Proteomic Identification of Different Isoforms of Phospholipases In Polybia paulista Venom (Hymenoptera, Vespidae)
Santos, LD1,3; Santos, KS2; Dias, NB1; Pinto, JRAS1; Souza, BM1; Castro, FM2,3; Cunha-Neto, E2,3; Kalil, J2,3; Palma, MS1,2,3
1Dept. of Biology-CEIS/UNESP, Rio Claro-SP (ldsantos@rc.unesp.br); 2Discipline of Allergy and Immunology/INCor, HC/FMUSP-SP; 3Institute of Immunological Investigations-CNPq/MCT
Hymenoptera stinging is quite common and can cause systemic, inflammatory and allergic reactions in the victimes of their stingings. P. paulista is one of the most abundant wasp specie in Southeast Brazil, causing many stinging accidents every year. The phospholipases (PLA) are recognized as one of the major allergens from these venoms. The purpose of this work is to identify the different isoforms of PLA in the venom of the social wasp P. paulista, through a proteomic approach. The crude venom was fractionated under FPLC by ion exchange chromatography and the fractions presenting PLA activity were submitted to 2D-SDS-PAGE with IPG strips from pI 3-10 in the first dimension. The protein spots were electrotransfered to Immobilon P membranes and submitted to N-terminal sequencing by Automatic Edman Degradation Chemistry. The proteins were identified by using the sequences in the protein engine search BLAST. The PLAs were distributed around the MW 16 KDa and in the pI range from 4-10. Different N-terminal sequences were reported. It is known that the PLAs from Vespidae venoms are originated from a single gene. However, the occurrence of post-translational modifications, such as side chain glycosylation and proteolytic cleavage at different sites of primary sequence, can result in different isoforms PLAs, presenting different immunogenicity levels. Immuno assays demonstrated that only three of these PLAs isoforms were immunogenic. The hemolytic activity assays revealed that the non immunogenic PLAs presented a reasonable hemolysis, and thus, these isoforms may be related to the inflammation processes caused by the venom. Meanwhile, the immunogenic isoforms did not present any hemolysis. The results above are suggesting that the venom of P. paulista may have evolved to scape from the immunological defense system of the mammals by using post-translational modifications of the major venom proteins, as strategy to create a series of different molecular structures with different levels of affinity by IgE specific. Supported by CAPES-CNPq-MCT/CAT-CEPID-FAPESP
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