EOSINOPHIL AND BASOPHIL GRANULE PROTEINS

描述：嗜酸性粒细胞和嗜碱性粒细胞现在被认为是关键细胞 在哮喘和其他超敏反应的病理生理学中的作用 疾病。因为它们有很强的生物活性，阳离子 嗜酸性粒细胞颗粒蛋白是嗜酸性粒细胞的中介物 功能。然而，关于嗜碱性粒细胞的信息很少。 颗粒蛋白。在这项资助下进行的先前研究 表征了主要颗粒蛋白的结构和功能 包括主要碱性蛋白(MBP)、嗜酸性粒细胞过氧化物酶(EPO)、 嗜酸性粒细胞衍生神经毒素(EDN)和嗜酸性粒细胞阳离子蛋白(ECP)。 在这里，我们确定了三条新的调查路线。首先，生产MBP 作为前分子(ProMBP)，转化为14 kDa MBP。初步 实验表明，组织蛋白酶L具有前肌动蛋白转换活性。 找出验证这一假设的实验。第二，立体化 14 kDa MBP和proMBP的结构将由X射线确定 结晶学。14 kDa的MBP已经结晶，一个 获得了2.8埃的原始数据集。然而，14 kDa MBP 晶体通常是孪晶的，可能有必要表达和 为了解决14 kDa MBP的结构问题，对proMBP进行了结晶。第三, 描述了鉴定新的嗜碱性粒细胞蛋白质的实验。 嗜碱性白血病患者的嗜碱性细胞通过以下方法获得 胞核分离和颗粒分离。高效液相色谱仪 颗粒裂解物显示存在大量蛋白质和 获得了8个部分序列。我们建议刺激脐带血 脐带单个核细胞分化为嗜酸性粒细胞/嗜碱性粒细胞杂交细胞和 从这些杂交种中产生一个cdna文库。将对混合动力车进行比较 IL-5刺激的脐带细胞的差异显示分析和 通过对随机选择的cDNA克隆进行测序。使用退化 差异显示的寡核苷酸和新DNA序列 对随机选择的克隆进行测序，我们将鉴定全长cdna 克隆和表达新的嗜碱性细胞蛋白，目的是生产 嗜碱性细胞的特异性抗体。总体而言，这些研究将提供 关于proMBP转化为 MBP，嗜酸性粒细胞颗粒蛋白的三维结构和 存在新的嗜碱性粒细胞相关蛋白。

DESCRIPTION: Eosinophils and basophils are now recognized as critical cells in the pathophysiology of bronchial asthma and other hypersensitivity diseases. Because of their potent biological activities, cationic eosinophil granule proteins are implicated as mediators of eosinophil functions. However, little information is available regarding basophil granule proteins. Prior studies under the auspices of this grant characterized the structure and function of the principal granule proteins including the major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Here, we identify three new lines of investigation. First, MBP is produced as a pro-molecule (proMBP) and converted to 14 kDa MBP. Preliminary experiments indicate that cathepsin L has proMBP converting activity, and we identify experiments to test this hypothesis. Second, the three-dimensional structures of 14 kDa MBP and proMBP will be determined by X-ray crystallography. The 14 kDa MBP have already been crystallized, and a native dataset at 2.8 angstrom has been obtained. However, 14 kDa MBP crystals are often twinned, and it may be necessary to express and crystallize proMBP in order to solve the structure of 14 kDa MBP. Third, experiments to identify novel basophil granule proteins are described. Basophils from a patient with basophilic leukemia were obtained by cytapheresis and granules isolated. High performance liquid chromatography of granule lysates revealed the presence of numerous proteins and the partial sequences of eight were obtained. We propose to stimulate umbilical cord mononuclear cells to differentiate to eosinophil/basophil hybrids and to produce a cDNA library from these hybrids. The hybrids will be compared to IL-5 stimulated umbilical cord cells by differential display analyses and by sequencing of randomly selected cDNA clones. Using degenerate oligonucleotides and novel DNA sequences from differential display and from sequencing of randomly selected clones, we will identify full-length cDNA clones and express the novel basophil proteins with the goal of producing antibody specific for the basophil. Overall, these studies will provide important new information regarding the mechanism of conversion of proMBP to MBP, the three dimensional structure of eosinophil granule proteins and the existence of novel basophil-associated proteins.