The Role of Defective CFTR Ion Transport on Mucin Sialylation and its Consequences on Mucus Physiology

项目总结 囊性纤维化是一种由囊性纤维化跨膜突变引起的遗传性疾病。 电导调节器(CFTR)，负责氯离子和碳酸氢盐运输的离子通道 穿过顶端细胞表面。慢性萎缩性胃炎影响多个器官，在肺部，导致粘液淤积，慢性 感染/肺损伤和死亡率。粘液淤积是CF的一个病理标志，许多形成凝胶的 呼吸道粘液的特性由凝胶形成的粘蛋白MUC5B和MUC5AC提供。这些凝胶的形成 粘蛋白是广泛的O-连接糖基化的，这些多糖的末端唾液酸化有助于它们的 负电荷和与CFTR建立的离子微环境的相互作用。O-连接的变化 因此，凝胶化粘蛋白的唾液酸化作用有望改变它们的物理化学性质。 早期证据表明，有缺陷的cftr可以减少粘蛋白唾液酸化，然而， 这一点，它与CFTR负离子转运受损的关系，以及它对粘液功能的影响尚不清楚。 此外，在多种呼吸道疾病中，低电荷粘蛋白糖型与病理性粘液有关， 但唾液酸化改变对粘蛋白结构和粘液生理的影响尚未得到证实。 下定决心。我们的初步数据显示唾液酸基转移酶在原发灶中的表达下调 体外和体内抑制唾液酸化可导致血管内皮细胞损伤 粘液纤毛运输不依赖粘液水合作用。通过这项拟议的研究，我们将确定 CFTR阴离子转运对粘蛋白唾液酸化的影响及粘蛋白改变对功能的影响 唾液酸化对粘蛋白结构和粘液转运的影响。我们的主要假设是受损的CFTR离子 转运下调了粘蛋白的唾液酸化，从而减少了它们的负电荷，从而产生了粘蛋白 CF的结构异常和粘液淤积。为了测试这一点，我们制定了两个具体目标。在目标1中， 我们将通过药物抑制OR来确定CFTR阴离子转运对粘蛋白唾液酸化的影响。 在体外和体内恢复阴离子转运，然后通过定量评估唾液酸化的变化 唾液酸转移酶表达、唾液酸转移酶蛋白水平和粘蛋白唾液酸化。在目标2中，我们将直接 下调或上调全局或特定唾液酸转移酶活性，并评估MUC5B或 MUC5AC的静电特性、构象和生理学，以定义改变的后果 在形成呼吸道粘蛋白的凝胶上进行唾液分析。这些研究将通过揭示一种新的机制来推动该领域的发展 CF的粘液淤积，同时确定治疗CF和可能的其他疾病的新的治疗靶点 粘液淤积。

PROJECT SUMMARY Cystic Fibrosis (CF) is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an ion channel responsible for the transport of chloride and bicarbonate across the apical cell surface. CF affects multiple organs, and in the lungs, results in mucus stasis, chronic infection/lung damage, and mortality. Mucus stasis is a pathologic hallmark of CF, and many of the gel-forming properties of airway mucus are provided by the gel-forming mucins, MUC5B and MUC5AC. These gel-forming mucins are extensively O-linked glycosylated, and terminal sialylation of these glycans contributes to their negative charges and interaction with the ionic microenvironment established by CFTR. Changes in O-linked sialylation of gel-forming mucins would therefore be expected to alter their physiochemical characteristics. Early evidence shows that defective CFTR can reduce mucin sialylation, however, the mechanistic basis for this, its relationship to impaired CFTR anion transport, and its consequences on mucus function are unknown. Furthermore, low charged mucin glycoforms have been linked to pathologic mucus in multiple airway diseases, but the consequences of altered sialylation on mucin structure and mucus physiology have not been determined. Our preliminary data shows that sialyltransferase expression is downregulated in CF primary human bronchial epithelial cells, and that inhibiting sialylation in vitro and in vivo leads to impairment of mucociliary transport independent of mucus hydration. Through this proposed study, we will identify the role of CFTR anion transport on mucin sialylation and determine the functional consequences of altered mucin sialylation on mucin structure and mucus transport. Our overarching hypothesis is that impaired CFTR ion transport downregulates the sialylation of mucins, which decreases their negative charge, resulting in mucin structural abnormalities and mucus stasis in CF. To test this, we have developed two specific aims. In aim 1, we will determine the effect of CFTR anion transport on mucin sialylation by pharmacologically inhibiting or restoring anion transport in vitro and in vivo, followed by assessing changes in sialylation by quantifying sialyltransferase expression, sialyltransferase protein levels, and mucin sialylation. In aim 2, we will directly downregulate or upregulate global or specific sialyltransferase activity and assess the changes in MUC5B or MUC5AC electrostatic properties, conformation, and physiology to define the consequences of altered sialylation on gel forming airway mucins. These studies will advance the field by revealing a new mechanism of mucus stasis in CF, while identifying novel therapeutic targets for treatment of CF and possibly other diseases of mucus stasis.