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Function of the CysD domain of the gel-forming MUC2 mucin
- Source :
- Biochemical Journal; Vol 436, Biochemical Journal
- Publication Year :
- 2011
-
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.
- 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
Subjects
Details
- Language :
- English
- ISSN :
- 02646021
- Volume :
- 436
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....4a8bf23ceff9561b204aa991b20e5ec7
- Full Text :
- https://doi.org/10.1042/bj20102066