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The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.

ABSTRACT: Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.

SUBMITTER: Ridley C

PROVIDER: S-EPMC6851316 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.

Ridley Caroline C Lockhart-Cairns Michael P MP Collins Richard F RF Jowitt Thomas A TA Subramani Durai B DB Kesimer Mehmet M Baldock Clair C Thornton David J DJ

The Journal of biological chemistry 20190930 45

Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, ...[more]

PMID: 31570524

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Project description:Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5-2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.

Project description:Objective:Paraquat (PQ), a widely used toxic herbicide, induces lung inflammation through mechanisms that remain incompletely understood. In a previous study, we found that the plasma MUC5B mucin level was implicated in PQ poisoning in patients. Here, we hypothesize that MUC5B is a critical mediator in PQ-induced cell inflammation. Methods:A mouse model of PQ-induced lung injury was used to examine the MUC5B expression level. A549 cells (alveolar epithelial cells line) were exposed to PQ in dose-dependent and time-dependent manners. Cell viability was detected by CCK-8 assays. The expression levels of MUC5B were examined by dot blot enzyme-linked immunosorbent assay (ELISA) and RT-qPCR. Western blotting was used to detect the levels of proteins in the MAPK and NF-?B pathways. Inflammatory factors in the cell culture medium were measured by ELISA. NF-?B and MAPK pathway inhibitors and MUC5B siRNA (siMUC5B) were used to determine the function of MUC5B. Finally, N-acetyl-cysteine (NAC) was added and its regulatory effect on the MAPK-NF-?B-MUC5B pathway was examined in PQ-induced cell inflammation. Results:MUC5B was significantly upregulated accompanying the increases in TNF-? and IL-6 secretion following PQ treatment in mouse and also in A549 cells after treatment with 50??M PQ at 24 hours. Furthermore, MAPK and NF-?B pathway inhibitors could dramatically decrease the expression of MUC5B and the secretion of TNF-? and IL-6. Importantly, siMUC5B could significantly attenuate the secretion of TNF-? and IL-6 induced by PQ. As expected, the addition of NAC efficiently suppresses the TNF-? and IL-6 secretion stimulated from PQ and also downregulated ERK, JNK, and p65 phosphorylation (ERK/JNK MAPK and NF-?B pathways) as well as MUC5B expression. Conclusion:Our findings suggest that MUC5B participates in the process of PQ-induced cell inflammation and is downstream of the NF-?B and MAPK pathways. NAC can attenuate PQ-induced cell inflammation at least in part by suppressing the MAPK-NF-?B-MUC5B pathway. These results nominate MUC5B as a new biomarker and therapeutic target for PQ-induced lung inflammation.

Dataset Information

The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.

Publications

The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.

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