title: Expression and characterization of a novel recombinant version of the secreted human mucin MUC5AC in airway cell lines.
abstract: Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins' size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease.
authors.affiliation: The School of Medicine and Medical Science, ‡Conway Institute of Biomolecular and Biomedical Research, and §School of Veterinary Medicine, University College Dublin , Dublin, Ireland.
authors.forename: Aindrias
authors.initials: A
authors.lastname: Ryan
authors.forename: Angeline
authors.initials: A
authors.lastname: Smith
authors.forename: Patrick
authors.initials: P
authors.lastname: Moore
authors.forename: Susan
authors.initials: S
authors.lastname: McNally
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Carrington
authors.forename: Colm J
authors.initials: CJ
authors.lastname: Reid
authors.forename: Marguerite
authors.initials: M
authors.lastname: Clyne
mesh_terms: D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D055271:Mucin 5AC / Q000096:biosynthesis* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*
chemical_list: C524678:MUC5AC protein, human; D055271:Mucin 5AC; D011994:Recombinant Proteins