title: Intestinal MUC2 mucin supramolecular topology by packing and release resting on D3 domain assembly.
abstract: MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.|Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
authors.forename: Harriet E
authors.initials: HE
authors.lastname: Nilsson
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden.|Mammalian Protein Expression Core Facility, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ambort
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Bäckström
authors.affiliation: Mammalian Protein Expression Core Facility, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Thomsson
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
authors.forename: Philip J B
authors.initials: PJB
authors.lastname: Koeck
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
authors.forename: Gunnar C
authors.initials: GC
authors.lastname: Hansson
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
authors.forename: Hans
authors.initials: H
authors.lastname: Hebert
mesh_terms: D000818:Animals; D016466:CHO Cells; D003106:Colon / Q000737:chemistry / Q000378:metabolism; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D007413:Intestinal Mucosa / Q000378:metabolism; D008854:Microscopy, Electron; D055262:Mucin-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C524625:MUC2 protein, human; D055262:Mucin-2; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
keywords: cystic fibrosis; electron microscopy; intestine; mucin; structure