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.

Determinación de la toxicidad retiniana de una mezcla de lidocaína: bupivacaína y hialuronidasa en conejos albinos de Nueva Zelanda / Determination of retina toxicity of a mixture of lidocaíne: bupivacaine and hialuronidase in albino rabbits of New Zealand

Con la finalidad de determinar la posible acción tóxica que pudiera producir sobre la integridad funcional y morfológica de la retina, la inyección de una mezcla de anestésicos (lidocaína 2 por ciento, bupivacaína 0,75 por ciento, hialuronidasa) en el globo ocular de conejos albinos de Nueva Zelanda, se utilizaron 12 conejos albinos a los cuales previa sedación con Clorhidrato de Ketamina se les realizó ERG, previo a la inyección, así como, 10 minutos y 4 horas posterior a la inyección, y estudio con microscopia de luz y electrónica posterior a la enucleación. Podemos infereir por lo observado que los cambios que se producen con la inyección de la mezcla utilizada en este estudio, ocasionan una alteración progresiva y más tardía que la observada con lidocaína o bupivacaína, donde apreciamos el inicio de la recuperación de la actividad electrorretinográfica a las cuatro (4) horas de realizada la inyección. Este efecto de alteración tardía y profunda producido con la mezcla podría deberse a la potenciación de la actividad del anestésico al ser administrado en forma combinada, aunado a la combinación de los mismos con la hialuronidasa. Las alteraciones observadas a la microscopia electrónica podrían sugerir la probable disrupción de la membrana limitante interna a nivel de las uniones de las células de MÜller con el paso de vítreo acumulándose en las capas más internas de la retina en forma de lagunas

Induction of vitreolysis and vitreous detachment with hyaluronidase and perfluoropropane gas

To simulate the posterior vitreous detachment (PVD) in the rabbit, 1 IU hyaluronidase and/or 0.2 ml of perfluoropropane gas was intravitreally injected. Ophthalmoscopic, light microscopic examination prepared by cryotechnique, electron microscopic examination, and electroretinogram were done on the 3rd and 28th postoperative days. As a result, the eyes undergone simultaneous intravitreal injection of 1 IU hyaluronidase and 0.2 ml perfluoropropane gas showed membranous structure split from the internal limiting membrane of the superior retina in 3 days after injection. The eyes also demonstrated membranous structure separated from the superior retina after 28 days, simulating vitreous detachment. On the contrary, neither agent alone induced vitreous detachment. No toxic retinal changes associated with simultaneous intravitreal injection of 1 IU hyaluronidase and 0.2 ml perfluoropropane gas were observed. Therefore, with a future support by histologic examination other than cryotechnique and by immunohistochemical analysis, the simultaneous intravitreal injection of perfluoropropane gas and hyaluronidase would be a promising method to induce vitreous detachment in non-vitrectomized eye.