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。粘液过多是致命性哮喘患者气流阻塞的重要原因。它也存在于轻到中度的疾病中，但对它的了解和治疗很少。哮喘的粘液过度产生与两种粘蛋白糖蛋白--MUC5AC和MUC5B的异常表达有关。MUC5AC增加是一致的发现，但MUC5B有所不同。它在一些患者中仍然稳定产生，但在另一些患者(&gt；90%)中被强烈抑制，导致MUC5AC比MUC5B多400倍。这种二分表达模式发生在对吸入性支气管收缩药乙酰甲胆碱(MCH)表现出最强烈急性反应的患者。这种呼吸道高反应性(AHR)特征可以通过过敏原暴露在小鼠身上建模。缺乏Muc5ac的基因敲除小鼠对AHR具有保护作用，这表明它是哮喘样气流阻塞的关键介质。因此，确定MUC5AC介导的AHR的具体机制可能揭示哮喘的重要阻塞机制和改善气流的潜在靶点。作为阐明MUC5AC和MUC5B在呼吸道中的功能的长期目标的一部分，MUC5AC和MUC5B基因敲除和过度表达的小鼠将被用来模拟人类哮喘中粘蛋白的二分表达，并测试每种粘蛋白对促进阻塞的粘液生物物理性质的特定影响。除了它们的差异表达外，新出现的数据表明MUC5AC和MUC5B具有不同的二硫键聚合物结构。它们也有特定的糖基化模式：MUC5B是唾液酸化的，MUC5AC是岩藻糖化的。岩藻糖基化增加了粘液的粘弹性，而催化粘蛋白�1，2-岩藻糖化的酶FUT2与哮喘恶化的风险有关。因此，推动这一建议的概念是，具有不同聚合物和多糖结构的粘蛋白建立了呼吸道粘液的阻塞潜力。因此，建议的研究验证了这样的假设，即MUC5AC通过特定的二硫键和岩藻糖基化机制介导AHR，这些机制通过促进粘液粘弹性而导致阻塞。提出了三个特定的目标：1)证明MUC5AC而不是MUC5B介导了过敏小鼠呼吸道的急性AHR；2)确定粘液粘弹性增加是否由MUC5AC特异性聚合和岩藻糖基化引起；3)确定MUC5AC依赖的二硫键和岩藻糖基化依赖的粘弹性特性是否是AHR和粘液堵塞的关键机制。野生型和粘蛋白突变小鼠将在基线和米曲霉提取物暴露引起的过敏性炎症条件下进行研究。将测量肺功能、粘液纤毛清除和粘液粘弹性。将确定聚合和岩藻糖化的药理、酶和遗传抑制的效果。这些研究的成功完成将促进我们对AHR机制的理解，并可能确定在哮喘和其他肺部疾病中预防阻塞同时保护呼吸道防御的新策略。

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.