1. Function of the CysD domain of the gel-forming MUC2 mucin
- Author
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Ute Krengel, Elisabeth Thomsson, Daniel Ambort, Gunnar C. Hansson, Jenny Mackenzie, Malin E. V. Johansson, and Sjoerd van der Post
- Subjects
Glycosylation ,CID, collision-induced dissociation ,BN-PAGE, blue native PAGE ,disulfide bonds ,Mucin 2 ,Biochemistry ,chemistry.chemical_compound ,Cricetinae ,CK domain, cystine-knot domain ,MS/MS, tandem MS ,Disulfides ,Peptide sequence ,0303 health sciences ,ESI, electrospray ionization ,AGC, automatic gain control ,LC, liquid chromatography ,Chemistry ,030302 biochemistry & molecular biology ,Covalent bond ,non-covalent dimer ,Electrophoresis, Polyacrylamide Gel ,Domain of unknown function ,Dimerization ,Research Article ,Stereochemistry ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Size-exclusion chromatography ,CHO Cells ,TCEP-HCl, tris(2-carboxyethyl)phosphine-HCl ,PTS domain, proline, threonine and serine domain ,03 medical and health sciences ,Cricetulus ,C-mannosylation ,FBS, fetal bovine serum ,mucus ,Animals ,Humans ,mass spectrometry (MS) ,Amino Acid Sequence ,mAb, monoclonal antibody ,Molecular Biology ,030304 developmental biology ,Mucin-2 ,EK, enterokinase ,Mucin ,IMDM, Iscove's modified Dulbecco's medium ,Cell Biology ,Fusion protein ,Protein Structure, Tertiary ,CHO, Chinese-hamster ovary - 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.
- Published
- 2011
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