Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption
Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption
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DOI：
10.1074/jbc.m513035200
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发表时间：
2006-03
影响因子：
4.8
通讯作者：
Po-long Wu;Shao‐Chen Lee;Chia-Chen Chuang;S. Mori;N. Akakura;Wen‐guey Wu;Y. Takada
中科院分区：
文献类型：
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作者：
Po-long Wu;Shao‐Chen Lee;Chia-Chen Chuang;S. Mori;N. Akakura;Wen‐guey Wu;Y. Takada
严重的组织坏死和伤口愈合过程延迟是眼镜蛇咬伤的主要症状。心脏毒素 (CTX) 是眼镜蛇毒液的主要成分，属于 Ly-6 蛋白家族，与组织损伤有关。台湾眼镜蛇的主要 CTX（即 CTX A3）与细胞膜中的硫苷脂的相互作用最近已被证明可诱导孔形成和细胞内化，并导致心肌细胞的细胞毒性（Wang, C.-H.、Liu, J.-H.、Lee, S.-C.、Hsiao, C.-D. 和 Wu, W.-g. (2006) J. Biol. Chem. (2006) J. Biol. Chem. 281、656-667）。我们在这里展示了来自台湾眼镜蛇的一种非细胞毒性CTX，即CTX A5或心脏毒素样碱性多肽，与αvβ3整合素特异性结合并抑制骨吸收活性。我们发现膜结合整合素和重组可溶性αvβ3整合素均以剂量依赖性方式特异性结合CTX A5。表面等离振子共振分析表明，人可溶性 αvβ3 与 CTX A5 结合，表观亲和力约为 0.3 μm。组成型表达 αvβ3 的小牛肺动脉内皮细胞显示出与膜结合和可溶性 αvβ3 整联蛋白相似的 CTX A5 结合谱，表明内皮细胞是 CTX 作用的潜在靶点。我们测试了 CTX A5 是否抑制破骨细胞分化和骨吸收，这一过程已知参与 αvβ3 结合并被含有 RGD 的肽抑制。我们证明，CTX A5 通过与破骨细胞中的鼠 αvβ3 整合素结合，在微摩尔范围内抑制这两种活性，并且 CTX A5 与 β3 整合素共定位。最后，在比较了CTX同源物之间的整合素结合亲和力后，我们提出CTX A5两个环附近的氨基酸残基参与整合素结合。这些结果确定 CTX A5 是一种非 RGD 整合素结合蛋白，具有作为整合素拮抗剂的治疗潜力。
Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.e. CTX A3, with sulfatides in the cell membrane has recently been shown to induce pore formation and cell internalization and to be responsible for cytotoxicity in cardiomyocytes (Wang, C.-H., Liu, J.-H., Lee, S.-C., Hsiao, C.-D., and Wu, W.-g. (2006) J. Biol. Chem. 281, 656-667). We show here that one of the non-cytotoxic CTXs, i.e. CTX A5 or cardiotoxin-like basic polypeptide, from Taiwan cobra specifically bound to αvβ3 integrin and inhibited bone resorption activity. We found that both membrane-bound and recombinant soluble αvβ3 integrins bound specifically to CTX A5 in a dose-dependent manner. Surface plasmon resonance analysis showed that human soluble αvβ3 bound to CTX A5 with an apparent affinity of ∼0.3 μm. Calf pulmonary artery endothelial cells, which constitutively express αvβ3, showed a CTX A5 binding profile similar to that of membrane-bound and soluble αvβ3 integrins, suggesting that endothelial cells are a potential target for CTX action. We tested whether CTX A5 inhibits osteoclast differentiation and bone resorption, a process known to be involved in αvβ3 binding and inhibited by RGD-containing peptides. We demonstrate that CTX A5 inhibited both activities at a micromolar range by binding to murine αvβ3 integrin in osteoclasts and that CTX A5 co-localized with β3 integrin. Finally, after comparing the integrin binding affinity among CTX homologs, we propose that the amino acid residues near the two loops of CTX A5 are involved in integrin binding. These results identify CTX A5 as a non-RGD integrin-binding protein with therapeutic potential as an integrin antagonist.