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

CFTR 离子传输缺陷对粘蛋白唾液酸化的作用及其对粘液生理学的影响

基本信息

批准号：
10734036
负责人：
Elex S Harris
金额：
$ 2.23万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10734036
关键词：
Affect Airway Disease Anions Apical Bicarbonates Biochemical Biogenesis Cell surface Cells Centrifugation Characteristics Charge Chlorides Chronic lung disease Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Disease Down-Regulation Electrostatics Environment Epithelial Cells Epithelium Functional disorder Gel Gene Silencing Genes Genetic Diseases Gland Human Hydration status Impairment In Vitro Infection Ion Channel Ion Transport Ions Lead Lectin Link Literature Lung MUC5AC gene MUC5B gene Mass Spectrum Analysis Messenger RNA Modeling Molecular Conformation Mucins Mucociliary Clearance Mucous body substance Mutation Normal Cell Optical Coherence Tomography Organ Pathologic Physiological Physiology Plasmids Polysaccharides Property Proteins Rattus Regulator Genes Research Training Respiratory System Role Serine Sialic Acids Sialyltransferases Small Interfering RNA Structural defect Structure Submucosa Surface System Testing Threonine Trachea Transmission Electron Microscopy Viscosity Western Blotting Work bronchial epithelium chronic infection cystic fibrosis mucus density disease-causing mutation gel electrophoresis glycosylation hydroxyl group improved in vivo in vivo Model inhibitor intermolecular interaction lung injury migration mortality mucus-associated lung diseases new therapeutic target novel overexpression pharmacologic sialylation targeted treatment translational potential

项目摘要

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.

项目概要囊性纤维化（CF）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)，负责传输氯离子和碳酸氢盐的离子通道穿过顶端细胞表面。 CF 影响多个器官，在肺部，会导致粘液瘀滞、慢性感染/肺损伤和死亡率。粘液停滞是 CF 的病理标志，许多凝胶形成物质气道粘液的特性由凝胶形成粘蛋白 MUC5B 和 MUC5AC 提供。这些凝胶形成粘蛋白被广泛的 O-连接糖基化，这些聚糖的末端唾液酸化有助于其负电荷以及与 CFTR 建立的离子微环境的相互作用。 O-连接的变化因此，凝胶形成粘蛋白的唾液酸化预计会改变其理化特性。早期证据表明，有缺陷的 CFTR 可以减少粘蛋白唾液酸化，然而，其机制基础其与 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尚未得到证实。决定。我们的初步数据表明，CF 原代细胞中唾液酸转移酶表达下调人支气管上皮细胞，在体外和体内抑制唾液酸化会导致粘液纤毛运输与粘液水合作用无关。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 CFTR 离子受损运输下调粘蛋白的唾液酸化，从而减少其负电荷，导致粘蛋白 CF 的结构异常和粘液淤滞。为了测试这一点，我们制定了两个具体目标。在目标 1 中，我们将通过药理学抑制或确定 CFTR 阴离子转运对粘蛋白唾液酸化的影响恢复体外和体内阴离子转运，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整体或特定唾液酸转移酶活性并评估 MUC5B 或 MUC5AC 静电特性、构象和生理学来定义改变的后果凝胶形成气道粘蛋白的唾液酸化。这些研究将通过揭示一种新的机制来推进该领域的发展 CF 中的粘液瘀滞，同时确定治疗 CF 和可能的其他疾病的新治疗靶点粘液瘀滞。