DISRUPTION AND EXPRESSION OF MAST CELL PROTEASE GENES

颗粒蛋白水解酶，代表肥大的主要蛋白质成分 细胞，已被用来鉴定不同的表型群体 纸巾。然而，人们对它们的功能和功能知之甚少 或者它们是如何在桅杆内外代谢的 手机。由于细胞因子/因子依赖的调节，小鼠肥大 细胞表达至少七种丝氨酸蛋白酶的不同组合 [指定小鼠肥大细胞蛋白水解酶(MMCP)1至7]和外肽酶 [指定的小鼠肥大细胞羧肽酶A(MMC-CPA)] 在中性pH条件下具有酶活性。据推测，病毒的数量和类型 每个小鼠肥大细胞表达的蛋白水解酶与数量和 这些效应细胞必须降解或激活的蛋白质类型 不同的组织环境。尽管许多MMCP高度 彼此同源，每种蛋白酶都有自己独特的一套 底物结合缝隙中的氨基酸。而离散函数 似乎很有可能，体内每个肥大细胞蛋白酶的特定底物 而整个超级家族之间的关系仍有待阐明。基因的克隆 现在编码这些小鼠肥大细胞颗粒蛋白酶的cDNA和基因 允许在此项目中使用两种互补的方法来解决 肥大细胞分泌颗粒的代谢和功能 蛋白酶。在特定的目标1中，14号染色体复合体中肥大 细胞糜酶(mMCP-1至mMCP-5)基因将被定位。转基因 将产生具有MMC-CPA、mMCP-7、mMCP-2和 可能是其他的乳糜酶基因被破坏了。烧蚀的后果 这些基因对转基因动物肥大细胞发育的影响 将通过形态测量和免疫组织化学分析进行评估。 功能效果的评估将通过确定桅杆是否 特定蛋白水解酶缺失的转基因动物素体中的细胞激活 对特定激动剂的增强反应的呼吸道，就像野生的 典型的动物。在特定目标2中，“亲”和“成熟”小鼠肥大细胞 蛋白水解酶将在昆虫细胞和大鼠嗜碱性白血病中表达 细胞。这些重组的蛋白酶将被提纯，然后用于 评估肥大细胞蛋白水解酶是如何被酶激活的 颗粒，在体外检测它们的底物特异性，并 确定它们在体外和体内代谢的机制 活着。

Granule proteases, which represent the major protein constituents of mast cells, have been used to identify distinct phenotypic populations in tissues. However, little is known about their functions, how they are activated, or how they are metabolized inside and outside of the mast cell. As a result of cytokine/factor-dependent regulation, mouse mast cells express varied combinations of at least seven serine proteases [designated mouse mast cell protease (mMCP) 1 to 7] and an exopeptidase [designated mouse mast cell carboxypeptidase A (mMC-CPA)] that are enzymatically active at neutral pH. Presumably, the number and type of proteases each mouse mast cell expresses are related to the number and type of proteins these effector cells must degrade or activate in their different tissue environments. Although many of the mMCPs are highly homologous with one another, each protease has its own unique set of amino acids in its substrate-binding cleft. While discrete functions seem likely, the specific substrates of each mast cell protease within and across superfamilies remain to be elucidated. The cloning of the cDNAs and genes that encode these mouse mast cell granule proteases now allows the use of two complementary approaches in this Project to address the metabolism and function of the mast cell's secretory granule proteases. In Specific Aim 1, the chromosome 14 complex where the mast cell chymase (mMCP-1 to mMCP-5) genes reside will be mapped. Transgenic mice will be generated that have the mMC-CPA, mMCP-7, mMCP-2, and possibly other chymase genes disrupted. The consequences of ablating such genes on the development of mast cells in the transgenic animals will be assessed by morphometric and immunohistochemical analyses. Functional effects will be assessed by determining whether or not mast cell activation in a particular protease-null, transgenic animal primes the airways for augmented response to a specific agonist as does a wild type animal. In Specific Aim 2, "pro" and "mature" mouse mast cell proteases will be expressed in insect cells and rat basophilic leukemia cells. These recombinant proteases will be purified and then used to assess how mast cell proteases are enzymatically activated in the granule, to examine their substrate specificities in vitro, and to determine the mechanisms by which they are metabolized in vitro and in vivo.