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Am J Physiol Lung Cell Mol Physiol. 2016 May 15;310(10):L993-L1002. doi: 10.1152/ajplung.00046.2016. Epub 2016 Mar 18.

Biosynthesis of the polymeric gel-forming mucin MUC5B.

Author information

1
Wellcome Trust Centre for Cell-Matrix Research, The University of Manchester, Manchester, United Kingdom; Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; and.
2
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina.
3
Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; and.
4
Wellcome Trust Centre for Cell-Matrix Research, The University of Manchester, Manchester, United Kingdom; Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; and dave.thornton@manchester.ac.uk.

Abstract

MUC5B is a major polymeric mucin in the airway mucus gel and is an essential component of innate defense of the respiratory epithelium. Knowledge of the synthesis and intracellular processing of MUC5B is incomplete. We investigated the molecular details of MUC5B assembly in primary human bronchial epithelial cells (HBECs) grown at an air-liquid interface (ALI). Electrophoretic and centrifugal separations of intracellular forms of MUC5B probed with antibodies specific for non-O-glycosylated and O-glycosylated forms of the mucin identified three major intracellular populations of MUC5B (non-O-glycosylated monomer and dimer, and O-glycosylated polymers). Biophysical analysis of recombinant MUC5B COOH-terminus (CT5B; D4-B-C-CK) expressed in 293-EBNA cells showed that MUC5B dimerizes by disulfide linkage. Pulse-chase studies in the HBEC ALI cultures showed that non-O-glycosylated MUC5B was synthesized within 20 min of metabolic labeling and O-glycosylated, polymeric mucin within 2 h. Radiolabeled O-glycosylated mucin polymers were secreted within 2 h and the majority were released by 48 h. These data indicate that MUC5B follows a similar assembly to the related glycoprotein, von Willebrand factor (vWF); however, unlike vWF the MUC5B polypeptide shows no evidence of major proteolytic processing of D-domains during the production of the mature secreted polymeric mucin in normal and cystic fibrosis (CF) primary bronchial epithelial cells. In contrast, MUC5B D-domains were modified by neutrophil elastase, a protease commonly found in CF sputum, demonstrating that proteolytic degradation of MUC5B is an extracellular event in CF sputum. These results define the pathway for synthesis of MUC5B in primary human goblet cells.

KEYWORDS:

cystic fibrosis; goblet cell; mucin; mucus; von Willebrand factor

PMID:
26993521
PMCID:
PMC4896102
DOI:
10.1152/ajplung.00046.2016
[Indexed for MEDLINE]
Free PMC Article

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