Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.     

Use of Proteases to Improve the Insecticidal Activity of Baculoviruses

Basement membranes that surround the tissues of lepidopterous larvae act as potential barriers to baculovirus movement and establishment of systemic infection. Hence, one potential approach to improving the insecticidal activity of baculoviruses is to perforate or eliminate the basement membranes of their hosts, thereby facilitating the process of infection. Toward this end, we constructed six recombinant clones of Autographa californica nucleopolyhedrovirus (AcMNPV) that express three proteases that digest basement membrane proteins: rat stromelysin-1, human gelatinase A, and flesh fly (Sarcophaga peregrina) cathepsin L. Expression of these proteases was directed from either the ie-1 promoter (in AcIE1TV3.STR1, AcIE1TV3.GEL, and AcIE1TV3.ScathL) or the p6.9 promoter (in AcMLF9.STR1, AcMLF9.GEL, and AcMLF9.ScathL). Recombinant proteases were detected in the culture medium of cells infected with recombinant viruses by either zymography or azocoll assay. AcMLF9.STR1 and AcMLF9.ScathL caused premature cuticular melanization of 5th instar Heliothis virescens. Melanization and fragmentation of internal tissues were observed in half of the larvae infected with AcMLF9.ScathL and not at all in larvae infected with AcMLF9.STR1 or wild-type AcMNPV. Lethal-concentration bioassays revealed no significant differences in virulence toward H. virescens among the protease-expressing recombinants and wild-type AcMNPV. However, in survival-time bioassays, AcMLF9.ScathL killed H. virescens approximately 30% faster than AcMLF9.LqhIT2, a virus expressing an insect-selective scorpion neurotoxin from the p6.9 promoter. Larvae infected with AcMLF9.ScathL consumed approximately 26-fold less lettuce than wild-type virus-infected larvae. These results highlight the potential of improving baculovirus efficacy through the expression of proteases.     

Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs

ScathL is a cathepsin L-like cysteine proteinase from Sarcophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs, through proteolysis of components of basement membranes. An expression system based on the methylotrophic yeast Pichia pastoris was used to produce recombinant ScathL. Although the expression construct contained the full proprotein coding sequence for ScathL, the proprotein was only detected in culture supernatant at early stages of expression by Western blotting. The purified recombinant protein contained only a polypeptide similar to mature ScathL, as a result of autocatalytic processing. After activation by reducing agents, the enzyme hydrolysed the cathepsin L substrate Z-Phe-Arg-AMC, with optimal activity at pH 5.5. ScathL showed decreasing activity with increasing ionic strength above 0.3M NaCl, and lost activity irreversibly at pH > or = 7.5. The enzyme showed limited activity towards protein substrates, digesting only to large fragments. ScathL was insecticidal towards larvae of the tomato moth, Lacanobia oleracera, following injection into the haemolymph. It caused melanisation, although no evidence of extensive proteolysis in haemolymph or gut was observed. Production of a inactive mutant form of ScathL showed that enzyme activity was necessary for the complete proprotein processing observed during production as a recombinant protein, and for insecticidal activity.     

Purification and characterization of an inhibitor of the cysteine protease from the hemolymph of Sarcophaga peregrina larvae

The hemolymph of Sarcophaga peregrina (flesh fly) larvae was found to contain multiple inhibitors of hemocyte cysteine protease. One of them, named sarcocystatin A, was purified and found to be a mixture of the components sarcocystatin A alpha and A beta in a molar ratio of 2:1. These components can exist in either the associated or dissociated form. The apparent heterogeneity of the protease inhibitors in the hemolymph was found to be partly due to association of sarcocystatin A alpha and A beta.     

Molecular cloning, sequencing, and characterization of cDNA for sarcotoxin IIA, an inducible antibacterial protein of Sarcophaga peregrina (flesh fly)

A cDNA clone for sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly) larvae [Ando, K., Okada, M., & Natori, S. (1987) Biochemistry 26, 226-230], was isolated and characterized. Sarcotoxin IIA was found to consist of 270 amino acid residues. Northern blot analysis showed that the sarcotoxin IIA gene was activated in response to injury of the body wall of the larvae. The gene was activated for much longer after injection of Escherichia coli into the abdominal cavity of larvae than after injection of saline alone. A common nucleotide sequence for mammalian inflammatory mediator protein cDNAs, TTATTTAT, was found in the 3'-untranslated region of sarcotoxin IIA cDNA, suggesting that this protein plays a role in the inflammatory response of this insect.     

Purification and characterization of a hemocyte proteinase of Sarcophaga, possibly participating in elimination of foreign substances.

We have reported that foreign protein injected into the abdominal cavity of Sarcophaga peregrina (flesh fly) larvae is degraded in the hemolymph by a proteinase secreted by hemocytes [Suzuki, T. and Natori, S. (1985) Comp. Biochem. Physiol. 81A, 191-193]. Here we report the purification and characterization of a proteinase from larval hemocytes. This enzyme is a cysteine proteinase consisting of 26-kDa and 29-kDa subunits with similar substrate specificity to mammalian cathepsin B. This enzyme was shown to be released from hemocytes into the hemolymph of larvae following injection of sheep red blood cells into the larvae, suggesting that it participates, at least in part, in elimination of foreign substances introduced into the body cavity.     

Involvement of EDTP, an egg-derived tyrosine phosphatase, in the early development of Drosophila melanogaster

Previously, we purified a novel protein tyrosine phosphatase from eggs of the flesh fly, Sarcophaga peregrina. This protein tyrosine phosphatase, named egg-derived tyrosine phosphatase (EDTP), is expressed during oogenesis and early embryogenesis but is rapidly degraded in middle embryogenesis by lysosomal cathepsin L. Here, we demonstrate the requirement of EDTP in the development of the fruit fly, Drosophila melanogaster. Deletion of the Drosophila EDTP gene using transposase-catalyzed imprecise excision resulted in homozygous lethals during embryogenesis. Additionally, germline clones generated using the FLP-FRT-ovo(D) system showed severe defects in ovarian development during oogenesis. These results indicate that the Drosophila EDTP gene is crucial in oogenesis and embryogenesis.     

A novel egg-derived tyrosine phosphatase, EDTP, that participates in the embryogenesis of Sarcophaga peregrina (flesh fly).

We have previously reported that cathepsin L mRNA is present in unfertilized eggs of Sarcophaga peregrina (flesh fly) as a maternal mRNA, which suggests that cathepsin L is required for embryogenesis. Now we have identified an egg protein, with a molecular mass of 100 kDa, that is extremely susceptible to cathepsin L digestion and which disappears rapidly as the embryos develop. We purified this protein to homogeneity, cloned its cDNA, and found that it contained a consensus sequence for the active site of tyrosine phosphatase. In fact this protein showed tyrosine phosphatase activity, indicating that it is a novel tyrosine phosphatase. The expression and subsequent disappearance of this protein, which we have named egg-derived tyrosine phosphatase (EDTP), may be indispensable for embryogenesis of Sarcophaga.     

Two subunits of the insect 26/29-kDa proteinase are probably derived from a common precursor protein

We previously identified the 26/29-kDa proteinase in the hemocytes of Sarcophaga peregrina (flesh fly) that appears to participate in elimination of foreign proteins in this insect [Eur. J. Biochem. 209, 939-944 (1992)]. Here, we report the cDNA cloning of this proteinase. The cDNA encodes a protein which includes both the 26- and 29-kDa subunit, strongly suggesting that the both subunits are derived from a single precursor protein. The 26- and 29-kDa subunit located at the amino-terminal and carboxyl-terminal of the precursor protein. The 29-kDa subunit itself appeared to be a proteinase, for this subunit had 52% sequence identity with Sarcophaga cathepsin L, while 26-kDa subunit had no significant similarity. We also showed that 26/29-kDa proteinase was insensitive to specific inhibitors of cathepsin L. These results indicate that this proteinase is a novel member of the papain family. We isolated similar cDNAs from Drosophila melanogaster and Periplaneta americana (cockroach), suggesting that this proteinase is conserved in a wide variety of insects and participates in their defense mechanisms.     

Identification and characterization of an antibacterial peptide of the 26-kDa protease of Sarcophaga peregrina with antibacterial activity

Previously, we purified a serine protease with a molecular mass of 26 kDa that exhibits potent antibacterial activity from a pupal extract of Sarcophaga peregrina (flesh fly). We divided this protease into 12 peptides and examined their antibacterial activity. A peptide corresponding to residues 155 to 174 (peptide 9) was found to exhibit antibacterial activity comparable to that of the 26-kDa protease. When Escherichia coli was treated with peptide 9, the permeability of both the outer and inner membranes increased, and substrates for beta-lactamase and beta-galactosidase entered the cells, but beta-galactosidase did not leak out of the cells under these conditions. It was suggested that residues 6 to 18 of peptide 9 form an amphiphilic alpha-helix under hydrophobic conditions with an N-terminal basic loop and then interact with acidic phospholipids in the bacterial membranes.     

Purification of a 29-kDa hemocyte proteinase of Sarcophaga peregrina.

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc-Leu-Leu-Val-Tyr-MCA and Z-Phe-Arg-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino-terminal sequence of this proteinase and that of cathepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes.     

Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae

The primary structure of sarcotoxin I, a potent bactericidal protein induced in the hemolymph of larvae of Sarcophaga peregrina (flesh fly), was investigated. Sarcotoxin I was a mixture of three proteins (sarcotoxins IA, IB, and IC) with almost identical primary structures. These proteins were found to consist of 39 amino acid residues and to differ in only 2-3 amino acid residues. The amino-terminal half of the molecules was rich in charged amino acids and was hydrophilic, whereas the carboxyl-terminal half was hydrophobic. It is suggested that the carboxyl-terminal half of sarcotoxin I penetrates into the bacterial membrane and that its amino-terminal half rich in basic amino acid residues interacts with acidic phospholipids in the bacterial membrane, resulting in perturbation of the membrane and loss of viability of the bacteria.     

Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina

Three antibacterial proteins were purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Sequencing studies showed that two of these proteins belong to the sarcotoxin I family, potent antibacterial proteins purified from the hemolymph of Sarcophaga larvae, whereas the other protein, named sapecin, is a new protein consisting of 40 amino acid residues including 6 cysteine residues. Unlike sarcotoxin I, sapecin preferentially represses the growth of various Gram-positive bacteria. The proteins of the sarcotoxin I family produced by this cell line were found to have carboxyl-terminal glycine, whereas sarcotoxin I in the hemolymph has amidated amino acids. This suggests that the embryonic cells lack an enzyme that cleaves off carboxyl-terminal glycine to form a new amidated carboxyl terminus.     

Degradation of a radiolabeled juvenile hormone analog using two insect species

A synthetic insect juvenile hormone analog (a juvenoid), ethylN-[2-[4-[[2,2-(ethylenedioxy)cyclohexyl]methyl]phenox]ethyl]carbamate, which has displayed high biological activity against different insect species and high stability under field conditions, was selected as a biologically active model compound for a study of a juvenile hormone analog degradation. The biologically active compound itself and its three diversely radiolabeled derivatives were applied to the flesh fly (Sarcophaga bullata) or the tsetse fly (Glossina palpalis), respectively. Monitoring of a fate of the applied juvenile hormone analog was carried out using a detection method of the radioactivity microdistribution within the whole insect body in combination with a radio high performance liquid chromatography (radio-HPLC), both of whole-body extracts made in different, but in advance scheduled, time intervals, and of extracts of insect excreta accumulated over an eight-day experiment.     

Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga peregrina.

Sarcotoxin II is a group of antibacterial proteins of Sarcophaga peregrina (flesh fly) with related primary structures. We have cloned three genes in this family. These genes formed a tandem array with about 2-kb intervals, and one of them was present in the opposite strand. The putative amino acid sequences of the proteins encoded by these genes were very similar except for a deletion in one of them. All of the genes were found to be activated transiently in the same way when the larval body wall was injured, suggesting that the encoded proteins are acute-phase-responsive proteins for protecting the insect from bacterial infection.     

Vitamin requirements of the cultured flesh fly cells, Sarcophaga peregrina (Diptera, Sarcophagidae)

Essential vitamins for the growth of a cell line derived from the flesh fly, Sarcophaga peregrina, were determined. By examining the survivability of continuous passages of the cells in the chemically defined medium lacking one vitamin, thiamine, riboflavin, pantothenate and either niacin or niacinamide were found to be essential for the continuous growth of the flesh fly cells in vitro. [Originally published in Volume 37, Archives of Insect Biochemistry and Physiology, 37:283-286 (1998).] Copyright 1998 Wiley-Liss, Inc.     

Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect

Sarcotoxin IA is a bactericidal peptide of 39 amino acids found in the common flesh fly, Sarcophaga peregrina. Many agronomically important bacteria in Japan are killed by this peptide at sub-micro molar levels, and the growth of tobacco and rice suspension cultured cells is not inhibited with less than 25 microM. Transgenic tobacco plants which overexpress the peptide, i.e. over 250 pmol per gram of fresh leaf, under the control of a high expression constitutive promoter showed enhanced resistance to the pathogens for wild fire disease (Pseudomonas syringae pv. tabaci) and bacterial soft rot disease (Erwinia carotovora subsp. carotovora).