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

Abstract

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 (...

Regulation of melanization by glutathione in the moth Pseudoplusia includens

“…Dopaquinone and dopaminequinone thereafter proceed through a series of unstable intermediates such as dopachrome and doaminechrome, which lead to formation of melanin (summarized by Kanost and Gorman, 2008). The standard method for monitoring the melanization process and PO activity in insect plasma is by measuring absorbance at or near 470 nm (see An et al, 2009). We note, however, this approach predominantly detects dopachrome and/or dopaminechrome, rather than melanin itself Aroca et al, 1992).…”

Structure-Function Analysis of Grass Clip Serine Protease Involved in Drosophila Toll Pathway Activation

“…The proteases of the first group display 15-17 residues between the two cysteines whereas those of the second group have 22-24 residues. This method failed to place Grass in any of the two groups as it has an unusually long sequence (29 residues) between Cys 3 and Cys 4 (16). Because Grass was the focus of our study, we undertook a new classification, taking into account the sequence and the structural data of the clip domains but also of the SP domains.…”

“…However, the isolation of proteases for further ex vivo molecular studies is hampered by the small size of Drosophila adults. Insects of larger size and of easily extractable hemolymph, such as Bombyx mori, Manduca sexta, and Tenebrio molitor, are better choices for biochemical characterization (12)(13)(14)(15)(16)(17). In particular, proteases and PRRs have been isolated from T. molitor, and a signaling cascade triggering the Toll activation has been reconstituted in vitro.…”

Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph

“…Extracelluar serpins exert control over serine proteinase cascades in vertebrate blood, including complement activation, blood clotting, and fibrinolysis (3,4). In insects, serine proteinase cascades initiated by microbial infection elicit antimicrobial peptide production and lead to activation of prophenoloxidase, which catalyzes the melanization response (5)(6)(7)(8)(9)(10)(11)(12)(13). Serpins inhibit proteinases required for prophenoloxidase activation in Manduca sexta (14 -17), Drosophila melanogaster (18 -21), Tenebrio molitor (22), and Anopheles gambiae (23).…”