Proteomic analysis of cleavage events reveals a dynamic two-step mechanism for proteolysis of a key parasite adhesive complex
Proteomic analysis of cleavage events reveals a dynamic two-step mechanism for proteolysis of a key parasite adhesive complex
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DOI：
10.1074/mcp.m300123-mcp200
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发表时间：
2004-06-01
影响因子：
7
通讯作者：
Carruthers, VB
中科院分区：
文献类型：
--
作者：
Zhou, XW;Blackman, MJ;Carruthers, VB
跨膜微线蛋白MIC 2及其配偶体M2 AP包含专性细胞内寄生虫刚地弓形虫（Toxoplasma gondii）快速侵入宿主细胞所需的粘附复合物。最近的研究表明，MIC 2/M2 AP复合物在入侵过程中在寄生虫表面经历了广泛的蛋白水解加工，包括未知蛋白酶对M2 AP的初级加工和被称为MPP 1的匿名蛋白酶对复合物的蛋白水解脱落。虽然已经表明MPP 1介导的切割对于有效的侵入是必需的，但是仍然不清楚粘附复合物是通过膜内还是膜内蛋白水解释放的。在这里，使用一个三阶段的战略分配切割位点的基础上完整的基质辅助激光解吸/电离质量，然后通过酶消化和抑制剂分析确认，我们证明了M2 AP是由两个寄生虫衍生的蛋白酶称为MPP 2和MPP 3处理。我们还使用荧光标签通过二维差异凝胶电泳定义了MPP 2的底物库。最后，我们使用互补的质谱技术，明确表明，MIC 2脱落的膜内切割其锚定结构域。基于该切割位点的性质，我们得出结论，脱落酶，MPP 1，可能是一个多通道膜蛋白酶的菱形家庭。我们的数据支持一种新的两步蛋白水解模型，该模型包括MIC 2/M2 AP复合物的初级加工，然后是次级切割，以在入侵的最后步骤中从寄生虫表面脱落复合物。
The transmembrane micronemal protein MIC2 and its partner M2AP comprise an adhesive complex that is required for rapid invasion of host cells by the obligate intracellular parasite Toxoplasma gondii. Recent studies have shown that the MIC2/M2AP complex undergoes extensive proteolytic processing on the parasite surface during invasion, including primary processing of M2AP by unknown proteases and proteolytic shedding of the complex by an anonymous protease called MPP1. While it was shown that MPP1-mediated cleavage is necessary for efficient invasion, it remained unclear whether the adhesive complex was liberated by juxtamembrane or intramembrane proteolysis. Here, using a three-phase strategy of assigning cleavage sites based on intact matrix-assisted laser desorption/ionization mass followed by confirmation by enzymatic digestion and inhibitor profiling, we demonstrate that M2AP is processed by two parasite-derived proteases called MPP2 and MPP3. We also define the substrate repertoire of MPP2 by two-dimensional differential gel electrophoresis using fluorescent tags. Finally, we use complementary mass spectrometric techniques to unequivocally show that MIC2 is shed by intramembrane cleavage within its anchoring domain. Based on the properties of this cleavage site, we conclude that the sheddase, MPP1, is likely a multipass membrane protease of the Rhomboid family. Our data support a novel two-step proteolysis model that includes primary processing of the MIC2/M2AP complex followed by secondary cleavage to shed the complex from the parasite surface during the final steps of invasion.