title: Biosynthesis of the polymeric gel-forming mucin MUC5B.
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.
authors.affiliation: 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.
authors.forename: Caroline
authors.initials: C
authors.lastname: Ridley
authors.affiliation: 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.
authors.forename: Sara
authors.initials: S
authors.lastname: Kirkham
authors.affiliation: 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.
authors.forename: Sally J
authors.initials: SJ
authors.lastname: Williamson
authors.affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina.
authors.forename: C William
authors.initials: CW
authors.lastname: Davis
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; and.
authors.forename: Philip
authors.initials: P
authors.lastname: Woodman
authors.affiliation: 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.
authors.forename: David J
authors.identifier: 0000-0001-7148-1970
authors.initials: DJ
authors.lastname: Thornton
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D019272:Leukocyte Elastase / Q000737:chemistry; D055257:Mucin-5B / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D059748:Proteolysis
chemical_list: C524578:MUC5B protein, human; D055257:Mucin-5B; D019272:Leukocyte Elastase
keywords: cystic fibrosis; goblet cell; mucin; mucus; von Willebrand factor