Structural and functional studies of WASP molecule in cellular basis

Wiskott-Aldrich综合征蛋白（WASP）是一种在非红系造血细胞中表达的细胞内蛋白，是WAS患者的缺陷蛋白。最近的研究表明，WASP与许多细胞内分子相互作用，在信号转导和肌动蛋白聚合调节中发挥关键作用，尽管从WASP缺乏的角度尚未完全阐明WAS患者观察到的各种临床症状。我们建立了流式细胞术（FCM-WASP）检测细胞内WASP的方法，通过FCM-WASP研究发现WAS患者淋巴细胞/单核细胞内WASP为零/极低水平，而正常人则检测到大量的WASP。我们将该方法应用于WAS患者和WAS携带者的筛选，以及WAS患者进行造血干细胞移植后混合嵌合体状态的评估。此外，在使用FCM-WASP进行WAS筛查的过程中，我们碰巧发现了一位WAS患者，他拥有少量淋巴细胞，其中遗传的WASP基因突变发生了自发的逆转。在这些FCM-WASP研究中，我们注意到对照个体的淋巴细胞呈双峰或宽峰阳性，而那些单核细胞总是呈尖峰阳性。为了研究FCM-WASP检测对照淋巴细胞出现双阳性峰（WASP^<高亮>和WASP^<低亮>）的依据，我们试图对这两个群体进行表征。FCM-WASP法双/三次染色，结果如下：T细胞CD4+和CD8+细胞均由WASP^<高亮度>和WASP^<低亮度>细胞组成。多数B细胞（CD20+）为WASP^<低亮>细胞，多数NK细胞（CD56+）为WASP^<高亮>细胞。进一步鉴定发现CD4+和CD8+细胞的CD45RA+细胞属于WASP^<低亮>细胞，CD4+和CD8+细胞的CD45RO+细胞属于WASP^<高亮>细胞。这些结果是通过a-WASP抗体3F3A5（由Dr.Nelson DL: NIH/USA提供）获得的，其表位为WASP的中间部分。如果使用另一种a-WASP抗体（Southern Biotech）。Inc)，其表位为WASP的n端，未检测到双阳性峰。为了研究WASP信息数量和蛋白水平的差异，我们对CD3+/CD45RA+细胞和CD3+/CD45RO+细胞进行纯纯化，然后进行RT-PCR和Western blotting分析。结果显示这些亚群细胞在WASP信息或蛋白水平上没有差异。近年来，WASP的分子间构象变化；报告了活性和非活性形式。我们推测FCM-WASP在正常个体淋巴细胞中观察到的WASP双阳性峰可以用这种情况来解释。为了证明这一假设，下面的实验正在进行中。我们构建了具有非常独特的L279P WASP突变体的逆转录病毒载体，该突变体被报道为WASP的组成活性形式。我们正在计划用FCM-WASP对含有突变型或野生型WASP的载体进行转导实验。活化后的WASP被招募到脂质筏中，重组细胞膜功能单元。利用共焦显微镜分析，我们计划分析WASP在WASP^<高亮>和WASP^<低亮>两个细胞中的位置。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