Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27517-7248.
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27517-7248.
Proc Natl Acad Sci U S A. 2021 Sep 28;118(39). doi: 10.1073/pnas.2104490118.
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 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，MUC5AC 是肺部主要的凝胶形成粘蛋白之一。然而，目前尚不清楚 MUC5AC 的结构和/或凝胶形成特性如何有助于健康状态下的固有肺防御，并导致疾病中粘性痰液的形成。为了了解这一点，我们在这里研究了 MUC5AC 与 MUC5B 的生物物理特性和高分子组装。为了研究每种天然粘蛋白，我们使用产生 MUC5AC 或 MUC5B 的 Calu3 单粘蛋白培养物。为了通过 N 端寡聚化来了解 MUC5AC 的大分子组装，我们表达了重组全长 N 端结构域（5ACNT）。扫描电子显微镜和原子力显微镜成像表明，两种粘蛋白在上皮细胞和实验表面上形成不同的网络；MUC5B 形成线性的、很少分支的多聚体，而 MUC5AC 形成紧密组织的网络，具有高度分支。石英晶体微天平耗散监测实验表明，与 MUC5B 相比，MUC5AC 与疏水面的结合显著增加，并且刚性更大、粘弹性更高。光散射分析确定 5ACNT 主要形成二硫键连接的共价二聚体和更高阶的寡聚体（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，MUC5AC 通过其 N 端形成二聚体和更高阶的寡聚体。总之，MUC5AC 独特的 N 端组织可能解释了分支的、高度网络化的 MUC5AC 凝胶具有更强的粘性和独特的粘弹性特性。这些特性可能为我们提供深入了解 MUC5AC 如何在慢性粘液阻塞性肺病中形成静态、“束缚”粘液层的原因/机制。
