Identification of plasma proteinase complexes with serpin-3 in Manduca sexta

Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.     

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

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.     

Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6

Insect immune responses include prophenoloxidase (proPO) activation and Toll pathway initiation, which are mediated by serine proteinase cascades and regulated by serpins. Manduca sexta hemolymph proteinase-6 (HP6) is a component of both pathways. It cleaves and activates proPO activating proteinase 1 (PAP1) and hemolymph proteinase-8 (HP8), which activates proSpätzle. Inhibitors of HP6 could have the capability of regulating both of these innate immune proteinase cascade pathways. Covalent complexes of HP6 with serpin-4 and serpin-5 were previously isolated from M. sexta plasma using immunoaffinity chromatography with serpin antibodies. We investigated the inhibition of purified, recombinant HP6 by serpin-4 and serpin-5. Both serpin-4 and serpin-5 formed SDS-stable complexes with HP6 in vitro, and they inhibited the activation of proHP8 and proPAP1. Serpin-5 inhibited HP6 more efficiently than did serpin-4. Injection of serpin-5 into larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced the bacteria-induced expression of attacin, cecropin and hemolin genes in fat body. Injection of serpin-4 had a weaker effect on antimicrobial peptide expression. These results indicate that serpin-5 may regulate the activity of HP6 to modulate proPO activation and antimicrobial peptide production during immune responses of M. sexta.     

A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta.

Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.     

hnRNP A1 and secondary structure coordinate alternative splicing of Mag

Myelin-associated glycoprotein (MAG) is a major component of myelin in the vertebrate central nervous system. MAG is present in the periaxonal region of the myelin structure, where it interacts with neuronal proteins to inhibit axon outgrowth and protect neurons from degeneration. Two alternatively spliced isoforms of Mag mRNA have been identified. The mRNA encoding the shorter isoform, known as S-MAG, contains a termination codon in exon 12, while the mRNA encoding the longer isoform, known as L-MAG, skips exon 12 and produces a protein with a longer C-terminal region. L-MAG is required in the central nervous system. How inclusion of Mag exon 12 is regulated is not clear. In a previous study, we showed that heteronuclear ribonucleoprotein A1 (hnRNP A1) contributes to Mag exon 12 skipping. Here, we show that hnRNP A1 interacts with an element that overlaps the 5′ splice site of Mag exon 12. The element has a reduced ability to interact with the U1 snRNP compared with a mutant that improves the splice site consensus. An evolutionarily conserved secondary structure is present surrounding the element. The structure modulates interaction with both hnRNP A1 and U1. Analysis of splice isoforms produced from a series of reporter constructs demonstrates that the hnRNP A1-binding site and the secondary structure both contribute to exclusion of Mag exon 12.     

Expression and characterization of recombinant Manduca sexta serpin- 1B and site-directed mutants that change its inhibitory selectivity

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P₁ residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P₁ residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P₁ alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.     

Cysteine proteinases substitute for serine proteinases in the midgut glands of Crangon crangon and Crangon allmani (Decapoda: Caridea)

The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by “trypsin-like” serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases.A comparison with the eucarid crustaceans Pandalus montagui, Pagurus bernhardus, Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba. E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba. In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus, C. pagurus and E. superba. The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail.     

Manduca sexta serpin-6 regulates immune serine proteinases PAP-3 and HP8. cDNA cloning, protein expression, inhibition kinetics, and function elucidation

Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in hemolymph. We recently isolated Manduca sexta serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387-395). To further characterize its structure and function, we cloned serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster serpin-5, an immune-responsive protein. We produced serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of serpin-6 is between Arg373 and Ser374. Serpin-6 is constitutively present in hemolymph of naive larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of serpin-6 and PAP-3 is 2.6 x 10(4) m(-1) s(-1), indicating that serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using serpin-6 antibody. These results suggest that serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.     

Related antipodes: a comparative study on digestive endopeptidases from Northern krill and Antarctic krill (Euphausiacea)

The Antarctic krill, Euphausia superba, and the Northern krill, Meganyctiphanes norvegica, are closely related species but occupy significantly different trophic and climatic environments. E. superba holds a key position as a phytoplankton grazer in the Southern Ocean. The omnivorous M. norvegica is an important member of plankton communities in the Northeast Atlantic. Both species expressed high proteolytic activities which were dominated by serine proteinases. In the stomachs of Antarctic krill, activities of total proteinase, trypsin, and chymotrypsin were significantly higher than in Northern krill. In the midgut glands, however, total proteinase and trypsin activities were similar in both species, but chymotrypsin activity was significantly higher in Antarctic krill. Moreover, Antarctic krill expressed four trypsin isoforms while only one isoform appeared in Northern krill. Chymotrypsin was present in either species as one single isoform. Antarctic krill adapted to the low and patchy distribution of food by elevated enzyme activities and the expression of trypsin isoforms with slightly different catalytic properties. Presumably, these enzymes facilitate in concerted action the efficient utilization of proteins from phytoplankton, the major food. Northern krill, in contrast, seems not to be equipped to face food limitation. It expresses a “simple” or “basic” set of digestive enzymes for utilizing abundant and easily digestible prey.     

Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases

Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses. We have isolated an immune-responsive serpin from the tobacco hornworm, Manduca sexta. This inhibitor, M. sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO). Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization. M. sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5-12 microg/ml and increases to 30-75 microg/ml after a microbial challenge. Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M. sexta. PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.     

Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3

The proteolytic activation of prophenoloxidase (proPO) is a critical defense mechanism in insects and crustaceans. We have isolated three prophenoloxidase-activating proteinases (PAPs) from cuticular extracts or hemolymph of Manduca sexta pharate pupae, which are negatively regulated by serpin-1J and serpin-3. To test if other serpins may also inhibit the PAPs, we fractionated the induced hemolymph by ammonium sulfate precipitation, gel filtration, and lectin affinity chromatography. A 47 kDa protein, designated M. sexta serpin-6, was identified in concanavalin A-bound fractions, which formed an SDS-stable complex with PAP-3. This inhibitor, not recognized by the serpin-1 or serpin-3 antibodies, was further purified on HPLC anion exchange and hydroxylapatite columns. The molecular mass and isoelectric point of serpin-6 were found to be 46,710 +/- 10 Da and 5.4. While its amino terminus was blocked, we obtained five internal peptide sequences, one of which is highly similar to M. sexta serpins-1, -2, and -3. Serpin-6 strongly inhibited PAP-3 but not PAP-1 or PAP-2, suggesting that the proPO activation by PAPs is differentially regulated by multiple serpins. When included in the reaction mixture containing proPO, PAP-3, and its cofactor, serpin-6 efficiently blocked the cleavage activation of proPO.     

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Identification of Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity

Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.     

Impact of transgenic oilseed rape expressing oryzacystatin-1 (OC-1) and of insecticidal proteins on longevity and digestive enzymes of the solitary bee Osmia bicornis

The risk that insect-resistant transgenic plants may pose for solitary bees was assessed by determining longevity of adult Osmia bicornis (O. rufa) chronically exposed to transgenic oilseed rape expressing oryzacystatin-1 (OC-1) or to the purified insecticidal proteins recombinant rOC-1, Kunitz soybean trypsin inhibitor (SBTI), Galanthus nivalis agglutinin (GNA), or Bacillus thuringiensis toxin Cry1Ab dissolved in sugar solution (at 0.01 and 0.1%, w:v, Cry1Ab only at 0.01%). Compared to control bees, longevity was significantly reduced by SBTI and GNA at both concentrations and by rOC-1 at 0.1%, but not by Cry1Ab or rOC-1 at 0.01%. Longevity on the OC-1 oilseed rape was not significantly different from the control plants. The effects of SBTI and rOC-1 on longevity were investigated through characterization of the digestive proteinases of O. bicornis and analysis of the response in proteinase profiles to ingestion of these proteinase inhibitors. A relatively complex profile of at least four types of soluble proteolytic enzymes was identified. Serine proteinases were found to be predominant, with metallo and especially cysteine proteinases making a smaller albeit significant contribution. The compensatory response to in vivo enzyme inhibition was similar for SBTI and rOC-1 although less pronounced for rOC-1. It consisted of a non-specific overproduction of native proteinases, both sensitive and insensitive, and the induction of a novel aspartic proteinase.