The structure and antimicrobial potential of wasp and hornet (Vespidae) mastoparans: A review

Wasp venom is a complex mixture of biologically active components, including high molecular weight proteins, small peptides, bioactive amines, and amino acids. Peptides comprise up to 70% of dried venom. In social wasp venoms, three of the major peptide types are mastoparans, which cause mast cell degranulation, chemotactic peptides, which promote chemotaxis of polymorphonucleated leukocytes, and kinin‐related peptides, which are known to produce pain and increase vascular permeability. Among these, the bioactive tridecapeptide mastoparan is the most common and may even have antimicrobial activity. Herein we summarize the results of studies on vespid mastoparans, focusing on hornets (Vespa spp.) identified following a systematic literature search for mastoparans of hornets in the genus Vespa, the most active mastoparan research taxon. The common features of hornet mastoparans are C‐terminal amidation, amphipathic helical structure, and multiple functions such as mast cell degranulation and hemolysis, as well as membrane permeabilization. Most interestingly, all tested hornet mastoparans have strong antimicrobial activities, suggesting that they can provide useful insights into and opportunities for development of novel antibacterial peptides.     

Recombinant allergens with reduced allergenicity but retaining immunogenicity of the natural allergens: hybrids of yellow jacket and paper wasp venom allergen antigen 5s

The homologous venom allergen Ag 5s from the yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis) have 59% sequence identity of their respective 204 and 205 amino acid residues, and they have low degrees of antigenic cross-reactivity in insect allergic patients and in animal models. Hybrids containing different segments of these two vespid Ag 5s were expressed in yeast. Circular dichroism spectroscopy suggests the hybrids to have the secondary structure of natural Ag 5. Inhibition ELISA with human and murine Abs suggests the hybrids to have the discontinuous B cell epitopes of the natural Ag 5 but with an altered epitope density. The hybrids were immunogenic in mice for B and T cell responses to both Ag 5s. The N-terminal region of Ag 5 was found to contain its dominant B cell epitope(s). Hybrids containing 10-49 residues of yellow jacket Ag 5 showed 100- to 3000-fold reduction in allergenicity when tested by histamine release assay with basophils of yellow jacket-sensitive patients. Our findings suggest that hybrids represent a useful approach to map the discontinuous B cell epitope-containing regions of proteins. They also suggest that Ag 5 hybrids may be useful immunotherapeutic reagents in man.     

The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.     

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus

Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.     

Major venom allergen of yellow jackets, Ves v 5: structural characterization of a pathogenesis-related protein superfamily.

Ves v 5 is one of three major allergens found in yellow-jacket venom: phospholipase A(1) (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis-related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 A. The majority of residues conserved between the pathogenesis-related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an alpha-beta-alpha sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to the related antigen 5's. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross-reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy.     

Hemolytic potency and phospholipase activity of some bee and wasp venoms

1. The action of crude venoms of four aculeate species: Apis mellifera, Vespa crabro, Vespula germanica and Vespula vulgaris on human erythrocytes was investigated in order to determine the lytic and phospholipase activity of different aculeate venoms and their ability to induce red blood cell hemolysis. 2. Bee venom was the only extract to completely lyse red blood cells at the concentration of 2-3 micrograms/ml. 3. Phospholipase activity in all of the examined vespid venoms was similar and the highest value was recorded in V. germanica. 4. Vespid venoms exhibited phospholipase B activity, which is lacking in honeybee venom. 5. In all membrane phospholipids but lecithin, lysophospholipase activity of vespid venoms was 2-6 times lower than the relevant phospholipase activity. 6. The incubation of red blood cells with purified bee venom phospholipase A2 was not accompanied by lysis and, when supplemented with purified melittin, the increase of red blood cell lysis was approximately 30%.     

Cardioactive effects of hornet venom, Vespa mandarinia

In the experiment of electrocardiogram, the crude venom of giant hornet (Vespa mandarinia) showed cardioactive effects on rat heart. The heart rate was accelerated within 5 min after injection of the venom intraperitoneally, then the heart beat was blocked, resulting in conduction delay. The cardioactive constituent was separated into two components by gel filtration. One which was high molecular species such as protein showed complete atrioventricular block. Another component, having a low molecular weight, was fractionated in 5 peaks, which accelerated heart rate.     

Intraocular pressure in cats is lowered by drops of hornet venom

1. Nine cats were given an intravenous injection of the Oriental hornet (Vespa orientalis, Vespinae; Hymenoptera) venom sac extract (VSE) and seven cats had the same VSE administered as eye drops. 2. When injected intravenously, the hornet VSE decreased the intraocular pressure in both eyes sharply during the first 20 min and with a slower rate later on until the end of the 3 hr experiment. The intraocular pressure dropped to zero in some cases. 3. VSE eye drops decreased the intraocular pressure only in the treated eye, while in the second eye (left as a control) the intraocular pressure remained the same throughout the experiment. 4. The decrease in the intraocular pressure was sharp during the first 20 min and slowed down afterwards until the end of the experiment. 5. The intraocular pressure did not reduce to zero. 6. This study shows that the active components of the hornet venom which caused a decrease in the intraocular pressure can cross the cornea and exert a hypotensive effect in the eye.     

A fatal effect of hornet venom on rat-brain cortical neurons

In a previous study, it was shown that the hornet venom or, more specifically, its venom sac extract (VSE) possesses deoxyribonuclease activity that exerts an effect both on insects as well as on mammals. We have now examined the effect of hornet VSE on primary culture of rat cortical neurons. Judging on the basis of our results, VSE induces a rapid cell death by a) permeabilizing the cell membrane, b) inducing DNA breaks, and c) cleaving the nuclear protein poly-ADP-ribose polymerase (PARP-1), thereby preventing DNA repair.     

European honeybee defense against Japanese yellow hornet using heat generation by bee‐balling behavior

The Japanese honeybee, Apis cerana japonica Radoszkowski, uses unique generation of heat by bee‐balling to defend against, overheat and kill predacious Japanese hornets. We have now observed the European honeybee, Apis mellifera Linnaeus, using similar bee‐balling behavior and heat generation against the Japanese yellow hornet, Vespa simillima xanthoptera Cameron. We monitored temperatures in the center of the bee‐ball and inside thoracic muscles of the captured hornet and found that the thoracic internal temperature (45.8 ± 2.32°C) was higher than that of the bee‐ball (44.0 ± 0.96°C). Although the thoracic temperature of captured hornets rose to the upper lethal level, defending European bees also showed some stinging attempts against the hornet, unlike the sympatric Japanese honeybee, which never stings during bee‐balling. The European honeybee bee‐balling behavior consists of three phases: (i) heating; (ii) heat‐retaining; and (iii) break up. Our results suggest that European honeybees kill hornets by raising the body temperature of hornets rapidly without stinging. The tactics of bee‐balling against hornets are complex and may be performed by extended division of labor.     

Age-related Variation in Snake Venom： Evidence from Two Snakes （Naja atra and Deinagkistrodon acutus） in Southeastern China

In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.     

Variation in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution

Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA.     

Venom from spiders of the genus Hippasa: biochemical and pharmacological studies

The venoms from female spiders of the genus Hippasa namely H. partita, H. agelenoides and H. lycosina are compared for biochemical and pharmacological properties. SDS-PAGE pattern revealed varied protein composition. Marked variability is seen with casein hydrolyzing enzymes in SDS-PAGE zymogram. H. partita venom was the only venom that hydrolyzed gelatin while the other two venoms did not. The venoms shared similar hyaluronidase activity, showing a single activity band in SDS-PAGE zymogram. The PLA2 activity varied as H. partita>H. agelenoides>H. lycosina venoms. Marked differences were noted in the ability to induce edema, cytotoxicity, myotoxicity and neurotoxicity, while hemorrhage was associated exclusively with H. partita venom.     

Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina

The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.     

A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms

Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.     

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.     

Vitellogenins Are New High Molecular Weight Components and Allergens (Api m 12 and Ves v 6) of Apis mellifera and Vespula vulgaris Venom

Background/Objectives

Anaphylaxis due to hymenoptera stings is one of the most severe clinical outcomes of IgE-mediated hypersensitivity reactions. Although allergic reactions to hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, venom immunotherapy is still hampered by severe systemic side effects and incomplete protection. The identification and detailed characterization of all allergens of hymenoptera venoms might result in an improvement in this field and promote the detailed understanding of the allergological mechanism. Our aim was the identification and detailed immunochemical and allergological characterization of the low abundant IgE-reactive 200 kDa proteins of Apis mellifera and Vespula vulgaris venom.

Methods/Principal Findings

Tandem mass spectrometry-based sequencing of a 200 kDa venom protein yielded peptides that could be assigned to honeybee vitellogenin. The coding regions of the honeybee protein as well as of the homologue from yellow jacket venom were cloned from venom gland cDNA. The newly identified 200 kDa proteins share a sequence identity on protein level of 40% and belong to the family of vitellogenins, present in all oviparous animals, and are the first vitellogenins identified as components of venom. Both vitellogenins could be recombinantly produced as soluble proteins in insect cells and assessed for their specific IgE reactivity. The particular vitellogenins were recognized by approximately 40% of sera of venom-allergic patients even in the absence of cross-reactive carbohydrate determinants.

Conclusion

With the vitellogenins of Apis mellifera and Vespula vulgaris venom a new homologous pair of venom allergens was identified and becomes available for future applications. Due to their allergenic properties the honeybee and the yellow jacket venom vitellogenin were designated as allergens Api m 12 and Ves v 6, respectively.     

Hyaluronidases--a group of neglected enzymes.

Hyaluronan is an important constituent of the extracellular matrix. This polysaccharide can be hydrolyzed by various hyaluronidases that are widely distributed in nature. The structure of some bacterial and animal enzymes of this type has recently been elucidated. It could be shown that the hyaluronidases from bee and hornet venom and the PH-20 hyaluronidase present on mammalian spermatozoa are homologous proteins.     

Characterization of hyaluronidase isolated from Agkistrodon contortrix contortrix (Southern Copperhead) venom

Snake venoms are a rich source of enzymes including many hydrolytic enzymes. Some enzymes such as phospholipase A2, proteolytic enzymes, and phosphodiesterases are well characterized. However many enzymes, such as the glycosidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine which venom was the best source for isolation of the enzyme, the hyaluronidase activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highest activity, this venom was used for purification of hyaluronidase. Molecular weight was determined by matrix-assisted laser desorption ionization mass spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 61,000 Da. Substrate specificity studies indicated that the snake venom enzyme was specific only for hyaluronan and did not hydrolyze similar polysaccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin. The enzyme is an endo-glycosidase without exo-glycosidase activity, as it did not hydrolyze p-nitrophenyl-beta-D-glucuronide or p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. The cleavage point is at the beta1,4-glycosidic linkage and not at the beta1,3-glycosidic linkage. Thus, snake venom hyaluronidase is an endo-beta-N-acetylhexosaminidase specific for hyaluronan.     

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C.