Dataset Information

Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph.

ABSTRACT: 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.

SUBMITTER: Yang F

PROVIDER: S-EPMC5997545 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

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:A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal β-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.

Project description:Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the tobacco hornworm, Manduca sexta. In this study, we purified M. sexta PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). M. sexta PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naïve larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglycans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation.

Project description:Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the β-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34-36% identical to M. sexta β-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system.

Dataset Information

Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets