Dataset Information

Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: organization, expression, and regulation by immune and hormonal signals.

ABSTRACT: Insect phenoloxidase (PO) participates in melanotic encapsulation, wound healing, and cuticle sclerotization. It is converted from prophenoloxidase (proPO) by a proPO-activating proteinase (PAP). Manduca sexta PAP-1, the final component of a serine proteinase cascade, cleaves proPO to generate active PO. In an effort to understand the transcriptional regulation, we isolated a genomic clone of the PAP-1 gene, determined its nucleotide sequence, and elucidated its exon-intron organization. Computer analysis revealed several immune and hormone responsive elements in the upstream region. Southern blot analysis suggested that the M. sexta genome contains a single copy of PAP-1 gene. Reverse transcription-polymerase chain reaction showed that PAP-1 was constitutively expressed in fat body, trachea, and nerve tissue of the fifth instar larvae. The mRNA levels in hemocytes and fat body markedly increased in response to a bacterial challenge. We also observed tissue-specific and developmental regulation of the gene's transcription. Treating M. sexta fat body culture with 20-hydroxyecdysone reduced the PAP-1 mRNA level. These data indicated that the expression of PAP-1 gene is under the dual control of immune and hormonal signals.

SUBMITTER: Zou Z

PROVIDER: S-EPMC2020815 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: organization, expression, and regulation by immune and hormonal signals.

Zou Zhen Z Wang Yang Y Jiang Haobo H

Insect biochemistry and molecular biology 20050317 6

Insect phenoloxidase (PO) participates in melanotic encapsulation, wound healing, and cuticle sclerotization. It is converted from prophenoloxidase (proPO) by a proPO-activating proteinase (PAP). Manduca sexta PAP-1, the final component of a serine proteinase cascade, cleaves proPO to generate active PO. In an effort to understand the transcriptional regulation, we isolated a genomic clone of the PAP-1 gene, determined its nucleotide sequence, and elucidated its exon-intron organization. Compute ...[more]

PMID: 15857768

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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

Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: organization, expression, and regulation by immune and hormonal signals.

Publications

Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: organization, expression, and regulation by immune and hormonal signals.

Similar Datasets

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