likely induced by Na⁺ entry and subsequent osmotic shock because juvenile hormone analog, pyriproxyfen, which activates Na⁺-K⁺ ATPase and induces subsequent cell shrinkage, antagonized the effect of eicosaniod on cell lysis. Furthermore, ouabain (a specific Na⁺ pump inhibitor) significantly inhibited oenocytoid lysis. These results suggest that eicosanoid mediates oenocytoid lysis by activating the intracellular PKC pathway.     

Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation

In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.     

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4–P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.     

Rhodnius prolixus infected with Trypanosoma rangeli: In vivo and in vitro experiments

Studies were carried out on the activation of the prophenoloxidase (proPO) in adults of Rhodnius prolixus infected by short and long epimastigote forms of Trypanosoma rangeli. The in vitro activation of the proPO cascade using l-DOPA as substrate was very low in the absence of fat body extract, hemolymph, and parasites. On the other hand, a higher PO activity was observed when short, but not long, epimastigotes of T. rangeli were incubated with fresh hemolymph, fat body extract, and l-DOPA. Supernatant from lysed long epimastigotes increased the PO activity at levels identical to those observed with supernatants from lysed short epimastigotes. Similarly, the PO activity of hemolymph obtained from inoculated insects with long epimastigotes of T. rangeli showed a very low activity when incubated with l-DOPA compared to the PO activity of hemolymph taken from insects inoculated with short epimastigotes of T. rangeli. Control insects inoculated with sterile PBS showed no PO activity. These data indicate the presence of (a) factor(s) in the hemolymph as well as in the fat body extract that may be released (or induced) by the presence of short epimastigotes of T. rangeli and which results in the activation of the R. prolixus proPO system. The implications of these findings are discussed in relation to the development of T. rangeli and its ability to overcome the proPO system, survive, and successfully colonize the hemolymph of R. prolixus.     

褐飞虱和桃蚜粗提液中酚氧化酶原激活系统的激活特点

采用研磨法制备褐飞虱Nilaparvata lugens (Stål)和桃蚜Myzus persicae (Sulzer)粗提液,用于研究几种化学因子对粗提液中酚氧化酶原激活系统(proPO-AS)的激活特点。结果表明, 在0.1～100 mmol/L的Ca²⁺浓度范围内,虱液和蚜液的酚氧化酶(PO)活性随Ca²⁺浓度升高而增强,并在30 mmol/L处PO活性达到最高,在此限之上PO活性反而下降。当昆布多糖浓度从10^-4 mg/mL升至10 mg/mL时,虱液和蚜液的PO活性也随之升高,但再提高昆布多糖浓度却未见PO活性的峰顶出现。在6种葡聚糖对PO的激活作用中,昆布多糖和酵母聚糖能显著增强PO活性,但curdlan对虱液和蚜液的PO无明显激活作用,而甘露聚糖、右旋糖苷和纤维素则降低PO活性。这些结果显示β-1,3-葡聚糖能有效激活proPO-AS。Ca²⁺和丝氨酸蛋白酶抑制剂PMSF加样顺序的不同也会影响PO活性。     

Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6

Insect immune responses include prophenoloxidase (proPO) activation and Toll pathway initiation, which are mediated by serine proteinase cascades and regulated by serpins. Manduca sexta hemolymph proteinase-6 (HP6) is a component of both pathways. It cleaves and activates proPO activating proteinase 1 (PAP1) and hemolymph proteinase-8 (HP8), which activates proSpätzle. Inhibitors of HP6 could have the capability of regulating both of these innate immune proteinase cascade pathways. Covalent complexes of HP6 with serpin-4 and serpin-5 were previously isolated from M. sexta plasma using immunoaffinity chromatography with serpin antibodies. We investigated the inhibition of purified, recombinant HP6 by serpin-4 and serpin-5. Both serpin-4 and serpin-5 formed SDS-stable complexes with HP6 in vitro, and they inhibited the activation of proHP8 and proPAP1. Serpin-5 inhibited HP6 more efficiently than did serpin-4. Injection of serpin-5 into larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced the bacteria-induced expression of attacin, cecropin and hemolin genes in fat body. Injection of serpin-4 had a weaker effect on antimicrobial peptide expression. These results indicate that serpin-5 may regulate the activity of HP6 to modulate proPO activation and antimicrobial peptide production during immune responses of M. sexta.     

Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.     

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.     

Purification of the prophenoloxidase from Hyalophora cecropia and four proteins involved in its activation

Prophenoloxidase (PPO) has been purified to homogeniety from hemolymph of Hyalophora cecropia. There are two forms of the enzyme with identical molecular weights (76 kDa). Four proteins directly involved in the activation of PPO have also been purified from the hemolymph. Active phenoloxidase is elicited by the addition of factor C1 and a serine protease (SPII), which alone cannot activate PPO. Purified SPII contains two proteins with M_r 43 and 53 kDa, the larger molecule may represent the unactivated enzyme. An inhibitor of the SPII catalyzed activation of PPO has been isolated. In addition a protein presumed to be dopa quinone imine conversion factor has been purified.     

Identification of two clip domain serine proteases involved in the pea aphid's defense against bacterial and fungal infection

Phenoloxidases (POs) are required for the pea aphid's defense against bacterial and fungal infection. Prophenoloxidases (PPOs) are proteolytically converted to its active form PO through a clip domain serine protease cascade. In this study, we identified five clip domain serine proteases in the pea aphids. The messenger RNA levels of two of them, Ap_SPLP and Ap_VP, were upregulated by Gram‐positive bacterium Staphylococcus aureus and fungus Beauveria bassiana infections. Double‐stranded RNA‐based expression knockdown of these two genes resulted in reduced PO activity of the aphid hemolymph, higher loads of S. aureus and B. bassiana in the aphids, and lower survival rates of the aphids after infections. Our data suggest that Ap_SPLP and Ap_VP are involved in PPO activation pathway in the pea aphid.     

Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Manduca sexta serpin-6 regulates immune serine proteinases PAP-3 and HP8. cDNA cloning, protein expression, inhibition kinetics, and function elucidation

Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in hemolymph. We recently isolated Manduca sexta serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387-395). To further characterize its structure and function, we cloned serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster serpin-5, an immune-responsive protein. We produced serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of serpin-6 is between Arg373 and Ser374. Serpin-6 is constitutively present in hemolymph of naive larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of serpin-6 and PAP-3 is 2.6 x 10(4) m(-1) s(-1), indicating that serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using serpin-6 antibody. These results suggest that serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.     

Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.     

Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p-nitrophenyl-p～-guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin-mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme-catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.