Role of Mucin in Lung Homeostasis and Pathophysiology

描述（由申请人提供）：哮喘对人类健康和经济有重大影响。过多的粘液是致命性哮喘中气流阻塞的重要原因。它也存在于轻度至中度疾病中，但对其了解和治疗甚少。哮喘中粘液的过度产生与两种粘蛋白糖蛋白MUC 5AC和MUC 5 B的表达失调有关。MUC 5AC增加是一致的发现，但MUC 5 B变化。它在一些患者中保持稳定产生，但在其他患者中被强烈抑制（>90%），导致MUC 5AC比MUC 5 B多400倍。这种二分表达模式发生在对吸入性支气管收缩剂乙酰甲胆碱（MCh）表现出最强急性反应的患者中。这种气道高反应性（AHR）特征可以通过过敏原暴露在小鼠中建模。缺乏Muc 5ac的敲除小鼠免受AHR的影响，表明它是哮喘样气流阻塞的关键介质。因此，确定MUC 5AC介导的AHR的特定机制可能揭示阻塞的重要机制和改善哮喘气流的潜在靶点。作为阐明MUC 5AC和MUC 5 B在气道中的功能的长期目标的一部分，Muc 5ac和Muc 5 b敲除和过表达小鼠将用于模拟人哮喘中的二分粘蛋白表达，并测试每种粘蛋白对促进阻塞的粘液的生物物理性质的特定作用。除了它们的差异表达之外，新出现的数据显示MUC 5AC和MUC 5 B具有不同的二硫键聚合物结构。它们还具有特定的糖基化模式：MUC 5 B是唾液酸化的，MUC 5AC是岩藻糖基化的。岩藻糖基化增加粘液粘弹性，而催化粘蛋白1，2-岩藻糖基化的酶FUT 2与哮喘急性发作风险相关。因此，推动这一提议的概念是具有不同聚合物和聚糖结构的粘蛋白建立了气道粘液的阻塞潜力。因此，所提出的研究检验了MUC 5AC通过特定的二硫化物和岩藻糖基化机制介导AHR的假设，所述机制通过促进粘液粘弹性引起阻塞。提出了三个具体目的：1）证明MUC 5AC而不是MUC 5 B介导过敏小鼠气道中的急性AHR; 2）确定粘液粘弹性增加是否由MUC 5AC特异性聚合和岩藻糖基化引起; 3）确定MUC 5AC二硫化物和岩藻糖基化依赖性粘弹性性质是否是AHR和粘液堵塞的关键机制。将在基线时以及在由黄芪提取物暴露诱导的过敏性炎症条件下研究野生型和粘蛋白突变小鼠。将测量肺功能、粘液纤毛清除率和粘液粘弹性。将确定聚合和岩藻糖基化的药理学、酶和遗传抑制作用。这些研究的成功完成将促进我们对AHR机制的理解，并可以确定预防阻塞的新策略，同时保留哮喘和其他肺部疾病的气道防御。

DESCRIPTION (provided by applicant): Asthma has significant human health and economic impacts. Excessive mucus is an important cause of airflow obstruction in fatal asthma. It is also present in mild to moderate disease, but is poorly understood and treated. Mucus overproduction in asthma is associated with the dysregulated expression of two mucin glycoproteins - MUC5AC and MUC5B. Increased MUC5AC is a consistent finding, but MUC5B varies. It remains stably produced in some patients, but is strongly repressed in others (>90%), resulting in as much as 400-times more MUC5AC than MUC5B. This dichotomous expression pattern occurs in patients who display the strongest acute responses to the inhaled bronchoconstricting agent methacholine (MCh). This airway hyperreactivity (AHR) feature can be modeled in mice through allergen exposure. Knockout mice lacking Muc5ac are protected from AHR, indicating that it is a critical mediator of asthma-like airflow obstruction. Thus, determining specific mechanisms of MUC5AC-mediated AHR may reveal important mechanisms of obstruction and potential targets for improving airflow in asthma. As part of a long term goal of elucidating the functions of MUC5AC and MUC5B in the airways, Muc5ac and Muc5b knockout and overexpressing mice will be used to model dichotomous mucin expression in human asthma, and to test the specific effects of each mucin on the biophysical properties of mucus that promote obstruction. In addition to their differential expression, emerging data show that MUC5AC and MUC5B have distinct disulfide polymer structures. They also have specific glycosylation patterns: MUC5B is sialylated, MUC5AC is fucosylated. Fucosylation increases mucus viscoelasticity, and FUT2, the enzyme that catalyzes mucin �1,2-fucosylation, is associated with asthma exacerbation risk. Thus, the concept driving this proposal is that mucins with distinct polymer and glycan structures establish the obstructive potential of airway mucus. Accordingly, the proposed studies test the hypothesis that MUC5AC mediates AHR through specific disulfide and fucosylation mechanisms that cause obstruction by promoting mucus viscoelasticity. Three Specific Aims are proposed: 1) demonstrate that MUC5AC, but not MUC5B, mediates acute AHR in allergic mouse airways; 2) determine whether increased mucus viscoelasticity is caused by MUC5AC-specific polymerization and fucosylation; 3) determine whether MUC5AC disulfide and fucosylation-dependent viscoelastic properties are critical mechanisms of AHR and mucus plugging. Wild type and mucin mutant mice will be studied at baseline, and under conditions of allergic inflammation induced by Apergillus oryzae extract exposure. Lung function, mucociliary clearance, and mucus viscoelasticity will be measured. The effects of pharmacological, enzymatic, and genetic inhibition of polymerization and fucosylation will be determined. Successful completion of these studies will advance our mechanistic understanding of AHR, and could identify novel strategies to prevent obstruction while preserving airway defense in asthma and other lung diseases.