Antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor.

Bee venom is a rich source of pharmacologically active substances. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent; however, this inhibitory ability was two-fold weaker than that of aprotinin. The fibrin(ogen)olytic activities of B. ignitus venom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism by which bumblebee venom affects the hemostatic system through the antifibrinolytic activity of Bi-KTI and through Bi-VSP-mediated fibrin(ogen)olytic activities, raising interest in Bi-KTI and Bi-VSP as potential clinical agents.

Fibrin(ogen)olytic activity of bumblebee venom serine protease.

Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis.

Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Molecular cloning and antimicrobial activity of bombolitin, a component of bumblebee Bombus ignitus venom.

Bombolitin is the most abundant component of bumblebee venom and shares structural and biological properties with melittin, a component of honeybee venom. Here, we describe the molecular cloning and antimicrobial activity of bombolitin isolated from the venom of the bumblebee Bombus ignitus. The B. ignitus bombolitin gene consists of 2 exons encoding 56-amino acid residues. The bombolitin isolated from B. ignitus venom is a 1.99 kDa mature peptide with 18-amino acid residues, and it is created by the cleavage of the probombolitin domain between Ala38 and Leu39. B. ignitus bombolitin exhibits venom gland-specific expression. We also investigated the antimicrobial properties of B. ignitus bombolitin against bacteria and fungi. The B. ignitus bombolitin showed high antibacterial activity against two Gram-positive and two Gram-negative bacteria. In addition, the B. ignitus bombolitin displayed antifungal activity against the plant pathogenic fungi Fulvia fulva and Alternaria radicina.

Molecular characterization of a phospholipid-hydroperoxide glutathione peroxidase from the bumblebee Bombus ignitus.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC 1.11.1.12) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln(82) and Trp(134). Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H(2)O(2) using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H(2)O(2) exposure, external temperature shock, and starvation. Under H(2)O(2) overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H(2)O(2).

Molecular cloning and characterization of 1-Cys and 2-Cys peroxiredoxins from the bumblebee Bombus ignitus.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated the regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that the loss of expression of BiPrx1 or BiTPx1 is compensated by the up-regulation of expression of the other peroxidase in response to H2O2 overload.

Expression profile of cathepsin B in the fat body of Bombyx mori during metamorphosis.

Proteolytic enzymes are involved in insect molting and metamorphosis, and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compare the expression profiles of B. mori cathepsins B (BmCatB) and D (BmCatD) during normal development and after RNA interference (RNAi)-mediated inhibition. BmCatB is induced by 20-OH-ecdysone, and is expressed in the fat body of B. mori during molting and the larval-pupal and pupal-adult transformations, where its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation, and BmCatB RNAi treatment resulted in an arrest of the larval-pupal transformation. RNAi-mediated BmCatB knockdown sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, when BmCatD was inhibited via RNAi, the expression of BmCatB was upregulated. Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis, and that BmCatB and BmCatD contribute to B. mori metamorphosis.

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus.

Phospholipase A(2) (PLA(2)) is one of the main components of bee venom. Here, we identify a venom PLA(2) from the bumblebee, Bombus ignitus. Bumblebee venom PLA(2) (Bi-PLA(2)) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA(2) gene. Bi-PLA(2) is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA(2) (136 amino acids) possesses features consistent with other bee PLA(2)s, including ten conserved cysteine residues, as well as a highly conserved Ca(2+)-binding site and active site. Phylogenetic analysis of bee PLA(2)s separated the bumblebee and honeybee PLA(2) proteins into two groups. The mature Bi-PLA(2) purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA(2). Immunofluorescence staining of Bi-PLA(2)-treated insect Sf9 cells revealed that Bi-PLA(2) binds at the cell membrane and induces apoptotic cell death.

Expression profile of the iron-binding proteins transferrin and ferritin heavy chain subunit in the bumblebee Bombus ignitus.

The iron-binding proteins, transferrin and ferritin, are involved in the processes of transport and storage in iron metabolism. Their expression is induced in response to iron overload. Here, we show the expression profile of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH) of the bumblebee Bombus ignitus in response to iron overload. Bi-Tf exhibits fat body-specific expression, whereas Bi-FerHCH is ubiquitously expressed and upregulated in various tissues, though in a similar manner, by iron overload. We also demonstrate their expression regulation via reduction of Bi-Tf or Bi-FerHCH levels in the fat body via RNA interference (RNAi). Under uniform conditions in which FeCl(3) was overloaded, the RNAi-induced Bi-Tf knock-down B. ignitus worker bees showed upregulated expression of Bi-FerHCH, and reciprocally, Bi-FerHCH RNAi knockdowns showed upregulated Bi-Tf expression in the fat body. This result indicates that, in case of the loss of Bi-Tf or Bi-FerHCH, the expression of Bi-FerHCH or Bi-Tf, respectively, is upregulated in response to iron overload.

Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus.

Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5'UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.

Molecular cloning and characterization of a glycosyl hydrolase family 9 cellulase distributed throughout the digestive tract of the cricket Teleogryllus emma.

A novel endogenous beta-1,4-endoglucanase (EG) gene belonging to the glycosyl hydrolase family 9 (GHF 9) that is distributed throughout the digestive tract of the cricket Teleogryllus emma was cloned and characterized. This gene, named TeEG-I, consists of eight exons encoding 453 amino acid residues and exists as a single copy in the T. emma genome. TeEG-I possesses all the features, including signature motifs and catalytic domains, of GHF 9 members, sharing high levels of identity with the termite, Mastotermes darwiniensis (64% protein sequence identity), and the cockroach, Panesthia cribrata (62%), GHF 9 cellulases. Recombinant TeEG-I, which is expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells, showed an optimal pH and temperature of pH 5.0 and 40 degrees C. The K(m) and V(max) values for digestion of carboxymethyl cellulose were 5.4 mg/ml and 3118.4 U/mg, respectively. Northern and Western blot analyses revealed that TeEG-I is present throughout the digestive tract, which correlated with the TeEG-I distribution and cellulase activity in the digestive tract as assayed by immunofluorescence staining and enzyme activity assay, respectively. These results indicate that TeEG-I is distributed throughout the entire digestive tract of T. emma, suggesting a functional role of endogenous TeEG-I in a sequential cellulose digestion process throughout the T. emma digestion tract.

Pr-lynx1, a modulator of nicotinic acetylcholine receptors in the insect.

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the gene itself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface as a GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by alpha3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing alpha3beta4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

Cloning and expression profiling of four antibacterial peptide genes from the bumblebee Bombus ignitus.

Four antibacterial peptide genes (apidaecin, hymenoptaecin, abaecin, and defensin) were cloned from the bumblebee Bombus ignitus, and cDNAs and their genomic structures were sequenced and characterized. Comparative analysis revealed that the four antibacterial peptides of B. ignitus had similar characteristics to other bee antibacterial peptides identified to date. The transcriptional expression profiles of the four antibacterial peptide genes in the fat body of B. ignitus workers revealed that all four antibacterial peptide genes were acutely induced in a similar manner by PBS injection or LPS stimulation, indicating that antibacterial peptides from various classes are simultaneously expressed in a single insect upon infection or injury.

Insect transferrin functions as an antioxidant protein in a beetle larva.

In insects transferrin is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone regulated protein. Here, a novel functional role for insect transferrin as an antioxidant protein is demonstrated. Stressors, such as heat shock, fungal challenge, and H(2)O(2) exposure, cause upregulation of the white-spotted flower chafer Protaetia brevitarsis (Coleoptera: Scarabaeidae) transferrin (PbTf) mRNA in the fat body and increases PbTf protein levels in the hemolymph. RNA interference (RNAi) treated PbTf reduction causes increased iron and H(2)O(2) levels in the hemolymph and results in induction of apoptotic cell death in the fat body during exposure to stress. The observed effects of PbTf RNAi suggest that PbTf inhibits stress-induced apoptosis by diminishing the Fenton reaction via the binding of iron, thus supporting an antioxidant role for PbTf in stress responses.

Molecular cloning and characterization of a transferrin cDNA from the white-spotted flower chafer, Protaetia brevitarsis.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.

Molecular cloning and expression of the C-terminus of spider flagelliform silk protein from Araneus ventricosus.

A cDNA coding for the C-terminus of spider flagelliform silk protein (AvFlag) was cloned from Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvFlag consists of 167 amino acids of a repetitive region and 87 amino acids of a C-terminal non-repetitive region. The peptide motifs found in spider flagelliform silk proteins, GPGGX and GGX,were conserved in the repetitive region of AvFlag. Phylogenetic analysis further confirmed that AvFlag belongs to the spider flagelliform silk proteins. The AvFlag cDNA was expressed as a 28 kDa polypeptide in baculovirus-infected insect cells. As a new expression approach for spider silk protein,the combination of polyhedrin and AvFlag creates a polyhedrin AvFlag fusion protein (61 kDa) that is produced as recombinant polyhedra; this provides a basis for the source of spider silk proteins for various applications.

Thioredoxin from the silkworm, Bombyx mori: cDNA sequence, expression, and functional characterization.

A thioredoxin (Trx) gene was cloned from the silkworm, Bombyx mori. The B. mori Trx (BmTrx) cDNA contains an open reading frame of 318 bp encoding 106 amino acid residues with a conserved active site (CGPC). Northern blot analysis revealed the presence of BmTrx transcripts in all tissues examined. The cDNA encoding BmTrx was expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells. The recombinant BmTrx proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from B. mori. When H2O2 or paraquat was injected into the body cavity of B. mori larvae, BmTrx mRNA expression was upregulated in the fat body tissue. In addition, the expression levels of BmTrx mRNA in the fat body were greatly increased when B. mori larvae were exposed to low or high temperatures, or injected with microorganisms. These results suggest that BmTrx possibly protects against oxidative stress caused by extreme temperatures and microbial infection as well as by intracellularly generated reactive oxygen species during metabolism.

A new technique for producing recombinant baculovirus directly in silkworm larvae.

A new technique for the direct production of recombinant baculovirus in the silkworm larvae is described. To assess the utility of this method, a combination of Bombyx mori nucleopolyhedroviral genome, transfer vector and Lipofectin was co-injected directly into newly ecdysed fifth instar, silkworm larvae. The recombinant virus was obtained from the hemolymph of injected larvae and the hemolymph then re-injected into the larvae as an inoculum. This resulted in a high-level production of foreign protein in the silkworm larvae. This technique produces easy and rapid recombinant protein production in silkworms.

Functional role of aspartic proteinase cathepsin D in insect metamorphosis.

BACKGROUND: Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. RESULTS: Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. CONCLUSION: Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.

Transferrin inhibits stress-induced apoptosis in a beetle.

Transferrin in insects is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone-regulated protein. We show here a novel functional role for insect transferrin. Stresses, such as iron overload, bacterial or fungal challenge, cold or heat shock, wounding, and H2O2 or paraquat exposure, cause upregulation of the beetle Apriona germari transferrin (AgTf) gene in the fat body and epidermis, and they cause increased AgTf protein levels. RNA interference (RNAi)-mediated AgTf reduction results in rapid induction of apoptotic cell death in the fat body during exposure to heat stress. The observed effect of AgTf RNAi indicates that AgTf inhibits heat stress-induced apoptotic cell death, suggesting a functional role for AgTf in defense and stress responses in the beetle.