GPI PHOSPHOLIPASE C OF T BRUCEI

非洲锥虫通过以下途径在哺乳动物宿主中维持感染 抗原变异，一种涉及一种抗原替换的过程 变异型表面糖蛋白(VSG)与第二(另一个)抗原性 独特的VSG。糖基磷脂酰肌醇(GGIs)是由 不同多肽序列的哪些VSG附着在寄生虫上 质膜。如果没有GPI，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。 布氏毛滴虫表达一种磷脂酶C(GPI-PLC)，它能与 高效率。该酶与GPI中间体共定位在 细胞膜的细胞质一侧，但令人惊讶的是没有出现 把他们分开。我们对研究监管机制很感兴趣 控制GPI-PLC在体内的这种自纯化以来的明显静止 酶在体外裂解GPI中间体。我们的假设是 GPI-PLC在体内的结构性激活将导致GPI缺陷 这反过来将导致与细胞相关的VSG的丢失。我们已经测试了 这一假设在利什曼原虫和克氏锥虫中发现是真的：A GPI缺乏导致主要的GPI锚定蛋白gp63和 SSP-4在这些寄生虫中分别表达。 一种有希望的治疗布氏毛滴虫的方法，它避开了 抗原变异的并发症包括破坏 GPI-PLC保持酶活性静止的机制 活细胞。因为GPI-PLC切割了VSG的GPI锚 与蛋白质序列无关(变异的来源是 抗原)，所有VSG与质膜的附着可以是 如果GPL-PLC在体内被结构性激活以切割GPI，则可防止 中间体。这样的布氏毛滴虫细胞系将是“无毛的”。如果 被引入哺乳动物宿主中，预测是这样的细胞 LINE将被宿主免疫反应消除。任何VSG获得 在缺乏GPI的布氏支原体中表达，很可能会被分泌，并且 如果且仅在以下情况下，才能成为“活疫苗接种”VSG池的来源， 这些细胞系是无毒的。 代替这一长期目标，我们的具体目标是：(I)揭开 体内调节GPI-PLC活性的机制及(II)研究 GPI-PLC的酶反应机理。

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.