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The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment.

Journal article
Authors Klaus Godl
Malin E V Johansson
Martin Lidell
Matthias Mörgelin
Hasse Karlsson
Fredrik J. Olson
James R Gum
Young S Kim
Gunnar C. Hansson
Published in The Journal of biological chemistry
Volume 277
Issue 49
Pages 47248-56
ISSN 0021-9258
Publication year 2002
Published at Institute of Medical Biochemistry
Pages 47248-56
Language en
Links dx.doi.org/10.1074/jbc.M208483200
Keywords Amino Acid Sequence, Animals, CHO Cells, Chromatography, Gel, Cricetinae, Dimerization, Disulfides, Electrophoresis, Polyacrylamide Gel, Genetic Vectors, Glycosylation, Hydrofluoric Acid, pharmacology, Immunoblotting, Microscopy, Electron, Molecular Sequence Data, Mucin-2, Mucins, chemistry, metabolism, Mutagenesis, Site-Directed, Peptides, chemistry, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trypsin, pharmacology
Subject categories Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Abstract

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.

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