Mucin granule-associated proteins in human bronchial epithelial cells: the airway goblet cell "granulome"

Abstract

BackgroundExcess 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.MethodsHere, 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.ResultsA 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."

Reprogrammed and transmissible intestinal microbiota confer diminished susceptibility to induced colitis in TMF −/− mice

“…This finding implies that TMF is required for mucus granule fusion, after granule translocation from the trans-Golgi compartment. These findings are corroborated by previous, independent proteomic studies, which indicated that TMF is part of the mucus granule complex and even associates with the granule membrane (22,23). To confirm this association, we used florescence microscopy to show colocalization of TMF with the MUC2 mucin in human colonic goblet cells.…”

IL-13–induced airway mucus production is attenuated by MAPK13 inhibition

“…Immunostaining was specific for CLCA1 and MUC5AC since there was no staining above background in the absence of primary antibody (Supplemental Figure 3). These findings are consistent with coordinated packaging of CLCA1 and MUC5AC within mucous granules as described previously (24,25) and, based on the present findings, concerted synthesis and secretion of CLCA1 and MUC5AC to the apical cell surface in response to IL-13-stimulation as well.…”

Protein composition of bronchoalveolar lavage fluid and airway surface liquid from newborn pigs

“…Thus it is anticipated that apocrine secretion could result in disruption of cell membranes and introduction of intracellular and membrane proteins into the ASL. In support of this possibility, a recent proteomic study demonstrated that membrane-bound granules secreted from airway epithelial goblet cells contain cytoskeletal and regulatory proteins as well as mucins, suggesting that such granules may be a source for some of the intracellular proteins detected in ASL (69). Additionally, several groups have reported the presence of exosomes, microvesicles implicated in host defense and cell communication, in human BAL and cell culture secretions (1,39).…”