Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Respir Res. 2011 Sep 6;12(1):118. doi: 10.1186/1465-9921-12-118.
Excess mucus in the airways leads to obstruction in diseases such as chronic bronchitis, asthma, and cystic fibrosis. Mucins, the highly glycosolated protein components of mucus, are stored in membrane-bound granules housed in the cytoplasm of airway epithelial "goblet" cells until they are secreted into the airway lumen via an exocytotic process. Precise mechanism(s) of mucin secretion, including the specific proteins involved in the process, have yet to be elucidated. Previously, we have shown that the Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein regulates mucin secretion by orchestrating translocation of mucin granules from the cytosol to the plasma membrane, where the granules dock, fuse and release their contents into the airway lumen. Associated with MARCKS in this process are chaperone (Heat Shock Protein 70 [HSP70], Cysteine string protein [CSP]) and cytoskeletal (actin, myosin) proteins. However, additional granule-associated proteins that may be involved in secretion have not yet been elucidated.
Here, we isolated mucin granules and granule membranes from primary cultures of well differentiated human bronchial epithelial cells utilizing a novel technique of immuno-isolation, based on the presence of the calcium activated chloride channel hCLCA1 (the human ortholog of murine Gob-5) on the granule membranes, and verified via Western blotting and co-immunoprecipitation that MARCKS, HSP70, CSP and hCLCA1 were present on the granule membranes and associated with each other. We then subjected the isolated granules/membranes to liquid chromatography mass spectrometry (LC-MS/MS) to identify other granule associated proteins.
A number of additional cytoskeletal (e.g. Myosin Vc) and regulatory proteins (e.g. Protein phosphatase 4) associated with the granules and could play a role in secretion were discovered. This is the first description of the airway goblet cell "granulome."
气道中过多的黏液会导致慢性支气管炎、哮喘和囊性纤维化等疾病的气道阻塞。黏蛋白是黏液的高度糖基化蛋白成分，储存在位于气道上皮“杯状”细胞细胞质中的膜结合颗粒中，直到通过胞吐作用将其分泌到气道腔中。黏蛋白分泌的确切机制（包括参与该过程的特定蛋白质）尚未阐明。以前，我们已经表明，豆蔻酰化丙氨酸丰富的蛋白激酶底物（MARCKS）蛋白通过协调黏蛋白颗粒从细胞质到质膜的易位来调节黏蛋白分泌，在质膜中，颗粒停靠、融合并将其内容物释放到气道腔中。在此过程中与 MARCKS 相关的是伴侣蛋白（热休克蛋白 70 [HSP70]、半胱氨酸串蛋白 [CSP]）和细胞骨架（肌动蛋白、肌球蛋白）蛋白。然而，尚未阐明可能参与分泌的其他颗粒相关蛋白。
在这里，我们利用基于钙激活氯离子通道 hCLCA1（鼠 Gob-5 的人同源物）存在于颗粒膜上的新型免疫分离技术，从分化良好的人支气管上皮细胞的原代培养物中分离出黏蛋白颗粒和颗粒膜，并通过 Western blot 和共免疫沉淀验证了 MARCKS、HSP70、CSP 和 hCLCA1 存在于颗粒膜上并相互关联。然后，我们将分离的颗粒/膜进行液相色谱-质谱联用（LC-MS/MS）以鉴定其他颗粒相关蛋白。
发现了一些与颗粒相关的额外细胞骨架（例如肌球蛋白 Vc）和调节蛋白（例如蛋白磷酸酶 4），它们可能在分泌中发挥作用。这是对气道杯状细胞“颗粒组”的首次描述。
