Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis.

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/ß)(7) barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients.

A simple, rapid method for the extraction of whole fire ant venom (Insecta: Formicidae: Solenopsis).

The invasive fire ant Solenopsis invicta is medically important because its venom is highly potent. However, almost nothing is known about fire ant venom proteins because obtaining even milligram-amounts of these proteins has been prohibitively challenging. We present a simple and fast method of obtaining whole venom compounds from large quantities of fire ants. For this, we separate the ants are from the nest soil, immerse them in dual-phase mixture of apolar organic solvent and water, and evaporate each solvent phase in separate. The remaining extract from the aqueous phase is largely made up of ant venom proteins. We confirmed this by using 2D gel electrophoresis while also demonstrating that our new approach yields the same proteins obtained by other authors using less efficient traditional methods.

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP-III at the level of endosomal membranes from rat mast cells.

It is well known that the activation of mast cells due to the binding of mastoparan to the G(α) subunit of trimeric G proteins involves exocytosis regulation. However, experimental evidence in the literature indicates that mastoparan can also activate certain regulatory targets of exocytosis at the level of the mast cell endosomal membranes that have not yet been identified. Therefore, the aim of the present investigation was the proteomic identification of these targets. To achieve these objectives, mast cells were activated by the peptide Protopolybia MP-III, and the proteins of the endosomal membranes were converted to proteoliposomes using sonication. Proteins were separated from one another by affinity chromatography using proteoliposomes as analytes and Protopolybia MP III-immobilized Sepharose 4B resin as the ligand. This experimental approach, which used SDS-PAGE, in-gel trypsin digestion and proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPase Cdc 42 and exocyst complex component 7 as components of the Ca(2+) -independent FcεRI-mediated exocytosis pathway, synaptosomal-associated protein 29, and GTP-binding protein Rab3D as components of the Ca(2+) -dependent FcεRI-mediated exocytosis pathway and Ras-related protein M-Ras, a protein that is related to the mediation of cell shaping and proliferation following exocytosis. The identification of the five proteins as targets of mastoparans may contribute in the near future to the use of this family of peptides as novel tools for dissecting the mechanism of exocytosis in mast cells.