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

项目摘要 囊性纤维化（CF）是一种遗传性疾病，由囊性纤维化跨膜突变引起。 电导调节器（CFTR），一种负责氯离子和碳酸氢根转运的离子通道 穿过顶端细胞表面。CF影响多个器官，并且在肺中，导致粘液淤滞，慢性 感染/肺损伤和死亡率。粘液淤滞是CF的病理标志，并且许多凝胶形成 气道粘液的性质由形成凝胶的粘蛋白MUC 5 B和MUC 5AC提供。这些凝胶形成 粘蛋白是广泛的O-连接的糖基化，这些聚糖的末端唾液酸化有助于它们的糖基化。 负电荷和与CFTR建立的离子微环境的相互作用。O-linked的变化 因此，预期形成凝胶的粘蛋白的唾液酸化会改变它们的生理化学特性。 早期的证据表明，有缺陷的CFTR可以减少粘蛋白唾液酸化，然而， 其与CFTR阴离子转运受损的关系及其对粘液功能的影响尚不清楚。 此外，低电荷粘蛋白糖型与多种气道疾病中的病理性粘液有关， 但唾液酸化改变对粘蛋白结构和粘液生理学的影响还没有被证实。 测定我们的初步数据表明，唾液酸转移酶表达下调CF原发性 人支气管上皮细胞，并且在体外和体内抑制唾液酸化导致 不依赖于粘液水合作用的粘液纤毛运输。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，并确定改变的粘蛋白的功能后果 唾液酸化对粘蛋白结构和粘液运输的影响。我们的首要假设是受损的CFTR离子 转运下调粘蛋白的唾液酸化，这减少了它们的负电荷，导致粘蛋白 CF结构异常和粘液淤滞。为了验证这一点，我们制定了两个具体目标。在目标1中， 我们将确定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，然后通过定量测定唾液酸化的变化来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 下调或上调总体或特异性唾液酸转移酶活性，并评估MUC 5 B或 MUC 5AC静电性质、构象和生理学以定义改变的MUC 5AC的结果。 形成气道粘蛋白的凝胶上的唾液酸化。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中的粘液淤滞，同时确定用于治疗CF和可能的其他疾病的新的治疗靶点 粘液淤滞。

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.