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Binding properties of the regulatory domains in Manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system.

ABSTRACT: Pathogen recognition and rapid initiation of defense responses are essential for the survival of host insects. In Manduca sexta, hemolymph proteinase-14 precursor (proHP14) senses non-self presence and triggers a branched serine proteinase pathway which leads to prophenoloxidase activation and melanin formation around the invading organisms. To understand functions of individual domains in HP14, we have produced a series of HP14 domains and truncation mutants and studied their interactions with microbial polysaccharides and beta-1,3-glucan recognition protein-1 (betaGRP1)-a biosensor for fungal and bacterial infection. These include: the low-density lipoprotein receptor class A repeats 1-5 (LDL(1-5)), Sushi domain, Wonton domain, and proteinase catalytic domain of HP14, as well as proHP14 missing 1-4 LDL repeats (DeltaLDL(1), DeltaLDL(12), DeltaLDL(1-3) and DeltaLDL(1-4)). LDL(1-5), Sushi, and Wonton domains specifically recognized Lys-type PG, whereas the latter two also bound betaGRP1. Wonton in addition bound to lipopolysaccharide (LPS), lipoteichoic acid (LTA), and meso-diaminopimelic acid (DAP)-type peptidoglycan (PG). The four N-terminally truncated proHP14 (DeltaL(x)) further confirmed specific interactions with LPS, LTA, DAP-PG, Lys-PG, laminarin, and betaGRP1. These binding data suggest a broad specificity of proHP14 in pattern recognition. Its role in mediating immune responses is anticipated to be influenced by other plasma factors and surface structures of invading pathogens.

SUBMITTER: Wang Y

PROVIDER: S-EPMC2813979 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Binding properties of the regulatory domains in Manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system.

Wang Yang Y Jiang Haobo H

Developmental and comparative immunology 20091129 3

Pathogen recognition and rapid initiation of defense responses are essential for the survival of host insects. In Manduca sexta, hemolymph proteinase-14 precursor (proHP14) senses non-self presence and triggers a branched serine proteinase pathway which leads to prophenoloxidase activation and melanin formation around the invading organisms. To understand functions of individual domains in HP14, we have produced a series of HP14 domains and truncation mutants and studied their interactions with ...[more]

PMID: 19900472

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Project description:Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the serpin superfamily. The enzyme system also leads to proteolytic processing of a Spätzle precursor. Binding of Spätzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spätzle to induce immunity-related genes. Here we provide evidence that M. sexta serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the serpin-12 core domain (serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both serpin-12 and HP14, suggesting that a covalent serpin-protease complex formed when serpin-12 inhibited HP14. Together, these data suggest that M. sexta serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.

Dataset Information

Binding properties of the regulatory domains in Manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system.

Publications

Binding properties of the regulatory domains in Manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system.

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