Back to Search
Start Over
Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.
Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.
- Source :
-
Journal of proteome research [J Proteome Res] 2014 Dec 05; Vol. 13 (12), pp. 6013-23. Date of Electronic Publication: 2014 Nov 25. - Publication Year :
- 2014
-
Abstract
- The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins.
- Subjects :
- Amino Acid Sequence
Animals
Binding Sites
CHO Cells
Chromatography, Liquid
Chymotrypsin metabolism
Cricetinae
Cricetulus
Electrophoresis, Polyacrylamide Gel
Glycosylation
Humans
Metalloendopeptidases metabolism
Molecular Sequence Data
Mucin-2 chemistry
Mucin-2 genetics
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase metabolism
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Subtilisin metabolism
Tandem Mass Spectrometry
Trypsin metabolism
Glycopeptides metabolism
Hydrolases metabolism
Mucin-2 metabolism
Polysaccharides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1535-3907
- Volume :
- 13
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Journal of proteome research
- Publication Type :
- Academic Journal
- Accession number :
- 25406038
- Full Text :
- https://doi.org/10.1021/pr500874f