Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain
Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain
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DOI：
10.1074/jbc.m413303200
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发表时间：
2005-03-04
影响因子：
4.8
通讯作者：
Trapani, JA
中科院分区：
文献类型：
--
作者：
Voskoboinik, I;Thia, MC;Trapani, JA
淋巴细胞孔形成蛋白穿孔素对于维持免疫平衡和有效防御细胞内病原体是必不可少的。到目前为止，还没有报道证实穿孔素结构域的功能，这些结构域完全基于与其他蛋白质结构域的初级序列相似性。在这份报告中，我们使用最近开发的全长穿孔素表达方式和强大的功能分析来研究穿孔素功能的一个特征：它绝对依赖钙来进行脂质结合和细胞溶解。我们首次提供了实验证据，证明预测的C-末端C2基序构成了一个负责穿孔素与膜结合的功能结构域。而429、435、483和485位的保守天冬氨酸残基对于钙依赖的质膜结合和细胞裂解是必不可少的，而Asp-491的贡献是有限的。最后，在实验验证了优化的三维模型后，我们预测了C2结构域的两个遗传穿孔素突变对钙依赖的脂质结合和细胞溶解的影响。
The lymphocyte pore- forming protein perforin is essential for maintaining immune homeostasis and for effective defense against intracellular pathogens. To date, there have been no reported structure- function studies to substantiate the function of any putative domains of perforin, which have been postulated totally on primary sequence similarities with domains in other proteins. In this report, we have used recently developed modalities for expressing full- length perforin and robust functional assays to investigate one of the hallmarks of perforin function: its absolute dependence on calcium for lipid binding and cell lysis. We provide, for the first time, experimental evidence that the predicted C- terminal C2 motif constitutes a functional domain that is responsible for membrane binding of perforin. Whereas conserved aspartate residues at positions 429, 435, 483, and 485 were essential for calcium- dependent plasma membrane binding and cell lysis, the contribution of Asp- 491 was limited. Finally, after experimentally verifying an optimized three- dimensional model, we have made predictions on the impact of two inherited perforin mutations of the C2 domain on calcium- dependent lipid binding and cell lysis.