| Snake venom is the richest natural resource for proteins, peptides, enzymes and other small molecules with biological activities. It has been widely studied for many decades. Up to now, lots of ingredients with biological activities have been purified from snake venom, including neurotoxin, cardiotoxin (also named as cytotoxin), myotoxin (or myonecrotic toxin), phospholipase A2, thrombin-like enzyme, platelet aggregation inhibitor, disintergrin, C-type lectin-line protein, neuron growth factor, cobra venom factor, etc. Many of them have been used as drugs to treat various diseases. A group of basic proteins with similar sequence but different activities have been found in cobra venom, which belong to three-finger protein family, including neurotoxin (cobrotoxin, and b, c), cardiotoxin (cardiotoxin I-VI), cardiotoxin-like basic protein (CLBP). Three-finger proteins have very similar sequence and tertiary structure but different functions. They are the ideal model for studying protein relationship of structure-function.In this study, a weak cardiotoxin had been isolated from Chinese cobra venom. The molecular weight and N-terminal sequence with 20 amino acid residues had been determined, it was confirmed to be cardiotoxin I(CTX I), which was found from cDNA library at first. CTX I showed typical sequence pattern of cardiotoxin family. However, to our surprise, this protein showed cytotoxicity, neurotoxic lethal activity, as well as analgesic effects. These biological activities show that CTX I should be transitional type of three-finger protein with great value for studying the evolution and differentiation of three-finger proteins.On relatively low concentration, i.e.2μ/ml,4μg/ml,6μg/ml,8μg/ml, CTX I showed no or not obvious inhibition to growth of Hela, HepG2, and low differentiated PC 12 cell lines; while it showed strong inhibition to growth of high differentiated PC 12, and the inhibition ratio were 28%,56%,67%, and 72% under these concentrations, leading to cell shrinking and death. Further studies suggested that CTX I showed concentration-dependent and time-dependent cytotoxicity to high differentiated PC 12 cell.By using high differentiated PC 12 cell as studying model, naloxone shows inhibition to the cytotoxicity of CTX I, but could not reverse the whole effects of CTX I. Further studies suggested that CTX I showed relative low analgesic effect. This is the first report that cardiotoxin possess analgesic activity, which was traditionally found in neurotoxins.For studying the cytotoxic mechanism of CTX I, the expression of cyclin D1, caspase-9 and caspase-3, oxidation-reduction system member TRX protein, and CDK5 were measured. The expression of cyclin D1 and caspase-3 decreased, meaning that CTX I may inhibit the growth of high differentiated PC12 cell via blocking the cell cycle and inducing the apotosis. While there is no effect on the expression of TRX, perhaps CTX I has little effect on the celluar status of oxidation-reduction, or other oxidation-reduction system plays a key role. Interestingly, CTX I showed no effect on the concentration of CDK5, suggesting that CTX I might has no addiction effect.In summary, CTX I is a transitional three finger protein, with three different kinds of biological activities, i.e. neurotoxicity, cytotoxicity, and analgesic effect. CTX I shows good potential for studying the structure-function relationship, as well as evolvement and differentiation of three finger proteins. |
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