GPI PHOSPHOLIPASE C OF T BRUCEI

非洲锥虫在哺乳动物宿主中持续感染， 抗原变异，一个过程，涉及一个替代 变体表面糖蛋白（VSG）与第二（另一）抗原性 不同的VSG。 糖基磷脂酰肌醇（GPIs）是锚， 不同多肽序列的VSG附着在寄生虫上 质膜 如果没有GPIs，抗原变异很可能是 由于VSG不能再附着在质膜上，因此被挫败。 T.布氏杆菌表达磷脂酶C（GPI-PLC），其切割GPI， 高效的 该酶与GPI中间体共定位在 细胞膜的细胞质侧，但令人惊讶的是， 把它们分开 我们有兴趣研究监管机制， 控制GPI-PLC在体内的这种明显静止，因为纯化的 酶在体外切割GPI中间体。 我们的假设是 GPI-PLC在体内的组成性激活将导致GPI缺陷 这又将导致与小区相关的VSG的丢失。 我们已经测试 利什曼原虫和T. cruzi并发现这是真的：a GPI缺陷导致主要GPI锚定蛋白gp 63和gp 64的丢失。 SSP-4，分别在这些寄生虫。 一种有希望的抗T.布鲁塞避开了 抗原变异的并发症包括破坏 保持GPI-PLC酶活性静止的机制， 活细胞 由于GPI-PLC切割VSG的GPI锚， 无论蛋白质序列如何（蛋白质序列的变异来源）， 抗原），所有VSG与质膜的附着可以是 如果GPL-PLC在体内被组成型激活以切割GPI， 中间体的 真是个T布氏细胞系将是“无涂层的”。 如果 引入哺乳动物宿主，预测是这样的细胞 线将消除宿主的免疫反应。 任何VSG， 在GPI缺陷型T.布氏杆菌很可能会分泌， 作为“活疫苗接种”的VSG库的来源，当且仅当， 细胞系是无毒的。 除了这个长远目标外，我们的具体目标是：（i）解决 体内调节GPI-PLC活性的机制，和（ii）研究 GPI-PLC的酶促反应机理。

African trypanosomes sustain an infection in a mammalian host by antigenic variation, a process which involves the replacement of one variant surface glycoprotein (VSG) with a second (another) antigenically distinct VSG. Glycosylphosphatidylinositols (GPIs) are the anchors by which VSGs of varying polypeptide sequences are attached to the parasite plasma membrane. Without GPIs antigenic variation is likely to be foiled since VSG can no longer be attached to the plasma membrane. T. brucei expresses a phospholipase C (GPI-PLC) which cleaves GPIs with high efficiency. The enzyme colocalizes with GPI intermediates on the cytoplasmic side of cellular membranes, but surprisingly does not appear to cleave them. We are interested examining the regulatory mechanisms governing this apparent quiescence of GPI-PLC in vivo since the purified enzymes cleaves GPI intermediates in vitro. Our hypothesis is that constitutive activation of GPI-PLC in vivo will cause a GPI deficiency that will in turn lead to loss of cell-associated VSG. We have tested this hypothesis in Leishmania and T. cruzi and found it to be true: a GPI deficiency causes loss of the major GPI-anchored proteins gp63 and Ssp-4, respectively, in these parasites. A hopeful therapeutic approach against T. brucei which sidesteps the complication of antigenic variation involves a disruption of the mechanisms that keep the enzymatic activity of GPI-PLC quiescent in living cells. Because GPI-PLC cleaves the GPI anchor of VSG irrespective of the protein sequence (the source of the variation in antigens), the attachment of all VSGs to the plasma membrane can be prevented if GPL-PLC were constitutively activated in vivo to cleave GPI intermediates. Such a T. brucei cell line will be "coat-less". If introduced into a mammalian host, the prediction is that such a cell line will be eliminated by host immune response. Any VSGs that get expressed in GPI-deficient T. brucei will most likely be secreted, and be a source of a pool of VSGs for "live vaccination" if, and only if, the cell line were avirulent. In lieu of this long term aim, our specific aims are to (i) unravel the mechanisms which regulate GPI-PLC activity in vivo, and (ii) study enzymatic reaction mechanism of GPI-PLC.