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Expression and characterisation of a novel recombinant version of the secreted human mucin MUC5AC in airway cell lines Aindrias Ryan, Angeline Smith, Patrick Moore, Susan McNally, Stephen D. Carrington, Colm J. Reid, and Marguerite Clyne Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi5011267 • Publication Date (Web): 05 Jan 2015 Downloaded from http://pubs.acs.org on January 12, 2015
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Biochemistry
A. Title
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Expression and characterisation of a novel recombinant version of the secreted human mucin
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MUC5AC in airway cell lines
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B. Funding Source Statement
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This work was supported by a PhD studentship award to Aindrias Ryan from Irish Research Council for
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Science, Engineering and Technology [Postgraduate Research Scholarship Scheme 2006], by a grant from
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the Childrens Medical and Research Foundation, Dublin, Ireland to Marguerite Clyne, Colm Reid and
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Stephen Carrington [R344], from Science Foundation Ireland [08/SRC/B1393] to Marguerite Clyne and
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Colm Reid and from the Cystic Fibrosis Association of Ireland and Health Research Board
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[MRCG/2011/10] to Marguerite Clyne.
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C. Byline
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Aindrias Ryan*†, Angeline Smith‡, Patrick Moore*†, Susan McNally‡, Stephen D. Carrington‡, Colm
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J. Reid‡§ and Marguerite Clyne*†§
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*The School of Medicine and Medical Science and †Conway Institute of Biomolecular and Biomedical
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Research, University College Dublin,
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‡
School of Veterinary Medicine, University College Dublin, Ireland. §
Joint senior authors
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To whom correspondence should be addressed. Dr. Marguerite Clyne, School of Medicine and Medical
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Science, University College Dublin, Belfield, Dublin 4, Ireland. Tel: 00353-1-7166619, Fax: 00353-1-
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7166649; Email:
[email protected].
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Biochemistry
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ABBREVIATIONS
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CF Cystic Fibrosis
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CFTR Cystic Fibrosis Transmembrane Conductance Regulator
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CK cysteine knot
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COPD chronic obstructive pulmonary disease
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DIG Digoxigenin
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ER endoplasmic reticulum
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GNA Galanthus nivalis agglutinin
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Histidine His
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HRP Horse Radish Peroxidase
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NR non-reduced
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PTS proline, threonine and serine
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PVDF Immobilon-P Polyvinylidene Difluoride
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R reduced
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TR tandem repeat
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VNTRs variable number tandem repeat regions
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vWF von Willebrand factor
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Biochemistry
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ABSTRACT
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Molecular manipulations and expression of mucins, large glycoproteins that provide the structural
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framework of mucus, is challenging due to their size and numerous domains including variable number
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tandem repeat regions (VNTRs), sites of O-glycosylation. Only individual human mucin domains have
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been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted
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mucin in the respiratory tract, encoding the N-terminus, C- terminus, N- and C- terminus together and N-
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and C-terminus interspersed with two native tandem repeat (TR) sequences in tracheal and bronchial cell
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lines. This latter protein contains all the functional domains required for the biosynthesis and secretion of
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glycosylated mucin. The N- terminus protein was found in a monomeric and higher molecular weight
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forms suggesting that secreted MUC5AC may form a branched net-like structure analogous to that
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described for MUC2. Proteins underwent cleavage at the C-terminus, polymerization and glycosylation.
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Thus, they appear to undergo pivotal processing steps in an analogous fashion to that for other individual
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recombinant mucin domains and as predicted for native MUC5AC. Secretion occurred when cells were
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grown on transwell filter inserts but not when they were grown on plastic indicating that the extracellular
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environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in
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molecular mass from the intracellular form indicating that additional processing occurred. These
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recombinant proteins expressed in different backgrounds offer the potential to address the role of different
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mucin domains on MUC5AC processing and function and the role of MUC5AC in health and disease.
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Mucins, a family of high molecular mass glycoproteins, may be membrane bound or secreted, forming the
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structural scaffolding for viscoelastic mucus gels at the apical surface of mucosal epithelia. They consist
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of an apomucin protein core containing proline, threonine and serine (PTS) rich sequences which are
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frequently organised in repeats or VNTR (Variable Number Tandem Repeat) domains that are sites of
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extensive O-glycosylation.1 Their heavy O-linked glycosylation along with further modifications
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including sulfation, imparts mucus with unique rheological, structural and chemical properties.2 The
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secreted gel forming mucins identified in humans are MUC2, MUC5AC, MUC5B, MUC6 and MUC19.2,3
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MUC5B and MUC5AC are the major secreted gel forming mucins in the airways.4-6 There is major
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interest in the role of these secreted mucins in respiratory diseases such as CF (Cystic Fibrosis) and
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COPD (chronic obstructive pulmonary disease), both characterized by hypersecretion of mucus,
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pulmonary obstruction, reduced mucocillary clearance and chronic bacterial infection.7
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Recombinant mucins have been an important tool in understanding the structure and the
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biological functioning of mucins.8-11 N- and C-termini of the human secreted mucin MUC2 and the C-
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terminus of MUC5AC have been expressed in a variety of cell lines including CHO-K1 hamster ovary
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cells and the LS174T human colonic cell line.8-11 More recently the N terminus of MUC5B and MUC6
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have also been cloned and expressed.12,13 Results with MUC5AC and MUC2 recombinant mucins have
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shown that dimerization of mucins occur at the C-terminus in an N-glycosylation dependent manner14 and
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both mucins have been shown to be post-translationally cleaved in the C-terminus cysteine rich region of
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the peptide.9-11 This autocatalytic cleavage occurs at low pH (
