Isolation, Functional Characterization and Proteomic Identification of CC2-PLA2 from Cerastes cerastes Venom: A Basic Platelet-Aggregation-Inhibiting Factor

Three-step chromatography and proteomic analysis have been used to purify and characterize a new basic phospholipase A(2) named CC2-PLA(2) from the venom of Cerastes cerastes. This phospholipase A(2) has been isolated to an extent of about 50-folds and its molecular weight was estimated at 13,534 Da. For CC2-PLA(2) identification and LC-MALDI-MS/MS analysis, the protein was reduced, alkylated and double hydrolyzed by lysine-C endopeptidase and trypsin. Tryptic fragments of LC-MS/MS analyzed CC2-PLA(2) showed sequence similarities with other snake venom PLA(2). This presents only 51 % (61/120 amino acid residues) sequence homology with the first PLA(2) (gi |129506|) previously purified from the same venom. The isolated CC2-PLA(2) displayed anti-aggregative effect on platelets and induced an inflammatory response characterized by leukocytosis in the peripheral blood. This inflammatory response is accompanied by a release of inflammatory mediators such as IL-6, eosinophil peroxidase and complement system. Obtained results indicate that CC2-PLA(2) induced a release of high level of pro-inflammatory (IL-6) cytokine and no effect on the level of anti-inflammatory cytokine (IL-10) in blood sera. Furthermore, eosinophil peroxidase activity and hemolytic complement effect increased in peripheral blood. Mononuclear and neutrophil cells were found predominant in the induced leucocytosis following CC2-PLA(2) administration into animals.