Cytotoxic Reactions Associated with Insect Immunity

Nappi, Anthony J.; Vass, Emily

doi:10.1007/978-1-4615-1291-2_33

Cited by 71 publications

(52 citation statements)

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“…In insects and crustaceans, limited proteolysis generates active phenoloxidase (PO) which catalyzes the formation of quinones (Ashida and Brey, 1998;Nappi and Vass, 2001;Jiang and Kanost, 2000). Quinones are utilized in encapsulating parasites with a melanin sheath.…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Wang¹,

Jiang²

2007

Insect Biochemistry and Molecular Biology

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Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal β-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila Snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with β-1,3-glucan and β-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu 152 and Ile 153 . Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg 355 and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys 153 and generated an amidase activity which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: β-1,3-glucan recognition -proHP14 autoactivation -proHP21 cleavage -PAP-2 generation -proPO activation -melanin formation.

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Section: Introductionmentioning

confidence: 99%

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Wang¹,

Jiang²

2007

Insect Biochemistry and Molecular Biology

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“…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).…”

mentioning

confidence: 99%

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

Tong

Kanost

2005

Journal of Biological Chemistry

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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“…The prophenoloxidase (proPO) 1 activation cascade is probably composed of several serine proteinases that are sequentially activated in response to microbial infection and lead to the proteolytic activation of proPO to phenoloxidase (PO). PO catalyzes the formation of quinones that are reactive intermediates for melanin synthesis (5,6). Quinones are also involved in cuticle sclerotization, wound healing, and sequestration of parasites or pathogens.…”

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confidence: 99%

A Pattern Recognition Serine Proteinase Triggers the Prophenoloxidase Activation Cascade in the Tobacco Hornworm, Manduca sexta

Wang

Guo

et al. 2004

Journal of Biological Chemistry

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A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.Similar to other invertebrates, insects lack an adaptive immune system and rely solely on their innate immune mechanisms to fight against invading microorganisms (1-3). These defense mechanisms are mediated by hemocytes (e.g. phagocytosis and encapsulation), fat body (e.g. induced synthesis of antimicrobial peptides), and plasma factors (e.g. hemolymph coagulation and melanization). Accumulating evidence indicates that a complex serine proteinase network in insect hemolymph coordinates some of these responses (4). The prophenoloxidase (proPO) 1 activation cascade is probably composed of several serine proteinases that are sequentially activated in response to microbial infection and lead to the proteolytic activation of proPO to phenoloxidase (PO). PO catalyzes the formation of quinones that are reactive intermediates for melanin synthesis (5, 6). Quinones are also involved in cuticle sclerotization, wound healing, and sequestration of parasites or pathogens.Although the physiological importance of the proPO activation in insect immune system has been appreciated for many years, the molecular characterization of the cascade components was reported only recently. We isolated three proPOactivating proteinases from cuticular extract or hemolymph of M. sexta prepupae, and they all required a protein cofactor for proPO activation (7-10). Similar results were obtained from the beetles Holotrichia diomphalia and Tenebrio molitor (11-13). In contrast, the silkworm proPO-activating enzyme does not appear to need any auxiliary factor for proPO activation (14). Limited by substrate availability, current biochemical research is focused on the components at the end of th...

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Cytotoxic Reactions Associated with Insect Immunity

Cited by 71 publications

References 122 publications

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

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

A Pattern Recognition Serine Proteinase Triggers the Prophenoloxidase Activation Cascade in the Tobacco Hornworm, Manduca sexta

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