Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester Academic Health Sciences Centre, Manchester, M13 9PT, UK.
School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Sciences Centre, Manchester, M13 9PT, UK.
Sci Rep. 2019 Nov 22;9(1):17350. doi: 10.1038/s41598-019-53768-0.
The polymeric mucin MUC5B provides the structural and functional framework of respiratory mucus, conferring both viscoelastic and antimicrobial properties onto this vital protective barrier. Whilst it is established that MUC5B forms disulfide-linked linear polymers, how this relates to their packaging in secretory granules, and their molecular form in mucus remain to be fully elucidated. Moreover, the role of the central heavily O-glycosylated mucin domains in MUC5B conformation is incompletely described. Here we have completed a detailed structural analysis on native MUC5B polymers purified from saliva and subsequently investigated how MUC5B conformation is affected by changes in calcium concentration and pH, factors important for mucin intragranular packaging and post-secretory expansion. The results identify that MUC5B has a beaded structure repeating along the polymer axis and suggest that these repeating motifs arise from distinct glycosylation patterns. Moreover, we demonstrate that the conformation of these highly entangled linear polymers is sensitive to calcium concentration and changes in pH. In the presence of calcium (Ca, 10 mM) at pH 5.0, MUC5B adopted a compact conformation which was lost either upon removal of calcium with EGTA, or by increasing the pH to 7.4. These results suggest a pathway of mucin collapse to enable intracellular packaging and mechanisms driving mucin expansion following secretion. They also point to the importance of the tight control of calcium and pH during different stages of mucin biosynthesis and secretion, and in the generation of correct mucus barrier properties.
聚合粘蛋白 MUC5B 为呼吸黏液提供了结构和功能框架，使这种重要的保护屏障具有粘弹性和抗微生物特性。虽然已经确定 MUC5B 形成二硫键连接的线性聚合物，但它们如何与分泌颗粒中的包装以及在黏液中的分子形式相关，仍有待充分阐明。此外，MUC5B 构象中核心高度 O-糖基化粘蛋白结构域的作用描述得还不完全。在这里，我们对从唾液中纯化的天然 MUC5B 聚合物进行了详细的结构分析，随后研究了 MUC5B 构象如何受到钙浓度和 pH 值变化的影响，这些因素对粘蛋白的颗粒内包装和分泌后扩张很重要。结果表明，MUC5B 具有沿聚合物轴重复的珠状结构，并表明这些重复基序源自不同的糖基化模式。此外，我们证明了这些高度缠结的线性聚合物的构象对钙浓度和 pH 值变化敏感。在 pH 5.0 时存在钙 (Ca，10 mM) 的情况下，MUC5B 采用紧凑的构象，当用 EGTA 去除钙或将 pH 值增加到 7.4 时，这种构象会丢失。这些结果表明了粘蛋白坍塌的途径，以实现细胞内包装，并在分泌后驱动粘蛋白扩张的机制。它们还表明，在粘蛋白生物合成和分泌的不同阶段，以及在产生正确的黏液屏障特性时，钙和 pH 值的严格控制非常重要。
