Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni

Two novel biologically active peptides, Eumenine mastoparan-OD and Orancis-Protonectin, were isolated from a solitary wasp, Orancistrocerus drewseni drewseni (Eumeninae, Vespidae). MALDI-TOF MS analysis of a small amount of the crude venom gave two intensive molecular-related ion peaks at m/z 1269.9 and 1552.9 that were expected to be novel based on a peptide database search. Purification of the crude venom by HPLC gave two peptide fractions, P-1 and P-2. The amino acid sequence of P-1 (GRILSFIKAGLAEHL-NH₂) and P-2 (ILGIITSLLKSL-NH₂) were determined by ESI-MS/MS, automated Edman degradation, and amino acid analysis. According to the high sequence homology with those of mastoparan and protonectin, P-1 and P-2 were labeled Eumenine mastoparan-OD and Orancis-Protonectin, respectively. Orancis-Protonectin is the first example of a protonectin analog isolated from the venom of a solitary wasp. The hemolytic activities of these new peptides were more potent than that of mastoparan.     

Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two‐dimensional gel electrophoresis and separation by ion‐exchange and reverse‐phase high‐performance liquid chromatography followed by mass spectrometry using tanden matrix‐assisted laser desorption/ionization with time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry and electrospray ionization‐quadrupole with time‐of‐flight (ESI‐Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10‐ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.     

Cloning and expression of the gene for an insect haemocyte anti‐aggregation protein (VPr3), from the venom of the endoparasitic wasp,Pimpla hypochondriaca

A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti‐aggregation activity in vitro and shares the same N‐terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full‐length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N‐terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1 mM) and growth of the bacteria at 37°C for 5 h, or at 24°C for 20 h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc.     

Display of wasp venom allergens on the cell surface of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast Saccharomyces cerevisiae preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp Vespula vulgaris venom: phospholipase A1, hyaluronidase and antigen 5 as the model.     

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host,Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom‐microinjected P. rapae pupae. Non‐parasitized and PBS‐microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom‐microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non‐parasitized and PBS‐microinjected pupae were significantly higher than that of parasitized and venom‐microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom‐microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non‐parasitized and PBS‐microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its host's endocrine system. © 2009 Wiley Periodicals, Inc.     

Isolation, cloning and characterization of Polistes dominulus venom phospholipase A1 and its isoforms

Polistes dominulus is known to be an allergenically important social wasp. Its venom has four major allergens (ziv. phospholipase A1, hyaluronidase, antigen 5 and a serine-protease). Amino acid sequences of its serine-protease and Antigen 5 have been published. In this paper, the partial amino acid sequence of its venom phospholipase native protein is reported. Also, we give an account to the complete nucleotide sequence of Polistes dominulus venom phospholipase A1 gene, its isoforms and their complete deduced amino acid sequences. Their similarity to the other phospholipases A1 of the family: Vespidae is discussed.     

Proteomic characterization of the hyaluronidase (E.C. 3.2.1.35) from the venom of the social wasp Polybia paulista

Polybia paulista wasp venom possesses three major allergens: phospholipase A1, hyaluronidase and antigen-5. To the best of our knowledge, no hyaluronidase from the venom of Neotropical social wasps was structurally characterized up to this moment, mainly due to its reduced amount in the venom of the tropical wasp species (about 0.5% of crude venom). Four different glycoproteic forms of this enzyme were detected in the venom of the wasp Polybia paulista. In the present investigation, an innovative experimental approach was developed combining 2-D SDS-PAGE with in-gel protein digestion by different proteolytic enzymes, followed by mass spectrometry analysis under collision-induced dissociation CID) conditions for the complete assignment of the protein sequencing. Thus, the most abundant form of this enzyme in P. paulista venom, the hyaluronidase-III, was sequenced, revealing that the first 47 amino acid residues from the N-terminal region, common to other Hymenoptera venom hyaluronidases, are missing. The molecular modeling revealed that hyaluronidase-III has a single polypeptide chain, folded into a tertiary structure, presenting a central (β/α)5 core with alternation of β-strands and α-helices; the tertiary structure stabilized by a single disulfide bridge between the residues Cys189 and Cys201. The structural pattern reported for P. paulista venom hyaluronidase-III is compatible with the classification of the enzyme as member of the family 56 of glycosidase hydrolases. Moreover, its structural characterization will encourage the use of this protein as a model for future development of "component-resolved diagnosis".     

Venom induces alarm behaviour in the social wasp Polybioides raphigastra (Hymenoptera: Vespidae): an investigation of alarm behaviour, venom volatiles and sting autotomy

Chemicals from the venom gland elicited alarm behaviour and attack in the Asian polistine wasp Polybioides raphigastra. When presented with crushed venom glands workers of this wasp respond with a mass stinging attack. Gas chromatography–mass spectrometry analyses show that the major volatiles in the venom gland are alkanes, monounsaturated alkenes and 2-alcohols. Several pyrazines, a spiroacetal and aromatics were also identified as trace compounds. The anatomy and morphology of the sting apparatus are reported, and we describe sting autotomy in this wasp. This is the first such report for the Ropalidiinae. The structures responsible for autotomy are likely to be large barbs present on the sting lancets, and a conspicuous tooth present on the medial side of the left lancet. Sting autotomy in P. raphigastra probably plays an important role in the localization of sites of attack by wasps defending the nest.     

Allergens of honey bee venom.

The allergenic activities of four purified components of honeybee venom were studied by using histamine release from leukocytes of bee sting-allergic patients. The components studied were hyaluronidase, phospholipase A₂, melittin and apamin with molecular weights, respectively, of about 50,000, 15,800, 2840 and 2038 d. In six of the seven patients studied, hyaluronidase and phospholipase were, respectively, on the average about two and eight times more active by weight than the venom. The situation was reversed in one patient in that hyaluronidase and phospholipase A₂ were, respectively, 90 and 0.5 times more active than the venom. With this single exception, hyaluronidase and phospholipase were about equally active on a molar basis as allergens. Melittin was on the average about one-tenth as active as the venom, and apamin was inactive as an allergen.Chemical modifications of phospholipase A₂ were carried out. Succinylation of eight of its eleven amino groups yielded a derivative that retained 4% of the enzymic activity of the native enzyme. Reduction and carboxymethylation of its four disulfide bonds or cyanogen bromide cleavage of its three methionyl bonds yielded enzymatically inactive derivatives. These derivatives showed varying decreases of allergenic activities when compared to the native enzyme. The results indicate that the antigenic determinants of phospholipase depend on the charge, the amino acid sequence and the conformation of the molecule.     

VENOM COMPONENTS OF Asobara japonica IMPAIR CELLULAR IMMUNE RESPONSES OF HOST Drosophila melanogaster

The endoparasitoid wasp Asobara japonica has highly poisonous venom: the host Drosophila larvae are killed by envenomation at a dose that is naturally injected by the female wasp at parasitism. This insecticidal venom is neutralized, however, because A. japonica introduces lateral oviduct components soon after venom injection at oviposition. Although the venom and lateral oviduct components of this parasitoid have been partially characterized, how the venom components favor successful development of wasp eggs and larvae in the host remains ambiguous. Here, we demonstrated that A. japonica venom did not affect host humoral immune responses, determined as expression of antimicrobial peptide (AMP) genes, but significantly diminished two cellular responses, spreading and phagocytosis, by host hemocytes. Moreover, venom components drastically elevated a serine protease‐like activity 4 h after its injection. The lateral oviduct components did not negate the detrimental effects of the venom on host cellular immunities, but significantly reduced the venom‐induced elevation of protease activity. Both active factors in venom and lateral oviduct components were roughly characterized as heat‐labile substances with a molecular mass of at least 10 kDa. Finally, venom of A. japonica, with a wide host range, was found to be much more toxic than that of Asobara rossica, which has a limited host range. These results reveal that A. japonica venom toxicity allows exploitation of a broader range of host insects because it is essential to overcome cellular immune responses of the host for successful parasitism.     

Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede <Emphasis Type="Italic">Scolopendra subspinipes</Emphasis> mutilans

Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was suc-cessfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5′ UTR and a 171 nt 3′ UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.     

The first report of kininogen from invertebrates

The hornet possesses highly toxic venom, which is rich in toxin, enzymes, and biologically active peptides. Several bradykinin-like peptides, vespakinins, have been found in wasp venoms since 1970s, but the mode of biosynthesis of these peptides is unknown. In the present study, a vespakinin M was purified from venom of Vespa magnifica. Its primary sequence was established as GRPPGFSPFRID. The cDNA encoding the vespakinin M was cloned from the cDNA library of V. magnifica venom gland. The cDNA structure of vespakinin M was found to contain a coding region of 168 nucleotides. The encoded precursor of vespakinin M is composed of a signal peptide, an acidic peptide, and a mature peptide of vespakinin M. This is the first kininogen from insects; it is also the first kininogen from invertebrates. The cDNA structure encoding vespakinin M suggests that the generation mode of bradykinin-related peptides in wasp is different from amphibian skin and mammalian blood system.     

Systematic analysis of a wasp parasitism arsenal

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus‐carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV‐mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus‐carrying wasp introduces into its host.     

Molecular,Immunological, and Biological Characterization of Tityus serrulatus Venom Hyaluronidase: New Insights into Its Role in Envenomation

Background

Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized.

Methods and principal findings

We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD₅₀ (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues.

Conclusions

Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.