title: Function of the CysD domain of the gel-forming MUC2 mucin.
abstract: The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, Medicinaregatan 9A, SE-413 90 Gothenburg, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ambort
authors.forename: Sjoerd
authors.initials: S
authors.lastname: van der Post
authors.forename: Malin E V
authors.initials: ME
authors.lastname: Johansson
authors.forename: Jenny
authors.initials: J
authors.lastname: Mackenzie
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Thomsson
authors.forename: Ute
authors.initials: U
authors.lastname: Krengel
authors.forename: Gunnar C
authors.initials: GC
authors.lastname: Hansson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D055262:Mucin-2 / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins
chemical_list: D004220:Disulfides; C524625:MUC2 protein, human; D055262:Mucin-2; D011993:Recombinant Fusion Proteins