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

• 批准号: 10464260
• 负责人: Elex S Harris
• 金额: $ 3.8万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10464260
• 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; Epithelial Cells; 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; Pharmacology; Physiological; Physiology; Plasmids; Polysaccharides; Property; Proteins; Rattus; Regulator Genes; Research Training; Respiratory System; Role; Serine; Sialic Acids; Sialyltransferases; Small Interfering RNA; Structural defect; Structure; Surface; System; Testing; Threonine; Trachea; Transmission Electron Microscopy; 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; new therapeutic target; novel; overexpression; sialylation; targeted treatment; translational potential

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.

项目摘要 囊性纤维化（CF）是由囊性纤维化跨膜突变引起的遗传疾病 电导调节剂（CFTR），该通道负责氯化物和碳酸氢盐的运输 穿过顶端细胞表面。 CF影响多个器官，在肺中会导致粘液停滞，慢性 感染/肺部损害和死亡率。粘液停滞是CF的病理标志，许多凝胶形成 气道粘液的特性由凝胶形成的粘蛋白MUC5B和MUC5AC提供。这些凝胶形成 粘蛋白是广泛的O连接糖基化的，这些聚糖的末端溶性有助于它们 负电荷和与CFTR建立的离子微环境相互作用。 O链接的变化 因此，预计将凝胶粘蛋白合同改变其生理化学特征。 早期的证据表明，有缺陷的CFTR可以减少粘蛋白溶性酶，但是，是机械基础 这是其与CFTR阴离子运输受损的关系及其对粘液功能的后果尚不清楚。 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， 但是，溶解酶对粘蛋白结构和粘液生理学的后果尚未 决定。我们的初步数据表明，CF主要的CF中乙烯基转移酶的表达被下调 人支气管上皮细胞，并且在体外抑制溶酶和体内会导致损害 与粘液保湿无关的粘膜钙转运。通过这项拟议的研究，我们将确定 CFTR阴离子在粘蛋白溶解上转运并确定粘蛋白改变的功能后果 对粘蛋白结构和粘液转运的溶解度。我们的总体假设是CFTR离子受损 转运下调粘蛋白的溶解度，从而降低其负电荷，导致粘蛋白 cf中的结构异常和粘液暂停。为了测试这一点，我们开发了两个具体目标。在AIM 1中， 我们将通过药理学抑制或 在体外和体内恢复阴离子的转运，然后通过量化评估溶解度的变化 乙烯基转移酶的表达，乙烯基转移酶蛋白水平和粘蛋白苷。在AIM 2中，我们将直接 下调或上调全球或特定的急证酶活性，并评估MUC5B或 MUC5AC静电特性，构象和生理学，以定义改变的后果 凝胶形成气道粘蛋白的溶解度。这些研究将通过揭示一种新的机制来推进现场 CF中的粘液停滞，同时识别用于治疗CF和可能其他疾病的新型治疗靶标 粘液停滞。