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An expansion of the dual clip-domain serine proteinase family in Manduca sexta: gene organization, expression, and evolution of prophenoloxidase-activating proteinase-2, hemolymph proteinase 12, and other related proteinases.

ABSTRACT: Prophenoloxidase-activating proteinases (PAPs) take part in insect defense responses including melanotic encapsulation and wound healing. To understand their gene structure and regulation, we screened a genomic library and isolated overlapping lambda clones for Manduca sexta PAP-2, hemolymph proteinase 12 (HP12), and HP24. Complete nucleotide sequence analysis indicated that all three genes encode polypeptides with two regulatory clip domains at the amino terminus, a linker region, and a catalytic serine proteinase domain at the carboxyl terminus. Each gene contains eight exons, with introns located at equivalent positions. Similar sequences are present in introns as well as exons, indicating that these genes arose from recent gene duplication and sequence divergence. We analyzed their 5' flanking sequences and identified putative immune and hormone responsive elements. Reverse transcription-polymerase chain reactions confirmed that PAP-2 and HP12 mRNA levels in the larval fat body and hemocytes increased after a bacterial challenge. However, HP24 expression was barely detected. PAP-2 transcripts in cultured fat body became less abundant after 20-hydroxyecdysone treatment. Thus, PAP-2, HP12, and HP24 mRNA levels are differentially regulated by immune and developmental signals. Comparison with HP15, HP23, and PAP-3 sequences suggested an evolutionary pathway of the dual clip-domain serine proteinases in M. sexta.

SUBMITTER: Wang Y

PROVIDER: S-EPMC2071929 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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An expansion of the dual clip-domain serine proteinase family in Manduca sexta: gene organization, expression, and evolution of prophenoloxidase-activating proteinase-2, hemolymph proteinase 12, and other related proteinases.

Wang Yang Y Zou Zhen Z Jiang Haobo H

Genomics 20051201 3

Prophenoloxidase-activating proteinases (PAPs) take part in insect defense responses including melanotic encapsulation and wound healing. To understand their gene structure and regulation, we screened a genomic library and isolated overlapping lambda clones for Manduca sexta PAP-2, hemolymph proteinase 12 (HP12), and HP24. Complete nucleotide sequence analysis indicated that all three genes encode polypeptides with two regulatory clip domains at the amino terminus, a linker region, and a catalyt ...[more]

PMID: 16324822

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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

An expansion of the dual clip-domain serine proteinase family in Manduca sexta: gene organization, expression, and evolution of prophenoloxidase-activating proteinase-2, hemolymph proteinase 12, and other related proteinases.

Publications

An expansion of the dual clip-domain serine proteinase family in Manduca sexta: gene organization, expression, and evolution of prophenoloxidase-activating proteinase-2, hemolymph proteinase 12, and other related proteinases.

Similar Datasets

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