Abstract
Severe tissue necrosis with a retarded wound healing process is a major symptom of the cobra snakebite. Cobra cardiotoxins (CTXs) are the major toxin peptides and constitute ~ 50% weight of the cobra venom. CTXs are believed to play a critical role in cobra venom toxicity. It is not clear whether there are cellular receptors for CTXs. In this study, we perform experiments spaning molecular biology, biophysical, cell-based and bone resorption in vitro studies to identify several Non-RGD integrin binding protein in Taiwan cobra venom and their biological implication on wound healing or the therapeutic potentials. Non-cytotoxic cobra CTX A5 binds to integrin {alpha}v{beta}3 with an apparent ~ 0.3 microM and inhibits both osteoclast differentiation and bone resorption in microM range with therapeutic potentials as an integrin antagonist. Although CTX A3 bind to {alpha}v{beta}3 integrin. Anti-{alpha}v{beta}3 integrin antibody LM609 can barely protect cell from CTX-induced cytotoxicity and neither {beta}3-CHO cells are more susceptible to CTX action. These results show that {alpha}v{beta}3 integrin are not involved well in CTX A3-induced cytotoxicity. SPR studies showed CTX A3 and CTX A5 also bind to {alpha}v{beta}1 integrin with an apparent dissociation constant of 1.8~2.5 microM. {alpha}v -CHO cells are more susceptible to CTX action compared with wildtype- and B2- variants. It implies that {alpha}v-integrins are involved in CTX-induced cytotoxicity. Fibroblasts lacking focal adhesion kinase (FAK), which is directly downstream of integrins, are much less sensitive to CTX A3 than those with FAK. These results establish that integrins are involved in CTX-induced cytotoxicity. Snake venom metalloproteases (SVMPs) are widely distributed in most viperid and crotalid venoms. We first cloned and purified a novol metalloprotease atragin from Naja atra venom. cDNA sequence analysis revealed that atragin as a novel PIII-type SVMPs, sharing highly sequence homolog (~97 %) with cobrin purified from Naja naja with conserved integrin binding motif XXCD compared with other SVMPs. It implied that atragin and cobrin come form the same ancestor. Atragin induced apoptosis of endothelium cells through digestion of extracellular matrix or cell surface receptor, therefore, lead to lost of cell adhesion. Atragin also inhibited fibroblast migration and showed synergistically inhibition effect with CTXA5. Apoatragin, which lacks metalloprotease activity, is unable to inhibit fibroblast migration. It suggested that SVMP might play an important role in the wound region with a retarded wound healing process. Finally, by comparing the integrin binding affinity among CTX homologues, we propose that the hydrophobic domain near the tip of loop I and the charged residues flanking the other hydrophobic loop II region may be involved in CTXs-integrin interaction.