Isolation and characterization of phospholipase A1 from Vespa tropica ducalis / 中草药

Objective: To find an effective method to isolated phospholipase from Vespa tropica ducalis and characterize its biological activities to support the pathogenic mechanism research and officinal value exploitation in the future. Methods: The component with phospholipase activity was isolated by successive gel filtration (Sephadex G-75, supferfine) and heparin affinity chromatography steps (Hitrap Heparin HP). The protein was identified by peptide mass fingerprinting, N-terminal amino acid determination and blast analysis, as well as phospholipase A1 (PLA1) activity monitor. Plasma recalification time test was employed to detect the effect of Vtp32 on coagulation. Results: A protein with phospholipase activity was orderly separated and purified from V. tropica ducalis venom using gel filtration and heparin affinity chromatography. The purified protein was homogenous on the SDS-PAGE gel with relative molecular mass of 32 000, so it was termed as Vtp32. Peptide mass fingerprinting assay and N-terminal amino acid sequence blast result revealed that Vtp32 showed high homologous with PLA1 from wasp of Vespa genus. In addition, Vtp32 hydrolyzed the sn-1 ester linkage of phospholipids. These results indicated that Vtp32 was PLA1 from V. tropica ducalis. Vtp32 hydrolyzed phosphatidylcholine, and the hydrolysis product can lyse human erythrocytes. Vtp32 delayed the recalification time of human plasma and hence had anti-coagulation activity. Conclusion: PLA1 is widely existed in the venom from V. tropica ducalis. Gel filtration followed by heparin affinity chromatography is an effective isolation strategy for the purification of PLA1. The results show that V. tropica ducalis PLA1 has hemolytic and anticoagulative activity.

Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica

Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (4297 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (/)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.

Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica

Background: Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (42-97 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (α/β)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.(AU)

Observations on the social wasp Ropalidia montana from peninsular India.

Four colonies of Ropalidia montana collected in August in the Nilgiri Hills of southern India had adult populations of 32,000-61,000. Queens represented 0·46–1·40% of the populations. All colonies contained males, but in widely varying proportions (1·47– 27.00%). The large adult populations and the fact that the nests were largely filled with brood in all stages of development indicate that the colonies were in a stage of active growth in this season. In December, however, colonies have been reported to have smaller adult populations and little brood. Thus brood production appears to be seasonal in southern India. Predation by the hornet Vespa tropica was observed in 4 of 31 active colonies. Ropalidia montana adults were defenseless in the face of these depredations, which appear to continue at a low level for days or weeks on a given nest.