Protein allergens of white-faced hornet, yellow hornet, and yellow jacket venoms.

White-faced hornet, yellow hornet, and yellow jacket venoms have very similar protein compositions; each contains mainly three basic proteins. Two of these proteins have hyaluronidase and phospholipase activities and the third one, designated as antigen 5, is of as yet unidentified biochemical function. These three proteins have molecular weights of about 45 000, 35 000, and 25 000, respectively. The three proteins of white-faced hornet venom have been purified to near homogeneity, while this is the case only for antigen 5 of yellow hornet and yellow jacket venoms. Strong antigenic cross-reaction of the hyaluronidase from these three vespid venoms was observed using specific rabbit anti-venom sera, while weak cross-reactions of phospholipases and of antigen 5s were observed. All three proteins are active as allergens to varying degrees in vespid sensitive individuals. With each vespid venom its antigen 5 seems to be the major allergen. The results help to clarify the commonly observed varying degrees of multiple sensitivity of people to different vespids.     

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.     

Can venom volatiles be a taxonomic tool for Polistes wasps (Hymenoptera,Vespidae)?

In the present study, we investigated whether venom volatiles have a species-specific composition and could possibly be used to discriminate between related paper wasp species. We compared venom volatile chemical profiles, obtained through gas chromatography-mass spectrometry analyses, of three non-parasitic European Polistes species ( P. dominulus , P. gallicus and P. nimphus ). The results show that the venom volatile composition is indeed species specific and can thus be a useful systematic tool.     

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.     

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.     

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%.     

Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast.

Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast alpha signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis).     

Expressions of Recombinant Venom Allergen, Antigen 5 of Yellowjacket (Vespula vulgaris) and Paper Wasp (Polistes annularis), in Bacteria or Yeast

Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast α signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis).     

胡蜂抗原5的研究进展

胡蜂抗原 5 (Ag5 )是胡蜂毒液中的一种主要变应原 ,对胡蜂过敏病人有变态反应作用。它一般由约 2 0 4个氨基酸残基组成 ,分子量约为 2 3kDa。不同的抗原 5之间有抗原交叉反应活性 ,且活性大小不同。它的cDNA已被克隆 ,并以融合蛋白的形式在大肠杆菌中进行表达。但Ag5的生物功能至今还不清楚。由于Ag5有抗原交叉反应活性 ,因此可将其用于免疫治疗。此外 ,Ag5还可用于植物病虫害防治     

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.     

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.     

莽山烙铁头蛇毒液蛋白质组学研究(英文)

目的收集一条中国特有品种莽山烙铁头蛇的新鲜毒液,对其蛇毒蛋白作生化分析。方法先以电泳胶片法分析比较莽山烙铁头蛇毒与中国大陆及台湾、日本琉球各地其它品种的烙铁头蛇和各地其他品种的烙铁头蛇的粗蛇毒,在凝胶电泳中展现之异同。然后利用高效液相色谱法(HPLC)将莽山烙铁头蛇毒中的磷脂酶分离,再以质谱仪等鉴定,并与以往相同方法分析莽山烙铁头蛇所得到的磷脂酶分析结果比较。结果我们的研究证实了莽山烙铁头蛇毒含唯一主要的磷脂酶,其含量大约占蛇毒总重量的58%,与中国其他烙铁头蛇品种及日本琉球烙铁头蛇的蛇毒某些碱性磷脂酶之氨基酸序列及蛋白结构特别相似。此碱性磷脂酶主要毒性是水肿、局部炎症及肌肉坏死。结论莽山烙铁头蛇咬伤的临床表现为抗凝与出血,局部炎症坏死等特点,亦可由其蛇毒成分得到印证与解释。     

Evidence of alarm pheromones in the venom of Polistes dominulus workers (Hymenoptera: Vespidae)

Abstract.  The active and coordinating capacity of defending the nest is a key feature of social insects. The present study investigates the presence of alarm pheromones in the venom of workers of the social wasp, Polistes dominulus . Laboratory experiments were performed with caged colonies of P. dominulus using a wind tunnel apparatus to test the behavioural response of workers to venom released by other workers and to venom extracts. Contrary to that previously reported for European paper wasps, the present results show that the venom is the source of alarm pheromones. Field experiments combining a visual (black target) and a chemical stimulus (venom extract) were performed to test the effect of the venom on the reaction of colonies. Wasps leave the nest, land on the visual target and attack the target significantly more once exposed to venom extract plus target than to solvent plus target. This work shows that the venom of P. dominulus workers elicits an alarm response, reduces the threshold for attack and acts as an attractant on targets. These results using P. dominulus indicate that, in both American and European species, colony defence is based on the same features, suggesting that chemical alarm is a widespread trait in the genus Polistes .     

A comparative study of the biological properties of some sea snake venoms.

1. The protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, phospholipase A, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, procoagulant, anticoagulant and hemorrhagic activities of ten samples of venoms from seven taxa of sea snakes were examined. 2. The results show that venoms of sea snakes of both subfamilies of Hydrophiinae and Laticaudinae are characterized by a very low level of enzymatic activities, except phospholipase A activity and, for some species, hyaluronidase activity. 3. Because of the low levels of enzymatic activities and the total lack of procoagulant and hemorrhagic activities, venom biological properties are not useful for the differentiation of species of sea snakes. Nevertheless, the unusually low levels of enzymatic activities of sea snake venoms may be used to distinguish sea snake venoms from other elapid or viperid venoms.     

The diversity of bioactive proteins in Australian snake venoms

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.     

Antimicrobial peptides from the venoms of Vespa bicolor Fabricius

Hornets possess highly toxic venoms, which are rich in toxins, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms. Vespa mastoparan (MP-VBs) and Vespa chemotatic peptide presenting antimicrobial action (VESP-VBs) were purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursors encoding VESP-VBs and MP-VBs were cloned from the cDNA library of the venomous glands. Analyzed by FAB-MS, the amino acid sequence and molecular mass for VESP-VB1 were FMPIIGRLMSGSL and 1420.6, for MP-VB1 were INMKASAAVAKKLL and 1456.5, respectively. The primary structures of these peptides are homologous to those of chemotactic peptides and mastoparans isolated from other vespid venoms. These peptides showed strong antimicrobial activities against bacteria and fungi and induced mast cell degranulation, but displayed almost no hemolytic activity towards human blood red cells.     

A Naturally Occurring Hypoallergenic Variant of Vespid Antigen 5 from Polybia scutellaris Venom as a Candidate for Allergen-Specific Immunotherapy

Stings by insects from the Hymenoptera order are known to cause life-threatening allergic reactions and impair life quality. Despite the effectiveness of conventional vespid venom immunotherapy, more standardized and safer allergy vaccines are required and recombinant hypoallergenic variants are important clinical tools. Antigen 5 is a major allergen of vespid venoms and it was previously reported that Antigen 5 from Polybia scutellaris (Poly s 5) could be a hypoallergenic variant. In this work we assess the immunological behavior and allergenic activity of Poly s 5 in order to explore its suitability for specific immunotherapy. With this aim, recombinant Poly s 5 was expressed in Pichia pastoris and the presence of cross-reactive epitopes with Pol a 5, a known allergenic Antigen 5, was investigated both at the IgG and IgE levels, by ELISA assays and a basophil-mediator release assay respectively. A molecular model was also built to better understand the relationship between immunological and structural aspects. In mice, Poly s 5 induced IgG antibodies which cross-reacted with Pol a 5. However, Poly s 5 induced only minimal amounts of IgE and was a poor inducer of basophil-mediator release, even when the cells were sensitized with Pol a 5-specific IgE. Moreover, Poly s 5-specific serum showed a specific protective activity and was able to inhibit the Pol a 5-induced basophil degranulation. Structural analysis from the molecular model revealed that a few amino acid substitutions in the N-terminal region of Poly s 5 should lead to an alteration of the surface topography and electrostatic potential of the epitopes which could be responsible for its hypoallergenic behavior. These findings, taken as a whole, show that Poly s 5 is likely a naturally occurring hypoallergenic Antigen 5 variant.     

Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.     

Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom

The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.     

A comparative study of the enzymatic and toxic properties of venoms of the Asian lance-headed pit viper (Genus Trimeresurus)

1. The lethalities, anticoagulant effects, hermorrhagic, thrombin-like enzyme, hyaluronidase, protease, arginine ester hydrolase, 5'-nucleotidase, L-amino acid oxidase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A activities of twenty-three samples of venoms from twelve species of Asian lance-headed pit vipers (genus Trimeresurus) were examined. 2. The results indicate that notwithstanding individual variations in venom properties, the differences in biological properties of the Trimeresurus venoms can be used for the differentiation of venoms from different species of Trimeresurus. 3. The results also suggest that differences in the biological properties of snake venoms are useful parameters in the classification of snake species. 4. Our results indicate that venoms from the species T. okinavensis exhibited biological properties markedly different from other Trimeresurus venoms examined. This observation supports the recently proposed reclassification of T. okinavensis as a member of the genus Ovophis, rather than the genus Trimeresurus.