RESUMO
Background:Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.Methods:The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.Results:V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4.Conclusion:The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.
Assuntos
Animais , Elementos Estruturais de Proteínas , Hialuronoglucosaminidase , Proteínas Recombinantes , Venenos de Vespas/químicaRESUMO
The profiles of high-performance gel filtration of venoms from Polybia paulista, Polybia ignobilis and Polybia occidentalis showed 13 peaks distributed among the three species. These profiles presented similarities that permitted the chromatographic characterization of the genus Polybia and differences that permitted the identification of each species studies. Thus, the comparative analysis of chromatographic profiles of high-performance gel filtration of venoms may be used as an auxiliary tool in taxonomic studies of Polybia wasps.