Regulation of Mast Cell Proteases

小鼠肥大细胞(MC)分泌颗粒的几乎全部重量由16种中性蛋白酶组成，它们与丝氨酸蛋白多糖(SGPG)离子结合，其中含有肝素(HP)或高度硫酸盐化的软骨素(CHS)链，如CHS-E。我们和其他人已经证明，这些蛋白酶/SGPG复合体中的一些在先天性和获得性免疫中发挥着有益的作用。例如，小鼠MC蛋白水解酶(MMCP)-6/HP复合体在肺部起到抗菌作用。 然而，现在很清楚，这些复合体中的一些参与了MC介导的临床疾病的病理过程。例如，胞外释放的mMCP-5/HP复合体在后肢缺血-再灌注缺氧损伤模型中引起肌肉横纹肌溶解。只有正确折叠的蛋白水解酶储存在颗粒中的翻译后机制，以及MC将相关蛋白水解酶的自溶降至最低的机制尚不清楚。根据细胞因子的不同，小鼠和人的巨噬细胞可将不同类型的糖胺多聚糖(GAG)合成到丝氨酸上。幽门螺杆菌显著改变类胰蛋白酶mMCP-6的底物专一性的发现增加了CHS-E对其他MC类胰蛋白酶和/或糜酶起类似调节作用的可能性。总的目标是 项目1是利用互补的生物化学、免疫学、细胞生物学、结晶学和分子生物学的方法来推断SGPG及其独特的GAG是如何控制不同MC颗粒蛋白水解酶的表达、颗粒储存、酶活性和细胞外代谢的。在特定的目标1中，我们将在存在和不存在MC衍生的GAG的情况下，用重组MC蛋白酶筛选我们的噬菌体展示肽库，以确定连接到SGPG的碳水化合物链的类型是否差异地改变了每个所研究的蛋白酶的酶活性。将研究SGPG在调节MC蛋白酶胞外代谢中的潜在作用，包括蛇毒和其他自然产生的蛋白酶抑制剂使其失活。我们还将确定与其首选的GAG络合的人MC类胰蛋白酶Beta1的晶体结构。在特定目标2中，我们将评估我们的HP和mMCP-5基因缺失小鼠(以及新创建的mMCP-6和CHS-E基因缺失小鼠)的能力 以对抗细菌和蠕虫感染。这些转基因小鼠也将被用来确定MC蛋白酶/SGPG复合体在基线和抗原诱导的气道反应性、纤维化和重塑中的作用。

Nearly the entire weight of a mouse mast cell's (MC's) secretory granules consists of 16 neutral proteases ionically bound to serglycin proteoglycans (SGPGs) that contain either heparin (HP) or highly sulfated chondroitin (ChS) chains such as ChS-E. We and others have shown that some of these protease/SGPG complexes play beneficial roles in innate and acquired immunity. For example, mouse MC protease (mMCP)-6/HP complexes play an anti-bacterial role in the lung. However, it is now clear that some of these complexes contribute to the pathology that occurs in MC-mediated clinical disorders. For example, exocytosed mMCP-5/HP complexes cause muscle rhabdomyolysis in a hindlimb ischemia-reperfusion model of hypoxia injury. The post-translational mechanism by which only properly folded proteases are stored in the granules and the mechanism by which MCs minimize autolysis of their associated proteases are unknown. Depending on the cytokine microenvironment, mouse and human MCs synthesize different types of glycosaminoglycans (GAGs) onto serglycin. The finding that HP dramatically alters the substrate specificity of the tryptase mMCP-6 raises the possibility that ChS-E plays a similar regulatory role for other MC tryptases and/or chymases. The overall objective of Project 1 is to use complementary biochemistry, immunology, cell biology, crystallographic, and molecular biology approaches to deduce how SGPGs and their unique GAGs control the expression, granule storage, enzymatic activity, and extracellular metabolism of the different MC granule proteases. In Specific Aim 1, we will screen our phage-display peptide libraries with recombinant MC proteases in the presence and absence of MC-derived GAGs to determine if the type of carbohydrate chain attached to a SGPG differentially alters the enzymatic activity of each investigated protease. The potential roles of SGPGs in the regulation of the extracellular metabolism of MC proteases, including their inactivation by serpins and other naturally occurring protease inhibitors will be investigated. We also will determine the crystal structure of human MC tryptase beta1 complexed to it's preferred GAG. In Specific Aim 2, we will evaluate the ability of our HP- and mMCP-5-null mice (as well as newly created mMCP-6- and ChS-E-null mice) to combat bacteria and helminth infections. These transgenic mice also will be use to ascertain the role of MC protease/SGPG complexes in baseline and antigen-induced airway reactivity, fibrosis, and remodeling.