Venom Acid Phosphatase Research Articles

Biopanning of allergens from wasp sting patients.

Objective: Wasp venom is a potentially important natural drug, but it can cause hypersensitivity reactions. The purpose of the present study was to systematically study the epitopes of wasp venom. Methods: Using a random 12-peptide phage library, we performed antibody-binding epitope panning on ten serum samples from wasp sting victims at 3 h and 4 days after the sting. The panning epitopes were identified by high-throughput sequencing and matched with wasp venom proteins by BLAST. The panned antibody-binding epitopes were verified by ELISA. Results: A total of 35 specific potential wasp venom epitopes in 4 days were identified. Amongst them, twelve peptide epitopes were matched with nine wasp venom proteins, namely, vitellogenin precursor, hexamerin 70b precursor, venom carboxylesterase-6 precursor, MRJP5, major royal jelly protein 8 precursor, venom acid phosphatase Acph-1 precursor, phospholipase A2, venom serine protease 34 precursor, and major royal jelly protein 9 precursor. The changes in serum IgM antibodies induced by wasp venom were confirmed by ELISA based on the 12 peptide epitopes. Conclusion: The nine wasp venom proteins are potential allergens, which should be excluded or modified in the potential biomedical applications of wasp venom.

De novo sequencing and transcriptome analysis of female venom glands of ectoparasitoid Bracon hebetor (Say.) (Hymenoptera: Braconidae)

Venom is a key-factor in the regulation of host physiology by parasitic Hymenoptera and a potentially rich source of novel bioactive substances for biotechnological applications. The limited study of venom from the ectoparasitoid Bracon hebetor, a tiny wasp that attacks larval pest insects of field and stored products and is thus a potential insect control agent, has not described the full complement and composition of these biomolecules. To have a comprehensive picture of genes expressed in the venom glands of B. hebetor, a venom gland transcriptome was assembled by using next generation sequencing technologies followed by de novo assemblies of the 10.81 M sequence reads yielded 22,425 contigs, of which 10,581 had significant BLASTx hits to know genes. The majority of hits were to Diachasma alloeum, an ectoparasitoid from same taxonomic family, as well as other wasps. Gene ontology grouped the sequences into molecular functions in which catalytic activity with 42.2% was maximum, cellular components in which cells with 33.8% and biological processes among which metabolic process with 30% had the most representatives. In this study, we highlight the most abundant sequences, and those that are likely to be functional components of the venom for parasitization. Full length ORFs of Calreticulin, Venom Acid Phosphatase Acph-1 like protein and arginine kinase proteins were isolated and their tissue specific expression was studied by RT-PCR. Our report is the first to characterize components of the B. hebetor venom glands that may be useful for developing control tools for insect pests and other applications.

Molecular characterization of a venom acid phosphatase from the Asiatic honeybee Apis cerana

Bee venom contains a variety of toxic components, including enzymes, peptides, and biogenic amines. An acid phosphatase Acph-1-like protein has been identified from Asiatic honeybee (Apis cerana) venom. However, no molecular information is currently available for acid phosphatases from A. cerana venom. In this study, an A. cerana venom acid phosphatase (AcVAP) was identified. The amino acid sequence analysis of the predicted AcVAP protein revealed high identity with other bee venom acid phosphatases. An anti-AcVAP antibody was produced against a recombinant AcVAP (46-kDa) expressed in in baculovirus-infected insect cells. Northern and Western blot analyses showed that AcVAP was expressed in the venom gland and was present as a 46-kDa protein in the secreted bee venom. The enzymatic properties of recombinant AcVAP were determined using p-nitrophenyl phosphate (p-NPP) as a substrate and exhibited the highest activity at pH4.8 and 45°C. Taken together, our data demonstrated that AcVAP functions as a bee venom acid phosphatase.

Bioinformatic and Functional Analysis of Venom from the Jewel Wasp Ampulex compressa

The parasitoid jewel wasp Ampulex compressa exploits its host, the American cockroach Periplaneta americana, as a food source for its young. The host is subdued through direct envenomation into the central nervous system, inducing a days‐long “zombie‐like state” referred to as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions intact. This condition is reversible, provided that egg deposition is prevented. To facilitate elucidation of biochemical mechanisms underlying venom‐induced hypokinesia, we developed a comprehensive inventory of the venom by combining: 1) next‐generation RNA sequencing, transcriptomic analysis of venom gland components, and 2) proteomics of venom apparatus extracts and whole milked venom via mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology. These analyses yielded a total of 201 proteins and peptides in the venom, 105 with Pfam domains, 146 similar to other proteins in the NCBI‐nr or Uniprot databases, and 45 novel proteins absent in global databases. A. compressa venom proteins include members of the M13 family of proteases (11), phospholipase A2 isoforms (10), along with hyaluronidase, chitinase, icarapin, venom acid phosphatase, serpins, and signaling peptides including tachykinin, corazonin, and myosuppressin. Differential expression analysis and proteomics of the two major tissues of the venom apparatus ‐ venom sac and venom gland ‐identified tissue‐specific expression of key venom components.We show that A. compressa venom tachykinins (AcVTK) are able to activate the cockroach brain tachykinin receptor with high affinity (EC50 ~1 nM) in vitro. Injection of synthetic AcVTK into the subesophageal ganglion, an established target of the wasp venom, increases escape response threshold for one hour post‐treatment. These results indicate that tachykinin may participate in the behavioral changes resulting from envenomation of cockroach cerebral ganglia.We isolated and characterized two novel, paralogous venom sac peptides found in milked venom. These peptides are alpha‐helical, amphipathic, and constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin‐1, into the subesophageal ganglion of cockroaches resulted in a short‐term increase in escape threshold.Our findings show that the venom of A. compressa is a rich collection of both well‐known and novel, pharmacologically diverse compounds. Our study provides new and comprehensive information on A. compressa venom contents that inform hypotheses regarding its mode of action.

Differential Properties of Venom Peptides and Proteins in Solitary vs. Social Hunting Wasps.

The primary functions of venoms from solitary and social wasps are different. Whereas most solitary wasps sting their prey to paralyze and preserve it, without killing, as the provisions for their progeny, social wasps usually sting to defend their colonies from vertebrate predators. Such distinctive venom properties of solitary and social wasps suggest that the main venom components are likely to be different depending on the wasps’ sociality. The present paper reviews venom components and properties of the Aculeata hunting wasps, with a particular emphasis on the comparative aspects of venom compositions and properties between solitary and social wasps. Common components in both solitary and social wasp venoms include hyaluronidase, phospholipase A2, metalloendopeptidase, etc. Although it has been expected that more diverse bioactive components with the functions of prey inactivation and physiology manipulation are present in solitary wasps, available studies on venom compositions of solitary wasps are simply too scarce to generalize this notion. Nevertheless, some neurotoxic peptides (e.g., pompilidotoxin and dendrotoxin-like peptide) and proteins (e.g., insulin-like peptide binding protein) appear to be specific to solitary wasp venom. In contrast, several proteins, such as venom allergen 5 protein, venom acid phosphatase, and various phospholipases, appear to be relatively more specific to social wasp venom. Finally, putative functions of main venom components and their application are also discussed.

Molecular characterization of a venom acid phosphatase Acph-1-like protein from the Asiatic honeybee Apis cerana

Bee venom contains a variety of peptides and enzymes, including acid phosphatases. An acid phosphatase has been identified from European honeybee (Apis mellifera) venom. However, although the amino acid sequence is known, no functional information is currently available for bee venom acid phosphatase Acph-1-like proteins. In this study, an Asiatic honeybee (Apis cerana) venom acid phosphatase Acph-1-like protein (AcAcph-1) was identified. The analysis of the predicted AcAcph-1 amino acid sequence revealed high levels of identity with other bee venom acid phosphatase Acph-1-like proteins. Recombinant AcAcph-1 was expressed as a 64-kDa protein in baculovirus-infected insect cells. The enzymatic properties of recombinant AcAcph-1, determined using p-nitrophenyl phosphate (p-NPP) as a substrate, showed the highest activity at 45°C and pH4.8. Northern and western blot analyses showed that AcAcph-1 was expressed in the venom gland and was present as a 64-kDa protein in bee venom. In addition, N-glycosylation of AcAcph-1 was revealed by tunicamycin treatment of recombinant virus-infected insect Sf9 cells and by glycoprotein staining of purified recombinant AcAcph-1. Our findings show that AcAcph-1 functions as a venom acid phosphatase. This paper provides the first evidence of the role of a bee venom acid phosphatase Acph-1-like protein.

Developing the venome of the parasitoid wasp Ampulex compressa (778.4)

The endoparasitoid wasp Ampulex compressa injects venom directly into the central nervous system of its host, the American cockroach (Periplaneta americana), to induce a long‐term behavioral state known as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions unaffected. To facilitate elucidation of the biochemical mechanism behind venom‐induced hypokinesia, we are developing a bioinformatic venome by combining next‐generation RNA sequencing and transcriptome analysis of venom gland tissue and mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology (MuDPIT) from extracts of whole milked venom. Preliminary analysis has yielded identification of more than 50 proteins and peptides, with putative identifications of venom components including endothelin‐converting enzyme‐like peptidase, icarapin, venom acid phosphatase, and tachykinin along with ~20 novel peptides and proteins. The action of the venom appears to be non‐cytolytic, specific, and reversible. Venome analysis of A. compressa may yield a rich collection of novel, pharmaceutically relevant compounds.

Proteomic analysis of the venom from the endoparasitoid waspPteromalus puparum(Hymenoptera: Pteromalidae)

Parasitoid venom is a complex mixture of active substances with diversified biological functions. Because of its range of activities, venom is an important resource with respect to potential application in agriculture and medicine. Only a limited number of peptides, proteins, and enzymes have been identified and characterized from parasitoid venom. Here we describe a proteomic analysis of the venom from the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae). Venom resolved by two-dimensional electrophoresis yielded 56 protein spots with major proteins in the pI range 4-7 and molecular mass range of 25-66.2 kDa. The amino acid sequences of the proteins were identified by mass spectrometry. Several venom proteins such as calreticulin, venom acid phosphatase, serine protease, arginine kinase, serine protease homolog, aminotransferase-like venom protein, and heat shock protein 70, were identified in silico based on their amino acid sequences. The full-length cDNAs of calreticulin and arginine kinase were cloned. Calreticulin showed 62% identity with calreticulin in the venom of Cotesia rubecula. Arginine kinase showed a high level of sequence identity (92%) with its counterpart in the venom of Cyphononyx dorsalis. RT-PCR analysis revealed that the transcript levels of calreticulin and arginine kinase were developmentally changed, suggesting a possible correlation with the oviposition process. This study contributes to our appreciation of a parasitoid wasp venom composition.

3-D Model of the bee venom acid phosphatase: Insights into allergenicity

The acid phosphatase Api m 3 is the major allergen of the honeybee venom. Except for the amino acid sequence, no other structural information for the enzyme is available. We applied homology modeling to assign the three-dimensional structure of Api m 3. The structure of the homodimeric human prostatic acid phosphatase was used to model the Api m 3 tertiary structure. IgE epitopes and antigenic sites were predicted using programs based on the structure of known epitopes and analysis of the 3-D model. The model of Api m 3 revealed an active site similar to those of the histidine—type acid phosphatases with conservation of the catalytically important residues. The observed substitutions in the phosphate ion binding site suggest differences in the substrate specificity in comparison to other acid phosphatases. The analysis of the Api m 3 three-dimensional model revealed a very likely mechanism of enzyme action.

Molecular cloning and characterization of acid phosphatase in venom of the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The present study describes cDNA cloning, sequencing, enzyme activity determination and localization, and mRNA expression of acid phosphatase in the venom apparatus of an endoparasitoid, Pteromalus puparum. This is the first report of cloning a venom acid phosphatase gene which has been described in parasitoid wasps. The cDNA consisted of 1378 bp with 1215 bp open reading frame and encoded a sequence of 405 amino acids. A 23 residues N-terminal signal peptide was followed by a short 15 residues (25–39) histidine acid phosphatases phosphohistidine signature and a long 302 residues (24–325) acid phosphatase family domain. The deduced amino acid sequence shared 32–88% identity to its counterparts from other insects. Enzyme activity was measured by using p-nitrophenyl phosphate ( p-NPP) as substrate, and a high level of acid phosphatase activity in venom was detected. Optimal pH and temperature for this enzyme activity was 4.8 and 45 °C, respectively. Ultracytochemical analyses further revealed that strong enzyme activity was located in the nuclei and secretory vesicles of the venom gland secretory cells. Expression of the acid phosphatase gene was observed to be regulated at different developmental stages by RT-PCR analysis as it expressed immediately with low abundance after adult emergence, then increased to the high level at 2–4 days, followed by a drop to the low abundance after 4 days. Compared to the mRNA expression, a time-course-related enzyme activity in an individual venom apparatus was also found.

Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca

Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, β-glucosidase, esterase, β-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/μg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC 50 4.2 × 10 − 4 M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.

Sequence and characterization of honeybee venom acid phosphatase

Sequence and characterization of honeybee venom acid phosphatase

Analysis of Differing Patterns of Cross-Reactivity of Honeybee and Yellow Jacket Venom-Specific IgE: Use of Purified Venom Fractions

Prior studies of sera from insect sting-allergic patients have analyzed the relationship of coexisting honeybee venom- and yellow jacket venom-specific IgE. Radioallergosorbent (RAST)-inhibition tests with these venoms revealed four different patterns of activity. In this present study, purified fractions prepared from these venoms were used to analyze these varying patterns. The hyaluronidases of yellow jacket venom and honeybee venom showed extensive cross-reaction. The phospholipases from these venoms showed minimal cross-reactivity; antigen 5 was restricted to yellow jacket venom. There was a high molecular weight component in yellow jacket venom with immunologic properties similar to honeybee venom acid phosphatase. Sera from individual patients showed quantitative and qualitative differences in the reactions to the major components of both venoms. The differences in the RAST-inhibition patterns in patients with elevated levels of both honeybee venom- and yellow jacket venom-specific IgE are accounted for by these differences as well as by differences in the cross-reactivity between the individual components.

Acid and alkaline phosphomonoesterase activities in snake venoms

1. 1. Acid and alkaline phosphatase activities of eight different snake venoms were determined quantitatively by using synthetic substrates, o-carboxyphenylphosphate and p-nitrophenylphosphate respectively. 2. 2. It was found that most of Elapidae venoms investigated had both acid and alkaline phosphatase activities. 3. 3. Three Crotalidae venoms investigated did not show any alkaline phosphatase activity. 4. 4. The strength of venom acid phosphatase activity is as follows: Agkistroden acutus > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca. 5. 5. The strength of venom alkaline phosphatase activity by using p-nitrophenylphosphate is in the order of Naja hannah > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca. When o-carboxyphenylphosphate was used as a substrate, the order of enzyme activity is Naja hannah > Naja haje > Naja naja samarensis > Naja melanoleuca > Naja naja atra. 6. 6. Acid phosphatase activity of all the Elapidae venoms was inhibited completely by fluoride. The alkaline phosphatase activity of Elapidae venoms was not inhibited by fluoride either using p-nitrophenylphosphate or o-carboxyphenylphosphate. 7. 7. The acid phosphatase of all the Elapidae venoms was not inhibited by zinc ion. However, most of the venom alkaline phosphatases were inhibited by zinc ion. 8. 8. Ethylenediaminetetraacetic acid (EDTA) had inhibitory action on venom phosphatase activity. However, tris-(hydroxymethyl)-aminoethane had a counter effect on the inhibitory action of EDTA. 9. 9. Optimum pH studies of the snake venom phosphatases showed that the acid phosphatases of the snake venoms had their highest activity in the range of pH 4–5. The alkaline phosphatases of the snake venoms had their optimum pH at 9. 10. 10. Comparable experiments were also conducted by using chicken intestine alkaline phosphatase and wheat germ acid phosphatase.