Biochemical Role of Airway Mucins in Cystic Fibrosis

描述(申请人提供)：囊性纤维化(CF)是最致命的 高加索人的遗传病，以过量生产为特征 病人呼吸道内粘性粘液分泌量。这会导致 呼吸道阻塞和慢性细菌感染，最终 导致呼吸衰竭。粘蛋白通过以下途径为上皮细胞提供保护 它们的糖类与细菌粘附素的相互作用。慢性殖民 铜绿假单胞菌被认为是慢性阻塞性肺疾病的主要死亡原因 病人。我们的实验室和其他实验室已经表明，铜绿假单胞菌 与正常气道相比，CF型呼吸道粘蛋白的结合亲和力强得多 粘液。这些观察结果暗示了CF粘蛋白糖基化的改变。的确， 已有关于CF粘蛋白异常糖基化的报道。然而，到目前为止， 铜绿假单胞菌与CF呼吸道相互作用增强的分子基础 粘蛋白还没有建立起来。我们假设改变了Cf的糖基化 粘蛋白是其与铜绿假单胞菌结合较强的原因。我们会 确定CF粘蛋白碳水化合物配体(S)的结构特征 通过制备糖肽和增加对铜绿假单胞菌的结合 从CF和对照粘蛋白中提取的单糖。糖肽(S) 显示对asialo-GM的高度抑制；与铜绿假单胞菌的结合将用于 分离0-连接的多聚糖用于进一步测试抑制活性和 使用最先进的高灵敏质量确定结构 光谱分析和酶促方法。含有选定粘蛋白的亲和凝胶 将用糖肽或粘蛋白糖纯化铜绿假单胞菌。 分别与呼吸道粘蛋白和糖脂相互作用的粘附素。这个 主要粘附素的一级结构将用分子方法确定 克隆技术。主要粘附素的结构表征将打开 防止铜绿假单胞菌与呼吸道结合的其他方法 CF患者的上皮细胞和粘蛋白。有关粘附素结合的信息 网站将允许分子建模，设计和合成有效的0-聚糖 铜绿假单胞菌感染的抑制剂。它的总体长期目标是 这项研究是为了预防和/或治疗CF患者的肺部感染。

DESCRIPTION (provided by applicant): Cystic Fibrosis (CF) is the most lethal genetic disease in Caucasians and is characterized by production of excessive amounts of viscous mucus secretions in the airways of the patient. This causes airway obstruction as well as chronic bacterial infections which eventually lead to respiratory failure. Mucins provide protection to epithelia through interaction of their saccharides with bacterial adhesins. Chronic colonization with Pseudomonas aeruginosa, is considered the principal cause of death in CF patients. Our laboratory and others have shown that P. aeruginosa had considerably stronger binding affinity for CF airway mucin than normal airway mucin. These observations implicate altered glycosylation of CF mucins. Indeed, aberrant glycosylation has been reported for CF mucin. However, to date, the molecular basis of increased interaction between P. aeruginosa and CF airway mucin has not been established. We hypothesize that altered glycosylation of CF mucin is responsible for its stronger binding with P. aeruginosa. We will determine structural features of the CF mucin carbohydrate ligand(s) that provide increased binding to P. aeruginosa by preparing glycopeptides and individual saccharides from CF and control mucins. The glycopeptide(s) which show high inhibition of asialo-GM; binding to P. aeruginosa will be used to isolate 0-linked glycans for further testing of inhibitory activity and structural determination using state-of-the-art highly sensitive mass spectrometry and enzymnatic methods. Affinity gels containing selected mucin glycopeptide or mucin saccharide will be used to purify the P. aeruginosa adhesins which interact with airway mucins and glycolipids, respectively. The primary structure of the major adhesins will be determined using molecular cloning techniques. Structural characterization of major adhesins will open additional approaches to prevent the binding of P. aeruginosa to airway epithelial cells and mucins of CF patients. Information on the adhesin binding sites will permit molecular modeling, design and synthesis of potent 0-glycan inhibitors of the P. aeruginosa infection. The overall long-term goal of this study is to prevent and/or treat lung infections in CF patients.