Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, “tethered” mucus layer in chronic muco-obstructive lung diseases.

MUC5AC（肺中主要的凝胶形成粘蛋白之一）水平升高与慢性阻塞性肺病（如慢性支气管炎和哮喘）密切相关。然而，目前尚不清楚 MUC5AC 的结构和/或凝胶制造特性如何有助于健康中的先天性肺防御并推动疾病中停滞粘液的形成。为了理解这一点，我们在这里研究了 MUC5AC 与 MUC5B 相比的生物物理特性和大分子组装。为了研究每种天然粘蛋白，我们使用了产生 MUC5AC 或 MUC5B 的 Calu3 单粘蛋白培养物。为了了解 MUC5AC 通过 N 端寡聚化的大分子组装，我们表达了一个重组的整个 N 端结构域 (5ACNT)。扫描电子显微镜和原子力显微镜成像表明，两种粘蛋白在上皮和实验表面形成了不同的网络；MUC5B 形成线性的、不常分支的多聚体，而 MUC5AC 形成具有高度分支的紧密组织的网络。石英晶体微量天平耗散监测实验表明，与 MUC5B 相比，MUC5AC 与疏水表面的结合明显更多，并且更硬且更具粘弹性。光散射分析确定 5ACNT 主要形成二硫键连接的共价二聚体和高级寡聚体（即三聚体和四聚体）。全长分子的中心糖基化区域的选择性蛋白水解消化证实了 MUC5AC 通过其 N 末端形成二聚体和高级寡聚体。集体，MUC5AC 独特的 N 端组织可以解释支链、高度网络化的 MUC5AC 凝胶更具粘性和独特的粘弹性。这些特性可能有助于了解 MUC5AC 为何/如何在慢性黏液阻塞性肺病中形成静态的“束缚”黏液层。