Structural and functional studies of WASP molecule in cellular basis

Wiskott-Aldrich综合征蛋白（WASP）是Wiskott-Aldrich综合征（WAS）患者的缺陷蛋白，是在非红系造血细胞中表达的细胞内蛋白。近年来的研究表明，WASP与细胞内的许多分子相互作用，在信号转导和肌动蛋白聚合的调节中起着关键作用，尽管WASP缺乏症的临床症状尚未完全阐明，但我们建立了流式细胞术检测细胞内WASP的方法结果表明，WAS患者的淋巴细胞/单核细胞的胞内WASP水平为零/非常低，而正常人的胞内WASP水平明显高于正常人。应用该方法对WAS患者和WAS携带者进行筛查，并对WAS患者造血干细胞移植后的混合嵌合体状态进行评估。此外，在WAS筛选过程中， 关于我们 我们用FCM-WASP研究时，发现一例WAS患者的淋巴细胞中有一小部分发生了遗传性WASP基因突变的自发性逆转，在这些研究中，我们注意到对照组的淋巴细胞呈双或宽阳性峰，而单核细胞均呈尖锐阳性峰。为了研究FCM-WASP检测对照淋巴细胞的双阳性峰（WASP^<high-bright>和WASP^<low-bright>）的基础，我们试图表征这两个群体。通过FCM-WASP双重/三重染色方法，显示了以下结果。T细胞，CD 4+和CD 8+细胞两者都由WASP+<high-bright>和WASP+<low-bright>细胞两者组成。大多数B细胞（CD 20+）是WASP^<low-bright>细胞，大多数NK细胞（CD 56+）是WASP^<high-bright>细胞。进一步的表征显示，CD 4+或CD 8+细胞的CD 45 RA+属于WASP+，<low-bright>而CD 4+或CD 8+细胞的CD 45 RO+细胞属于WASP<high-bright>+。这些结果是通过使用α-WASP抗体3F 3A 5（由纳尔逊DL博士提供：NIH/USA）获得的，其表位是WASP的中间部分。在使用另一种α-WASP抗体（Southern Biotech. Inc），其表位为WASP的N端，从未检测到双阳性峰。为了研究WASP信息量或蛋白水平的差异，我们纯化了CD 3 +/CD 45 RA+细胞和CD 3 +/CD 45 RO+细胞，然后进行RT-PCR和Western印迹分析。结果表明，这些亚群细胞之间的WASP信息或蛋白水平的任何差异。最近，WASP的分子间构象变化，活性和非活性形式的报道。我们推测，正常人外周血淋巴细胞中WASP的双阳性峰可以用这种情况来解释。为了证明这一假设，以下实验正在进行中。我们构建了具有非常独特的WASP突变体L279 P的逆转录病毒载体构建体，该突变体被报道为WASP的组成型活性形式。我们正在计划用FCM-WASP分析含有突变型WASP或野生型WASP的载体的转导实验。也有报道称活化的WASP被募集到脂筏，重组细胞膜功能单位。使用共聚焦显微镜分析，我们计划分析WASP在属于WASP^和WASP^的两种细胞中的位置<high-bright><low-bright>。少

The Wiskott-Aldrich syndrome protein (WASP), which is defect in Wiskott-Aldrich syndrome (WAS) patients, is intracellular protein expressed in non-erythroid hematopoietic cells. Recent studies revealed that WASP interacts with numbers of intracellular molecules and plays key roles in the signal transduction and the regulation of actin polymerization, although varied clinical symptoms observed in WAS patients has not been fully elucidated from a viewpoint of WASP-deficiency.We have established the methods to detect intracellular WASP by flow cytometry (FCM-WASP) and revealed that WAS patients showed null/very low level of intracellular WASP of lymphocytes/monocytes by FCM-WASP studies, while significant amount of WASP was detected in normal individuals. We applied the methods for the screening for WAS patient and WAS carrier, and the evaluation of the mixed chimera status in WAS patients who underwent hematopoietic stem cell transplantation. In addition, during the course of a WAS scree … More ning using FCM-WASP, we happened to find a patient with WAS who possessed a small population of lymphocytes in which a spontaneous reversion of the inherited WASP gene mutation had taken place.During these FCM-WASP studies, we have noticed that lymphocytes of control individuals showed double or a broad positive peak while those monocytes invariably showed a sharp positive peak. To study the basis for the double positive peaks (WASP^<high-bright> and WASP^<low-bright>) of control lymphocytes detected by FCM-WASP, we tried to characterize the two populations. By double/triple staining FCM-WASP methods, it was revealed following results. T cells, both CD4+ and CD8+ cells are composed of both WASP^<high-bright> and WASP^<low-bright> cells. Most of B cells (CD20+) are WASP^<low-bright> cells, most of NK cells (CD56+) are WASP^<high-bright> cells. Further characterization revealed that CD45RA+ of either CD4+ or CD8+ cells belong to WASP^<low-bright> and CD45RO+ cells of either CD4+ or CD8+ cells belong to WASP^<high-bright> cells. These results were obtained by using a-WASP antibody 3F3A5 (provided by Dr.Nelson DL : NIH/USA), whose epitopes are middle part of WASP. In case of using another a-WASP antibody (Southern Biotech. Inc), whose epitopes are N-terminal of WASP, no double positive peaks were never detected. To study any difference in quantity of WASP message or protein level, we purely purified CD3+/CD45RA+cells and CD3+/CD45RO+ cells, and then RT-PCR and Western blotting analysis were performed. The results revealed any difference in WASP message or protein level between these subpopulation cells.Recently, the intermolecular conformational change of WASP ; active and inactive form was reported. We presumed that double positive peak of WASP observed in normal individual lymphocytes by FCM-WASP could be explained by this scenario. To prove the hypothesis, following experiments are in progress. We constructed retrovirus vector construct with very unique WASP mutatnt of L279P, which was reported as constitutively active form of WASP. We are planning transduction experiment of vector containing the mutant WASP or wild WASP to analyze using FCM-WASP. It was also reported that activated WASP is recruited to lipid raft, reorganized cell membrane functional unit. Using con-focal microscopic analysis, we are planning to analyze location of WASP in both cells belong to WASP^<high-bright> and WASP^<low-bright>. Less