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Changes in composition and levels of hemolymph proteins during metamorphosis of Manduca sexta.

ABSTRACT: The tobacco hornworm, Manduca sexta, is a lepidopteran model species widely used to study insect biochemical processes. Some of its larval hemolymph proteins are well studied, and a detailed proteomic analysis of larval plasma proteins became available in 2016, revealing features such as correlation with transcriptome data, formation of immune complexes, and constitution of an immune signaling system in hemolymph. It is unclear how the composition of these proteins may change in other developmental stages. In this paper, we report the proteomes of cell-free hemolymph from prepupae, pupae on day 4 and day 13, and young adults. Of the 1824 proteins identified, 907 have a signal peptide and 410 are related to immunity. Drastic changes in abundance of the storage proteins, lipophorins and vitellogenin, for instance, reflect physiological differences among prepupae, pupae, and adults. Considerably more proteins lacking signal peptide are present in the late pupae, suggesting that plasma contains relatively low concentrations of intracellular components released from remodeling tissues during metamorphosis. The defense proteins detected include 43 serine proteases and 11 serine protease homologs. Some of these proteins are members of the extracellular immune signaling network found in feeding larvae, and others may play additional roles and hence confer new features in the later life stages. In summary, the proteins and their levels revealed in this study, together with their transcriptome data, are expected to stimulate focused explorations of humoral immunity and other physiological systems in wandering larvae, pupae, and adults of M. sexta and shed light upon functional and comparative genomic research in other holometabolous insects.

SUBMITTER: Cao X

PROVIDER: S-EPMC7704632 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R366 at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R410 as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg366 by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg410 for trypsin-like proteases, Gly406 and Ala409 for the elastase and Thr404 for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0-50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.

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.

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Changes in composition and levels of hemolymph proteins during metamorphosis of Manduca sexta.

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