Manduca sexta Serpin-3 Regulates Prophenoloxidase Activation in Response to Infection by Inhibiting Prophenoloxidase-activating Proteinases

Zhu, Yifei; Wang, Yan; Gorman, Maureen J.; Jiang, Haobo; Kanost, Michael R.

doi:10.1074/jbc.m309682200

Cited by 169 publications

(190 citation statements)

References 43 publications

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“…Silver staining was performed according to Ausubel et al (33). Immunoblot analysis was carried out using 1:3000 diluted antisera (28). Antibody binding was visualized using alkaline phosphate-conjugated goat anti-rabbit IgG and alkaline phosphate substrates kit (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

“…For assaying PAPs, 5 mM CaCl 2 was included in the buffer. Thrombin activity was measured in 10 mM Tris-HCl, 50 mM NaCl, pH 8. tive PAP-1 and PAP-3 were isolated during purification of the recombinant pro-PAPs expressed in Sf9 cells (28,37).…”

Section: Methodsmentioning

confidence: 99%

“…Its physiological function remains unknown. The serpin-3 gene encodes an immune-responsive serpin (28) that is expressed in the fat body after bacterial challenge. Serpin-3 is present at a low level in larval plasma and increases dramatically in concentration after microbial challenge.…”

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Manduca sexta Serpin-4 and Serpin-5 Inhibit the Prophenol Oxidase Activation Pathway

Tong

Kanost

2005

Journal of Biological Chemistry

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108

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Infection stimulates the innate immune responses of insects, including activation of prophenol oxidase (pro-PO) in plasma as the last step of a serine protease cascade. To investigate the roles of protease inhibitors in regulating this pathway, we cloned cDNAs for two new serpins (serpin-4 and serpin-5) from the tobacco hornworm, Manduca sexta. Serpin-4 and serpin-5 mRNAs are constitutively expressed at a low level in larval hemocytes and fat body and increased dramatically upon bacterial challenge. These serpins are present in larval plasma at ϳ3 (serpin-4) and ϳ1 g/ml (serpin-5) and increased 3-8-fold by 24 h after injection of bacteria or fungi. Recombinant serpin-4 and serpin-5 decreased pro-PO activation when added to plasma, but they did not directly inhibit the pro-PO-activating proteases. Instead, they apparently regulate the pathway by inhibiting one or more target proteases upstream of the pro-PO-activating proteases.Prophenol oxidase (pro- PO) 1 activation is an innate immune response in arthropods. Upon injury or infection, a pro-PO zymogen in plasma is activated by a specific protease. Phenol oxidase (PO) hydroxylates monophenols to o-diphenols and then oxidizes o-diphenols to quinones, which can polymerize to form melanin at the injury site or around invading organisms (1, 2). Quinones may also be involved in the production of cytotoxic molecules such as superoxides and hydroxyl radicals that could help kill the invading organisms (1, 3).A serine protease cascade functions in pro-PO activation (2, 4, 5). The final protease in the pathway, pro-PO-activating protease (PAP) (also called pro-PO-activating enzyme or factor) has been identified from several insects, including the tobacco hornworm, Manduca sexta (6 -8), the silkworm, Bombyx mori (9), a beetle, Holotrichia diomphalia (10, 11), and from a crustacean, the crayfish Pacifastacus leniusculus (12). These PAPs contain one or two clip domains at their amino terminus and a serine protease domain at their carboxyl terminus (13). PAPs are synthesized as zymogens, which are activated by a specific proteolytic cleavage between the clip domain and the protease domain. However, protease(s) that activate PAPs have not yet been characterized.Serpins present in plasma regulate the pro-PO activation pathway (4,5,(13)(14)(15)(16). Serpins are serine protease inhibitors, about 400 amino acid residues long (17-20), with an exposed reactive center loop near their carboxyl terminus (21,22). The P1 residue located in this loop determines the primary specificity of inhibition. Four serpins from M. sexta have been characterized previously. The serpin-1 gene encodes 12 variants with different inhibitory selectivity, due to alternative splicing of exons that encode the reactive center loop (23-25). Serpin-1 mRNA is constitutively expressed in the larval fat body and at a lower level in hemocytes (26). One of the reactive site variants, serpin-1J, inhibits pro-PO activation in plasma by inhibiting PAPs (8, 25). Serpin-2 is an intracellular protein (27), expressed in ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Manduca sexta Serpin-4 and Serpin-5 Inhibit the Prophenol Oxidase Activation Pathway

Tong

Kanost

2005

Journal of Biological Chemistry

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108

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“…Other proteins required for proPO activation are clip-domain serine proteinase homologs (SPHs), whose catalytic serine is replaced with glycine and, therefore, lack proteolytic activity (26, 27). Serine proteinase inhibitors, including members of the serpin superfamily, regulate the activation of proPO by inhibiting the activating proteinases (28,29).Drosophila clip-domain serine proteinases Persephone, Grass, Spirit, and spätzle-processing enzyme (SPE) participate in the activation of Toll pathway, stimulating synthesis of antimicrobial peptides as an innate immune response (18, 30 -32). Although genetic evidence indicates that Persephone and Spirit are upstream of SPE in the cascade, the substrate(s) of Persephone and Spirit have not been identified, and which proteinase directly activates SPE is unknown.…”

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confidence: 99%

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Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Ishibashi

Ragan

et al. 2009

Journal of Biological Chemistry

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Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.Innate immune systems of mammals and arthropods include extracellular serine proteinase cascade pathways, which rapidly amplify responses to infection and stimulate killing of pathogens. These proteinase-driven processes include the complement system of vertebrates (1, 2) and pathways in arthropods involving proteinases containing amino-terminal clip domains (3). Clip domain proteinases function in blood coagulation (4, 5), activation of prophenoloxidase (proPO) that leads to melanin synthesis (6 -9), and stimulation of the Toll pathway to promote synthesis of antimicrobial peptides/proteins (AMPs) 2 secreted into the hemolymph (10, 11).The serine proteinase systems best characterized in arthropods are the horseshoe crab hemolymph coagulation pathway and the cascade leading to activation of the Toll pathway in dorsal-ventral development in Drosophila (12-14). Recent research also has led to better characterization of the proPO activation pathway in Manduca sexta (7,15,16) and the Tollsignaling pathway in the Drosophila immune response (17, 18) and to both the proPO and Toll pathways in the beetle Tenebrio molitor (11,19).In the proPO activation pathway, soluble pattern recognition proteins initially recognize pathogen-associated molecular patterns such as bacterial peptidoglycan or fungal ␤-1,3-glucan (...

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Serpin7 controls egg diapause of migratory locust (Locusta migratoria) by regulating polyphenol oxidase

et al. 2020

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Diapause is a state of arrested growth, which allows insects to adapt to diverse environments. Serine protease inhibitors (serpins) play an important role in various physiological processes, including blood coagulation, fibrinolysis, development, complement activation and extracellular matrix remodeling. We hypothesized that serpin may affect energy metabolism and thereby control diapause of migratory locust (Locusta migratoria) embryos by regulating protease cascades. A total of seven nonredundant serpin genes (named serpin1–serpin7) of L. migratoria were obtained through transcriptomic analysis. We further performed label‐free proteomic sequencing and analysis of diapause and nondiapause eggs of L. migratoria, revealing significant differences in serpin7 expression. A significant reduction in diapause rate under the short photoperiod was observed in insects treated with serpin7 double‐stranded RNA. Furthermore, knockdown of the serpin7 gene resulted in significant upregulation of the activity of polyphenol oxidase. We therefore propose that the observed serpin7 gene plays a crucial role in diapause, suggesting that control of energy metabolism may have potential as a future strategy for the reproductive control of insect pests.

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Manduca sexta Serpin-3 Regulates Prophenoloxidase Activation in Response to Infection by Inhibiting Prophenoloxidase-activating Proteinases

Cited by 169 publications

References 43 publications

Manduca sexta Serpin-4 and Serpin-5 Inhibit the Prophenol Oxidase Activation Pathway

Manduca sexta Serpin-4 and Serpin-5 Inhibit the Prophenol Oxidase Activation Pathway

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Serpin7 controls egg diapause of migratory locust (Locusta migratoria) by regulating polyphenol oxidase

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