Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi
Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi
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DOI：
10.1042/bj20041842
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发表时间：
2005-04-15
影响因子：
4.1
通讯作者：
Heise, N
中科院分区：
文献类型：
--
作者：
Figueiredo, JM;Dias, WB;Heise, N
IPC（肌醇磷酸神经酰胺）合成酶是真菌生存所必需的酶，也是有效抗真菌化合物如锈菌菌素和金霉素A的靶标。与真菌和其他一些低等真核生物类似，原生动物寄生虫克氏锥虫能够合成游离或蛋白质连接的含IPC的糖肌醇磷脂。作为理解克氏锥虫IPC合成的重要性和机制的第一步。我们研究了锈菌菌素和金担子素A对寄生虫不同生命周期阶段增殖的影响。这些化合物不干扰外鞭毛体的纯性生长，但金担子素A以剂量依赖性方式降低了感染小鼠腹腔巨噬细胞中锥鞭毛体的释放和细胞内无鞭毛体的数量。我们已经证明了第一个什一税，所有形式的T。cruzi表达IPC合酶活性，其能够将肌醇磷酸从磷脂酰肌醇转移到C-6-NBD-cer {6-[N-（7-体外-2，1，3-苯并恶二唑-4-基）-氨基]己酰基神经酰胺}，形成肌醇磷酸-C-6-NBD-cer，将其纯化并通过其在TLC和HPLC上的色谱行为进行表征，对磷脂酰肌醇特异性磷脂酶C的敏感性和对温和碱性水解的抗性。与酿酒酵母IPC合成酶不同，克氏锥虫酶受Triton X-100刺激，但不受二价阳离子、CHAPS或无脂肪酸BSA刺激，并且不受锈菌菌素或aureobasidin A或两者组合的抑制。进一步的研究表明，金担子素A对巨噬细胞的作用不依赖于感染T。cruzi细胞这些结果表明T. cruzi合成其自身的IPC，但其机制不受锈菌菌素和金芽孢杆菌素A的影响。
IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T cruzi. we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first tithe that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C-6-NBD-cer {6-[N-(7-vitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C-6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.